WO2011031977A1 - Intermittent and pulse lithium treatments for scar revision and wound healing - Google Patents

Intermittent and pulse lithium treatments for scar revision and wound healing Download PDF

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WO2011031977A1
WO2011031977A1 PCT/US2010/048439 US2010048439W WO2011031977A1 WO 2011031977 A1 WO2011031977 A1 WO 2011031977A1 US 2010048439 W US2010048439 W US 2010048439W WO 2011031977 A1 WO2011031977 A1 WO 2011031977A1
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lithium
skin
treatment
wound
acid
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PCT/US2010/048439
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French (fr)
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William D. Ju
Stephen M. Prouty
Shikha P. Barman
Scott C. Kellogg
Eric Schweiger
Seth Lederman
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Follica, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/18—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing inorganic materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
    • A61K8/20—Halogens; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/36—Carboxylic acids; Salts or anhydrides thereof
    • A61K8/365—Hydroxycarboxylic acids; Ketocarboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B18/00—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body
    • A61B18/18—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves
    • A61B18/20—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves using laser
    • A61B18/203—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves using laser applying laser energy to the outside of the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B18/00—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body
    • A61B2018/00315—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body for treatment of particular body parts
    • A61B2018/00452—Skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/81—Preparation or application process involves irradiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/0613—Apparatus adapted for a specific treatment
    • A61N5/0616—Skin treatment other than tanning

Definitions

  • the invention relates to intermittent lithium treatments, or a single pulse lithium treatment for scar revision and wound healing in human subjects.
  • Uses of compositions containing compounds that liberate lithium ions are described, including adjuvants and devices for administration.
  • the intermittent treatment protocol involves multiple courses of lithium treatment interrupted by lithium treatment "holidays.”
  • a dose of lithium is administered over a short period of time.
  • the lithium treatment(s) can be used in combination with other treatments for scar revision, wound healing, and hair follicle neogenesis.
  • Such combination treatments may involve mechanical or physical treatments that modulate scar revision or wound healing, or that cause integumental perturbation ⁇ e.g.
  • the combination treatment(s) may be administered concurrently with, or during the "holidays" between, cycles of intermittent lithium treatments; or concurrently with, or before and/or after the pulse lithium treatment.
  • Wound healing or wound repair, is an intricate process in which the skin (or some other organ) repairs itself after injury.
  • the epidermis (outermost layer) and dermis (inner or deeper layer) exist in a steady-state equilibrium, forming a protective barrier against the external environment. Once the protective barrier is broken, the physiologic process of wound healing is immediately set in motion.
  • the classic model of wound healing is divided into three or four sequential, yet overlapping, phases: (1) hemostasis (not considered a phase by some authors); (2) inflammation; (3) proliferation; and (4) maturation and remodeling.
  • fibrin clot When tissue is wounded, blood platelets (thrombocytes) aggregate at the injury site to form a fibrin clot. This clot acts to control active bleeding (hemostasis). Fibrin and fibronectin crosslink to form a plug that traps proteins and particles and prevent further blood loss. This fibrin-fibronectin plug, also called the extracellular matrix, is also the main structural support for the wound until collagen is deposited. Migratory cells use this plug as a matrix to crawl across, and platelets adhere to it and secrete factors. The clot is eventually lysed and replaced with granulation tissue and then later with collagen.
  • the contact of blood with collagen during wounding triggers platelets to begin secreting inflammatory factors, extracellular matrix proteins, cytokines, and growth factors.
  • the proinflammatory factors released by platelets like serotonin, bradykinin, prostaglandins, prostacyclins, thromboxane, and histamine, increase cell proliferation and migration to the area, cause blood vessels to initially constrict to prevent further blood loss, and then cause blood vessels to become dilated and porous.
  • Vasoconstriction lasts five to ten minutes and is followed by vasodilation, a widening of blood vessels, which peaks at about 20 minutes post- wounding.
  • the main factor involved in vasodilation is histamine, which also causes blood vessels to become porous, allowing the tissue to become edematous. Increased porosity of blood vessels facilitates the entry of inflammatory cells into the wound site from the bloodstream.
  • PMNs polymorphonuclear neutrophils
  • helper T cells which secrete cytokines to cause more T cells to divide and to increase inflammation and enhance vasodilation and vessel permeability. T cells also increase the activity of macrophages.
  • Gamma-delta T cells are enriched in epidermis and become activated upon wounding, secreting growth factors that induce keratinocyte proliferation (Havran and Jameson, 2010).
  • Macrophages are essential to wound healing. They replace PMNs as the predominant cells in the wound by two days after injury. Attracted to the wound site by growth factors released by platelets and other cells, monocytes from the bloodstream enter the area through blood vessel walls. Numbers of monocytes in the wound peak one to one and a half days after the injury occurs. Once they are in the wound site, monocytes mature into macrophages. The main role of macrophages is to phagocytose bacteria and damaged tissue, and they also debride damaged tissue by releasing proteases.
  • Macrophages also produce factors that induce and speed angiogenesis; secrete a number of factors such as growth factors and other cytokines, especially during the third and fourth post-wounding days, which attract cells involved in the proliferative stage of healing; and stimulate cells that re-epithelialize the wound, create granulation tissue, and lay down a new extracellular matrix.
  • factors such as growth factors and other cytokines, especially during the third and fourth post-wounding days, which attract cells involved in the proliferative stage of healing; and stimulate cells that re-epithelialize the wound, create granulation tissue, and lay down a new extracellular matrix.
  • inflammation As inflammation dies down, fewer inflammatory factors are secreted, existing ones are broken down, and numbers of neutrophils and macrophages are reduced at the wound site. Because inflammation plays a role in fighting infection, clearing debris and inducing the proliferative phase, it is a necessary part of healing. However, inflammation can lead to tissue damage if it lasts too long. Thus the reduction of inflammation is frequently a goal in therapeutic settings. Inflammation lasts as long as there is debris in the wound. Thus the presence of dirt or other objects can extend the inflammatory phase for too long, leading to a chronic wound.
  • the proliferative phase is characterized by the overlapping steps of angiogenesis, collagen deposition, granulation tissue formation, epithelialization, and wound contraction.
  • angiogenesis neovascularization
  • new blood vessels are formed by vascular endothelial cells.
  • angiogenesis is also imperative for other stages in wound healing.
  • Endothelial growth and proliferation is stimulated by the presence of lactic acid in the wound and by hypoxia.
  • Endothelial stem cells from uninjured blood vessels attracted to the wound by fibronectin found on the fibrin scab and chemotactically by angiogenic factors released by other cells, migrate through the extracellular matrix (ECM) into the wound site to establish new blood vessels.
  • ECM extracellular matrix
  • the tissue in which angiogenesis has occurred typically looks red (is erythematous) due to the presence of capillaries.
  • Granulation tissue functions as rudimentary tissue, and begins to appear in the wound during the inflammatory phase, two to five days post wounding, and continues growing until the wound bed is covered.
  • Granulation tissue consists of new blood vessels, fibroblasts, inflammatory cells, endothelial cells, myofibroblasts, and the components of a new, provisional ECM.
  • the provisional ECM is different in composition from the ECM in normal tissue. Its components - fibronectin, hyaluronan, collagen, glycosaminoglycans, elastin, glycoproteins and proteoglycans - originate from fibroblasts. Later this provisional matrix is replaced with an ECM that more closely resembles that found in non-injured tissue.
  • Fibroblasts begin entering the wound site two to five days after wounding and their numbers peak at one to two weeks post-wounding. In the first two or three days after injury, fibroblasts mainly proliferate and migrate, while later, they are the main cells that lay down the collagen matrix in the wound site. Fibroblasts from normal tissue migrate into the wound area from its margins. Initially fibroblasts use the fibrin scab formed in the inflammatory phase to migrate, adhering to fibronectin. Fibroblasts then deposit ground substance into the wound bed, and later collagen, which they can adhere to for migration.
  • Growth factors e.g., PDGF, TGF- ⁇
  • fibronectin encourage proliferation, migration to the wound bed, and production of ECM molecules by fibroblasts.
  • Hypoxia also contributes to fibroblast proliferation and secretion of growth factors (e.g. , growth factors that attract epithelial cells to the wound site), though too little oxygen inhibits proliferation and deposition of ECM components, which may lead to excessive, fibrotic scarring. Fibroplasia ends two to four weeks after wounding.
  • Fibroblasts begin secreting appreciable collagen by the second or third post-wounding day, and its deposition peaks at one to three weeks. Collagen deposition is important because it increases the strength of the wound; before it is laid down, the only thing holding the wound closed is the fibrin- fibronectin clot, which does not provide much resistance to traumatic injury. Also, cells involved in inflammation, angiogenesis, and connective tissue
  • Keratinocytes migrate without first proliferating. Migration of keratinocytes over the wound site is stimulated by lack of contact inhibition and by chemicals such as nitric oxide. Migration can begin as early as a few hours after wounding, although the time of onset of migration is variable. Epithelial cells require viable tissue to migrate across, so if the wound is deep it must first be filled with granulation tissue. Cells on the wound margins proliferate on the second and third day post-wounding in order to provide more cells for migration.
  • Epithelial cells climb over one another in order to migrate. This growing sheet of epithelial cells is often called the epithelial tongue. Basal and suprabasal cells become mobilized and both migrate and get “pushed” into the wound site (Usui et al, 2005). The more quickly this movement occurs, the less of a scar there will be.
  • Fibrin, collagen, and fibronectin in the ECM may further signal cells to divide and migrate.
  • migrating keratinocytes use the fibronectin cross-linked with fibrin that was deposited in inflammation as an attachment site to crawl across. As keratinocytes migrate, they move over granulation tissue but underneath the scab (if one was formed), separating it from the underlying tissue. Epithelial cells have the ability to phagocytose debris such as dead tissue and bacterial matter that would otherwise obstruct their path. Because they must dissolve any scab that forms, keratinocyte migration is best enhanced by a moist environment, since a dry one leads to formation of a bigger, tougher scab.
  • keratinocytes To make their way along the tissue, keratinocytes must dissolve the clot, debris, and parts of the ECM in order to get through. They secrete plasminogen activator, which activates plasminogen, turning it into plasmin to dissolve the scab. Cells can only migrate over living tissue, so they must secrete collagenases and proteases to dissolve damaged parts of the ECM in their way, particularly at the front of the migrating sheet. Keratinocytes also dissolve the basement membrane, using instead the new ECM laid down by fibroblasts to crawl across.
  • Keratinocytes continue migrating across the wound bed until cells from either side meet in the middle, at which point contact inhibition causes them to stop migrating. When they have finished migrating, the keratinocytes secrete proteins that form the new basement membrane and become anchored once again to the basement membrane. Basal cells begin to divide and differentiate in the same manner as they do in normal skin to re-establish the strata found in re-epithelialized skin.
  • Contraction Around a week after wounding, fibroblasts differentiate into myofibroblasts and the wound begins to contract. In full thickness wounds, contraction peaks at 5 to 15 days post wounding. Contraction can last for several weeks and continues even after the wound is completely re-epithelialized. If contraction continues for too long, it can lead to
  • Contraction reduces the size of the wound.
  • a large wound can become 40%-80% smaller after contraction.
  • Wounds can contract at a speed of up to 0.75 mm per day, depending on how loose the tissue in the wounded area is.
  • Contraction usually occurs along an "axis of contraction," which allows for greater organization and alignment of cells with collagen.
  • contraction occurs without myofibroblast involvement.
  • fibroblasts stimulated by growth factors, differentiate into myofibroblasts.
  • Myofibroblasts which are similar to smooth muscle cells, are responsible for contraction.
  • Attracted by fibronectin and growth factors they move along fibronectin linked to fibrin in the provisional ECM in order to reach the wound edges, where they form connections to the ECM and to each other.
  • actin in the myofibroblast is linked across the cell membrane to molecules in the extracellular matrix like fibronectin and collagen.
  • Myofibroblasts have many such adhesions, which allow them to pull the ECM when they contract, reducing the wound size. In this part of contraction, closure occurs more quickly than in the first, myofibroblast-independent part. As the actin in myofibroblasts contracts, the wound edges are pulled together. Fibroblasts lay down collagen to reinforce the wound as myofibroblasts contract. The contraction stage ends as myofibroblasts stop contracting and commit apoptosis. The breakdown of the provisional matrix leads to a decrease in hyaluronic acid and an increase in chondroitin sulfate, which gradually triggers fibroblasts to stop migrating and proliferating. These events signal the onset of the maturation stage of wound healing.
  • the maturation and remodeling phase of tissue repair is said to have begun.
  • the maturation phase can last for a year or longer, depending on the size of the wound and whether it was initially closed or left open.
  • type III collagen which is prevalent during proliferation, is gradually degraded and the stronger type I collagen is laid down in its place.
  • disorganized collagen fibers are rearranged, cross-linked, and aligned along tension lines.
  • the tensile strength of the wound increases, with the strength approaching 50% that of normal tissue by three months after injury and ultimately becoming as much as 80% as strong as normal tissue. Since metabolic activity at the wound site is reduced, the scar loses its red appearance as blood vessels that are no longer needed are removed by apoptosis.
  • wounds may heal by primary intention.
  • Such wounds may be referred to as "closed wounds.” These wounds are usually surgically closed in layers along tissue planes by a physician. In primary intention, a linear scar results at the intersection of the approximated tissues.
  • Scarring is often minimal, but can be variable depending on the size and location of the wound, the tension on tissue and other factors. Most surgical wounds are sutured closed, so they heal by primary intention. In primary intention, wound closure is usually performed with sutures, staples, or an adhesive. Other examples of wounds that heal by primary intention are well repaired lacerations, well reduced bone fractures, and wounds that heal after flap surgery (the edges of which tend to appear scarred).
  • wounds are referred to as "open wounds.”
  • wounds formed by blast injury, shrapnel e.g., from improvised explosive devices ("IEDs")
  • blunt trauma e.g., blunt trauma
  • dental wounds e.g., gingivectomy, gingivoplasty, tooth extraction sockets
  • poorly reduced fractures e.g., gingivectomy, gingivoplasty, tooth extraction sockets
  • third degree burns heal by secondary intention.
  • Healing by secondary intention follows the same basic steps as wounds that heal by primary intention, i.e., inflammation, proliferation, and remodeling, but each sequence may take much longer, especially the proliferative phase.
  • Wound care must be performed daily to encourage wound debris removal to allow for granulation tissue formation. Depending on the size and location of the wound, placement of a partial or full-thickness skin graft may be considered if no infection is present and the area is of sufficient size that healing may not be complete for at least 2 or 3 weeks. Infection and inflammation of the wound can dysregulate repair and transform the wound into a clinically non-healing wound.
  • wound healing by tertiary intention the wound is initially cleaned, debrided, and observed, and typically 4 or 5 days elapse before closure.
  • the wound is purposely left open. Examples include healing of wounds by use of tissue grafts.
  • a major component of wound healing in humans is scar formation.
  • a scar (“cicatrix”; plural, “cicatrices”) is an area of fibrous tissue that forms as part of the healing process to replace normal skin after injury.
  • a hallmark of scars is altered extracellular matrix, notably a reduction of elastin fibers (De Vries et al, 1995).
  • Scars result from damage to the dermis, and with the exception of very minor lesions, every wound results in some degree of scarring. Scars generally form in proportion to the extent of damage.
  • Scar tissue is also usually of inferior functional quality.
  • a scar is a collagen-rich, elastin-poor dermal matrix with a simple stratified epithelial covering. Deposition of such a collagen-rich matrix in the neo- dermis is prone to contracture, loss in elasticity, and reduced tensile strength. Scars in the skin are also less resistant to ultraviolet radiation. For example, scars from skin transplants are typically dysfunctional, discolored, etc. Skin flaps and grafts are common methods of achieving rapid closure of large defect wounds. Not only do these methods tend to result in scarring at the donor site, but the sites of apposition of flap or graft edges to the wound edges can also result in linear scars.
  • TGFs Transforming Growth Factors
  • RSK Ribosomal s6 kinase
  • Scarring is likely to be extensive when wounds heal by secondary intention.
  • the wound heals by granulation, wherein epithelial cells grow over the wound from all sides of the normal skin, which results in a shiny layer of epithelial cells and fibrous tissue that is rich in collagen but does not contain underlying structures ("adnexal structures," including hair follicles).
  • adnexal structures including hair follicles
  • the scar also lacks the suppleness of normal skin. This type of scar can result in contractures when it occurs over the mouth or eyes or on the skin around joints, and can be disfiguring.
  • atrophic scars In addition to scars that form by secondary intention, there are numerous other types of scars that we distinguish, including atrophic scars, hypertrophic scars, keloid scars, hypopigmented scars, hyperpigmented scars, depressed scars (which, compared with atrophic scars, also have contour abnormality, while "atrophic" scars are implied to have only thinning), ice-pick scars (a type of depressed scar), spread scars (scars that widen due to tension over a time period, and which may become somewhat atrophic in the center), fineline scars, widespread (or stretched) scars, scar contractures, and other "intermediate" types of scars that are difficult to categorize.
  • types of scars see Bayat et al, 2003, BMJ. 326:88-92, the contents of which are incorporated herein by reference in its entirety.
  • Atrophic scars are characteristic of scars that are too thin, and typically form below the plane of the skin. Atrophic scars have a pitted appearance, and are caused when underlying structures supporting the skin, such as fat or muscle, are lost. This type of scarring is commonly associated with acne, but can also be caused by chickenpox, surgery or an accident. Acne scars and striae (scars from stretched skin) are exemplary of atrophic scars. Striae are caused when the skin is stretched rapidly (for instance during pregnancy, significant weight gain, or adolescent growth spurts), or when skin is put under tension during the healing process, usually near joints. This type of scar usually improves in appearance after a few years.
  • Keloid scars are raised above the surface of the skin, but by definition they grow beyond the boundaries of the original wound. Keloids can be viewed as a tumorous (although benign) growth. Keloid scars can occur on anyone, but they are most common in dark-skinned people. Keloid scars can be caused by surgery, an accident, by acne or, sometimes, from body piercings. In some people, keloid scars form spontaneously. Although they are primarily a cosmetic problem, keloid scars can be itchy or painful in some individuals. They tend to be most common on the shoulders, chest and earlobes.
  • Human skin appendages also referred to as "adnexal” structures, include hair and hair follicles, sebaceous glands (which secrete sebum onto hair follicle to oil the hair), eccrine and apocrine sweat glands, and nails.
  • the skin of an adult human is essentially covered with hair follicles and contains approximately five million hair follicles, with approximately 100,000 - 150,000 covering the scalp. Only a minority of human hair follicles produce a hair fiber that can be appreciated visibly (a "terminal hair") and these specialized follicles are localized on specific regions of skin.
  • the portions of human skin that lack visible hair contain, for the most part, hair follicles that produce "vellus hair” while certain other hair follicles may contain or produce no hair (see Figure 1).
  • Hair follicles and particularly human hair follicles, are crypt structures comprised of distinct components, each comprised of several different specialized cells (see Figures 2 and 3).
  • the vast majority of hair follicles contain units called sebaceous glands (which produce sebum), and a minority (follicles located in specialized areas of the skin) also contain apocrine glands (which produce sweat used primarily for olfactory cues).
  • sebaceous glands which produce sebum
  • apocrine glands which produce sweat used primarily for olfactory cues.
  • the structures of the hair follicle include the follicular papilla (FP) and the germinative epithelium (GE) (together, the bulb).
  • the FP is comprised of mesenchymal cells (and connective tissue).
  • the other cells of the follicle are epithelial and include at least 8 cellular lineages including the outer root sheath (ORS), the companion layer (CL), the internal root sheath Henle's layer (He), internal root sheath Huxley's layer (Hu), the cuticle of the internal root sheath (Csth), the cuticle of the hair shaft (Csft), the cortex of the hair shaft, and the medulla of the shaft (Med).
  • ORS outer root sheath
  • CL the companion layer
  • He internal root sheath Henle's layer
  • Huxley's layer Hu
  • the cuticle of the internal root sheath Csth
  • Csft cuticle of the hair shaft
  • the mechanism for the switch from regeneration to repair with age in mammals is not fully known, but involves cytokines and an immune response that promotes rapid epidermal closure, dermal fibroplasia and increased collagen deposition.
  • hair follicles do not form, thus contributing to the lack of adnexal structures in a scar.
  • the abnormal structure of scars contributes to the their associated morbidity. Due to lack of eccrine glands, there is poor thermoregulation. Increased fibroplasia and collagen can lead to contracture and loss of mobility in affected areas of the body.
  • hair follicle keratinocytes contribute to epidermal closure, and hair follicle dermal sheath fibroblasts play central roles in dermal healing. See, Ito et al, 2005; Jahoda & Reynolds, 2001; Stenn & Paus, 2001, Physiol. Revs. 81 :449-494.
  • the only skin tissue (aside from scar tissue) that normally lacks hair follicles is the glabrous skin on palmar and plantar aspects of hands and feet, respectively, and the lips and labia.
  • human glabrous skin lacks hair follicles, it is rich in eccrine sweat glands.
  • Wound healing studies in pig have shown that sweat glands, by themselves, are capable of regenerating epidermis, which likely accounts for lack of scarring in glabrous skin wounds that spare the base of sweat glands.
  • palmar Barret at al, 2000
  • plantar Barret & Herndon, 2004 wounds can result in scarring, a sequelae related to increased depth of wound and delayed wound healing.
  • hair follicles were not understood to be capable of neogenesis and bulge cells were not proven to be a source of stem cells for hair follicle neogenesis.
  • Fathke then turned the focus of their investigation to the -catenm-independent Wnts expressed in the skin - which are not activated by lithium chloride.
  • the prolonged expression of the ⁇ -catenin independent Wnt yielded the same results - mature hair follicles were not generated in the mice and large epithelial cysts formed in the wounds.
  • Fathke interpreted their data as evidence for the restoration of tissue patterning in the adult mammalian wound epithelium - a feature not normally seen in adult cutaneous wound healing - they provided neither evidence of hair follicle neogenesis, nor the concomitant hair growth and enhanced wound healing that would be expected in connection with it.
  • mice are a poor model for human wound healing and scar formation. Mice tend to heal most wounds rapidly, with little or no scarring. In humans, however, severe wounds and burns are usually associated with cutaneous repair that results in scar tissue, no hair follicles, and the loss of regenerative capability that hair follicles may provide ⁇ see, Fathke et al, 2006, BMC Cell Biol. 7:4).
  • One reason for the difference in wound healing capability between humans and mouse may be that the biology of hair follicles in humans and mice differs in several significant respects. In the mouse, a thick fur coating is essential to healthy life (because hair plays roles in
  • Acute treatment of wounds is generally focused on hemostasis and antimicrobial considerations. The treatment depends on the type, cause, and depth of the wound as well as whether other structures beyond the skin are involved. Treatment of acute lacerations involves examination, cleaning, and closing the wound. If the laceration occurred some time ago, it may be allowed to heal by secondary intention due to the high rate of infection associated with immediate closure. Minor wounds like bruises tend to heal on their own with skin discoloration that usually disappears within 1 -2 weeks. Abrasions usually require no active treatment except keeping the area clean with soap and water, although scarring may occur. Puncture wounds may be prone to infection depending on the depth of penetration.
  • a greater amount of exudate and necrotic tissue in a wound increases likelihood of infection by serving as a medium for bacterial growth away from the host's defenses. Since bacteria thrive on dead tissue, wounds are often surgically debrided to remove the devitalized tissue. Debridement and drainage of wound fluid are an especially important part of the treatment for diabetic ulcers, which may create the need for amputation if infection gets out of control. Mechanical removal of bacteria and devitalized tissue is also the idea behind wound irrigation, which is accomplished using pulsed lavage.
  • maggot therapy the intentional introduction by a health care practitioner of live, disinfected maggots into nonhealing wounds.
  • Maggots dissolve only necrotic, infected tissue; disinfect the wound by killing bacteria; and stimulate wound healing.
  • Maggot therapy has been shown to accelerate debridement of necrotic wounds and reduce the bacterial load of the wound, leading to earlier healing, reduced wound odor and less pain. The combination and interactions of these actions make maggots an extremely potent tool in chronic wound care.
  • Negative pressure wound therapy is a treatment that improves ischemic tissues and removes wound fluid used by bacteria. This therapy, also known as vacuum- assisted closure, reduces swelling in tissues, which brings more blood and nutrients to the area, as does the negative pressure itself. The treatment also decompresses tissues and alters the shape of cells, causes them to express different mRNAs and to proliferate and produce ECM molecules.
  • Persistent chronic pain associated with non-healing wounds is caused by tissue (nociceptive) or nerve (neuropathic) damage and is influenced by dressing changes and chronic inflammation.
  • Chronic wounds take a long time to heal and patients can suffer from chronic wounds for many years.
  • Chronic wound healing may be compromised by coexisting underlying conditions, such as venous valve backflow, peripheral vascular disease, uncontrolled edema and diabetes mellitus.
  • Underlying ischemia may also be treated surgically by arterial revascularization, for example in diabetic ulcers, and patients with venous ulcers may undergo surgery to correct vein dysfunction.
  • Diabetics that are not candidates for surgery may also have their tissue oxygenation increased by Hyperbaric Oxygen Therapy, or HBOT, which can compensate for limitations of blood supply and correct hypoxia.
  • HBOT Hyperbaric Oxygen Therapy
  • higher oxygen content in tissues speeds growth factor production, fibroblast growth, and angiogenesis.
  • increased oxygen levels also means increased production of ROS.
  • Antioxidants molecules that can lose an electron to free radicals without themselves becoming radicals, can lower levels of oxidants in the body and have been used with some success in wound healing.
  • chronic wound healing may be speeded by replacing or stimulating those factors and by preventing the excessive formation of proteases like elastase that break them down.
  • VEGF vascular endothelial growth factor
  • IGF insulin-like growth factor 1-2
  • PDGF transforming growth factor- ⁇
  • EGF epidermal growth factor
  • Other treatments include implanting cultured keratinocytes into the wound to re-epithelialize it and culturing and implanting fibroblasts into wounds.
  • SLPI Secretory leukocyte protease inhibitor
  • Some patients are treated with artificial skin substitutes that have fibroblasts and keratinocytes in a matrix of collagen to replicate skin and release growth factors.
  • skin from cadavers is grafted onto wounds, providing a cover to keep out bacteria and preventing the buildup of too much granulation tissue, which can lead to excessive scarring.
  • the allograft skin transplanted from a member of the same species
  • it encourages cellular proliferation and provides a structure for epithelial cells to crawl across.
  • allografts may not work, requiring skin grafts from elsewhere on the patient, which can cause pain and further stress on the patient's system.
  • Collagen dressings are another way to provide the matrix for cellular proliferation and migration, while also keeping the wound moist and absorbing exudate.
  • Skin grafts do not fully address the need for effective scar revision because (1) there is a limited supply of donor tissue (typically buttocks, abdomen, legs, in-front or behind the ear, etc.); (2) scarring occurs at donor sites and contractures ⁇ i.e., around the eyes and mouth); and (3) skin grafts typically (but not always) (with the possible exception of scalp) lack qualities of the donor site (porosity, vascularity, color, pigmentation, thickness, texture and overall cosmetic appearance, etc.).
  • the scar is serially excised, and a balloon is implanted at the wound site, which pushes the tissue around the scar to expand.
  • Surgical excision of hypertrophic or keloid scars is often used with other methods such as pressotherapy or silicone gel sheeting (see below). Lone excision of keloid scars shows a high recurrence rate, close to 45%.
  • Surgical excision in combination with the immunomodulator imiquimod 5% cream may also have a benefit on scar reduction.
  • Other intralesional injections can also be used.
  • collagen injections or other soft tissue fillers can be used to raise sunken scars to the level of surrounding skin. Its effects are temporary, however, and it needs to be regularly repeated. There is also a risk in some people of an allergic reaction.
  • Silicone scar treatments improve scar appearance and are often used to prevent and treat hypertrophic scarring.
  • the exact mechanism of action is unknown, though some studies suggest a manipulation of local ionic charges or a decrease in production of "proinflammatory" substances like TGF 2.
  • Dimethicone silicone gel appears to be is as effective as silicone sheeting in improving scar appearance.
  • Mustoe TA, 2008 "Evolution of silicone therapy and mechanism of action in scar management," Aesth Plast Surg 32:82-92. Polyurethane bandages are also used.
  • Pressure garments are used under supervision by a medical professional. They are most often used for burn scars that cover a large area, and is only effective on recent scars. Pressure garments are usually custom-made from elastic materials, and fit tightly around the scarring. They work best when they are worn 24 hours a day for six to twelve months. It is believed that they work by applying constant pressure to surface blood vessels and eventually causing scars to flatten and become softer.
  • Needling is an inexpensive process where the scarred area is continuously needled to promote collagen formation. Once needled the area is allowed to fully heal, and needled again if required depending on the intensity of the scar. Scarring needles and needling rollers are available for home use; however, needling should not be done on parts of the face or areas where major nerves are located without professional medical supervision. Needling at home must also be done in line with hygienic and sterilization requirements.
  • Dermabrasion involves the removal of the surface of the skin with specialist equipment and usually involves a general anesthetic. It is useful with raised scars, but is less effective when the scar is sunken below the surrounding skin.
  • Low-dose, superficial radiotherapy is used to prevent re-occurrence of severe keloid and hypertrophic scarring. It is usually effective, but only used in extreme cases due to the risk of long-term side effects.
  • Vitamin E causes contact dermatitis in up to 33% of users and in some cases it may worsen scar appearance. See, Baumann & Spencer, 1999, "The effects of topical vitamin E on the cosmetic appearance of scars," Dermatol Surg. 25:31 1-315; and Jenkins et al, 1986, “Failure of topical steroids and vitamin E to reduce postoperative scar formation following reconstructive surgery,” J Burn Care Rehabil 7: 309-312.
  • vitamin C normalizes collagen production and encourages the production of an organized, healthy collagen framework, which improves scar appearance.
  • Vitamin C and some of its esters also fade the dark pigment associated with some scars. See, Fitzpatrick & Rostan, 2002, “Double-blind, half-face study comparing topical vitamin C and vehicle for rejuvenation of photodamage,” Dermatol Surg 28:231-236; and Farris PK, 2005, “Topical vitamin C: a useful agent for treating photoaging and other dermatologic conditions,” Dermatol Surg 31 :814-818.
  • the intermittent and pulse lithium treatments can be administered in situ to acute wounds, chronic wounds, to scars, and/or surrounding skin.
  • the intermittent or pulse lithium treatments can be administered to the wound site or surrounding skin before, at the time of, and/or subsequent to, either acute wounding or, more typically, the wounding that is induced in scar revision.
  • the intermittent and pulse lithium treatments may also be administered to skin-derived cells or skin tissue ex vivo.
  • an intermittent or pulse lithium treatment may be used to enhance hair follicle neogenesis or enhance the re-association of dissociated hair follicle cells into follicles and facilitate their growth and expansion either in situ, or, alternatively, in culture for their implantation into fresh wounds and scar revisions.
  • hair follicles can be introduced to the wound by migration or de novo hair follicle neogenesis, or by transplanting one or more of the following skin elements: full skin (xeno-; autologous human), follicular units, dissociated cells (donor dominance; recipient effects), ex vivo-expanded skin and/or follicular units, or human skin equivalents in vivo (universal donors).
  • Engineered human skin, or human skin equivalents can also be used for hair follicle neogenesis and scar revision platforms.
  • Intermittent lithium treatments or a single pulse lithium treatment are used to revise scars and heal wounds in human subjects.
  • Any pharmaceutically acceptable compound that releases the lithium ion also referred to herein as lithium cation, Li+, or ionized lithium
  • lithium ion also referred to herein as lithium cation, Li+, or ionized lithium
  • compounds include, but are not limited to lithium gluconate, lithium succinate, and other organic salts/acids; and lithium chloride and other inorganic salts/acids, as described in Section 5.1, infra.
  • the intermittent lithium treatment protocol involves multiple courses of lithium treatment interrupted by lithium treatment "holidays" (periods during which no lithium treatment is administered).
  • a lithium treatment holiday is a period of time during which the patient stops the lithium treatment with the intent of resuming treatment.
  • a dose of lithium is administered over a short period of time.
  • the lithium treatment can be administered topically, transdermally, intradermally, cutaneously, subcutaneously, intramuscularly, intravenously, orally, sublingually, or can be bucchal.
  • Topical lithium treatment is a preferred embodiment because high local concentrations can be achieved while minimizing systemic exposure.
  • lithium gluconate 8% weight/weight (w/w) gel e.g., Lithioderm 8% gel
  • w/w gel commercially available in France for the treatment of seborrheic dermatitis
  • lithium is formulated into a modified release form that allows controlled release, over time, into the skin.
  • the lithium is formulated as part of a mesh scaffold that delivers lithium into the skin. More details on these and other lithium formulations and delivery methods for use in the treatment methods described herein are described in Sections 5.1-5.3 infra.
  • the intermittent and pulse lithium treatments can be administered alone to wounded skin (e.g., prior to, during, or subsequent to scar revision, or acute skin wounding, or chronic skin wounding) or in combination with other treatments to enhance wound healing or scar revision.
  • the intermittent and pulse lithium treatments can also be administered in combination with other treatments that facilitate hair follicle development and deposition into the wounded skin.
  • Embodiments of the invention include combination therapies, involving the addition of other treatment(s) concurrently with, or during the breaks between, the cycles of intermittent lithium treatments; or the addition of other treatment(s) concurrently with, or before and/or after the pulse lithium treatment.
  • Such combination therapies can include, but are not limited to, the concurrent or sequential use of other chemical agents, or mechanical or physical treatments including but not limited to, laser ⁇ e.g., Fraxel), dermatome planing, laser abrasion, electrology, intense pulsed light, or surgical treatments ⁇ e.g., skin graft or follicular unit extraction (FUE), etc.) that promote scar revision or wound healing.
  • laser e.g., Fraxel
  • dermatome planing e.g., dermatome planing
  • laser abrasion e.g., electrology, intense pulsed light
  • surgical treatments ⁇ e.g., skin graft or follicular unit extraction (FUE), etc.
  • intermittent lithium treatments or pulse lithium treatments in combination with perturbation e.g., debriding, peeling, or wounding
  • perturbation e.g., debriding, peeling, or wounding
  • methods such as laser treatment, dermabrasion, needling (using, e.g., microneedles), electromagnetic disruption, electroporation, or sonoporation; chemically ⁇ e.g., to induce inflammation); or by any other method described herein or known in the art, prior to or concurrent with administration of a lithium formulation described herein.
  • the integumental perturbation procedure can be any combination with perturbation (e.g., debriding, peeling, or wounding) of the skin and/or other tissues of the integumentary system by methods such as laser treatment, dermabrasion, needling (using, e.g., microneedles), electromagnetic disruption, electroporation, or sonoporation; chemically ⁇ e.g., to induce inflammation); or by any other method described herein or known in
  • wounding procedure used for scar revision.
  • the procedure can be controlled to limit perturbation to the epidermis, or extend deeper into the dermis and/or hypodermis.
  • the occurrence of pinpoint bleeding would indicate removal of the epidermis and portions of the upper layer of the dermis.
  • the occurrence of increased bleeding would indicate deeper penetration (and thus perturbation) into the dermis layer.
  • lasers particularly fractional lasers, and skin graft, follicular unit, and skin component transplant technologies have the capacity to induce regenerative changes in skin that mimic wounding and have applications in revision of scars.
  • laser techniques may "mimic" the plastic, embryonic-like, state of the epidermis created by other wound signals, but with laser's precision, versatility, and demonstrated efficacy in small scars.
  • intermittent lithium treatments and pulse lithium treatments administered concurrently or in sequential/alternating combination with other agents or treatments that modulate the wound healing process.
  • the intermittent and pulse lithium treatments may be administered with treatments that either promote or delay the wound healing process, such as described in Section 5.4.3 infra.
  • the intermittent lithium treatments or pulse lithium treatments described herein can be administered concurrently or alternating sequentially with one or more of the following treatments that prevent follicle senescence, for example, anti-oxidants such as glutathione, ascorbic acid, tocopherol, uric acid, or polyphenol antioxidants); inhibitors of reactive oxygen species (ROS) generation, such as superoxide dismutase inhibitors; stimulators of ROS breakdown, such as selenium; mTOR inhibitors, such as rapamycin; or sirtuins or activators thereof, such as resveratrol, or other SIRT1, SIRT3 activators, or nicotinamide inhibitors.
  • anti-oxidants such as glutathione, ascorbic acid, tocopherol, uric acid, or polyphenol antioxidants
  • ROS reactive oxygen species
  • stimulators of ROS breakdown such as selenium
  • mTOR inhibitors such as rapamycin
  • sirtuins or activators thereof such as resveratrol
  • the intermittent lithium treatments or a pulse lithium treatment provided herein can also be administered concurrently or alternating sequentially with one or more of the following treatments that promote hair growth, in order to enhance formation of new hair follicles: minoxidil, finasteride, bimatoprost (Latisse), CaCl 2 , or adenosine, or techniques of integumental perturbation such as, e.g. , by mechanical means, chemical means,
  • electromagnetic means e.g. , using a laser such as one that delivers ablative, non-ablative, non-fractional, superficial, or deep treatment, and/or are CCh-based, or Erbium- YAG-based, etc.
  • irradiation irradiation, radio frequency (RF) ablation, or surgical procedures (e.g., hair
  • Treatments that promote hair growth, or, alternatively, treatments that prevent hair growth may also be used in combination with the intermittent lithium treatments or a pulse lithium treatment described herein in order to promote the establishment of desired hair patterning in the healed wound or revised scar, thereby improving the appearance of the treated skin.
  • treatments that regulate gender-specific specialized human hair follicles including those under the influence of sex-steroid regulation, or that regulate the differentiation of stem cells into gender-specific specialized human hair follicles, possibly resulting in follicles having features that are different from natural follicles in the target location of skin (e.g., normal sized follicles with terminal hair where previously miniaturized follicles with vellus hair were present) may be administered.
  • treatment of grafted skin with a combination of lithium and a modulator of specific hair patterning may reduce donor dominance and enhance the ability of the graft to acquire properties of the recipient site.
  • intermittent lithium treatments or a pulse lithium treatment may be used concurrently or in sequential combination with either a treatment that enhances hair growth (described above) or a cytotoxic drug, a hair growth retardant, such as eflornithine HC1 (Vaniqa), 5-fluorouracil (5-FU) (e.g., Efudex 5% cream), or other epilation or depilation methods to prevent or reduce hair growth.
  • Success of a pulse or intermittent lithium treatment described herein can be measured by one or more of the following outcomes:
  • Human subjects who are candidates for the pulse or intermittent lithium treatments described herein include any subject in need of improved wound healing, particularly wound healing without scarring, or scar revision.
  • Human subjects who are candidates for such treatments include any subject for whom improved wound healing or scar revision is desired.
  • Such human subjects include, but are not limited to, subjects with photodamaged skin, acne scars, chicken pox scars, scarring (cicatricial) alopecia, chronic non-healing wounds or scars due to, e.g., diabetes, venous or arterial disease, old age or senescence, infection, medication, chemotherapy, trauma, burns, stress, autoimmune disease, malnutrition, or endocrine dysfunction.
  • Surgical subjects who are candidates for such treatments include, but are not limited to, patients with skin graft, hair transplantation, skin cancer surgery, or Mohs surgery.
  • Subjects who are candidates for such treatments also include subjects with any other form of wounding or scarring or disease or disorder associated with wounding or scarring as discussed infra and/or known in the art.
  • the subject has a wound or scar on a cosmetically sensitive location, such as the face or neck.
  • the invention is based in part on the recognition that the timing of the administration of lithium is important for it to function as an effective modulator of wound healing (and thus, scar revision) in human subjects.
  • lithium treatment results, indirectly, in increasing Wnt signaling, but agents that increase Wnt signaling have had conflicting effects on hair follicle development and wound healing.
  • they stimulate follicle morphogenesis but also induce hair follicle tumors (Gat et al. , 1998, Cell 95: 605-614), and lead to decreased hair growth (Millar et al., 1999, Dev. Biol. 207: 133-149).
  • the invention is also based, in part, on the principle that human skin is replenished by bone-marrow derived and tissue-derived stem cells throughout life.
  • the lithium treatment(s) is used in combination with methods that mobilize tissue stem cells (e.g., using integumental perturbation) and/or methods that mobilize bone marrow-derived stem cells (e.g., growth factors such as G-CSF and/or chemical agents such as plerixafor (Mozobil®)).
  • the lithium treatments described herein are used together with methods that regulate the differentiation of these stem cells into specialized human hair follicles in order to facilitate the desired hair patterning at the acceptor site, using agents such as finasteride, fluconazole, spironolactone, flutamide, diazoxide, 11 -alpha-hydroxyprogesterone, ketoconazole, RU58841, dutasteride, fluridil, or QLT-7704, an antiandrogen oligonucleotide, cyoctol, topical progesterone, topical estrogen, cyproterone acetate, ru58841, combination 5 alpha reductase inhibitors, oral contraceptive pills, and others in Poulos & Mirmirani, 2005, Expert Opin.
  • agents such as finasteride, fluconazole, spironolactone, flutamide, diazoxide, 11 -alpha-hydroxyprogesterone, ketoconazole, RU58841, du
  • Investig. Drugs 14: 177-184 incorporated herein by reference, or any other antiestrogen, an estrogen, or estrogen-like drug (alone or in combination with agents that increase stem cell plasticity; e.g., such as valproate), etc. , known in the art.
  • Such combination treatments can further include the use of agents that modulate hair growth or that modulate wound healing.
  • Anagen Growth stage of the hair Follicle Cycle.
  • Bulb Lowermost portion of the hair follicle, containing rapidly proliferating matrix cells that produce the hair.
  • Catagen Stage of the hair cycle characterized by regression and involution of the follicle.
  • Cicatricial (scarring) Alopecia Abnormal hair loss with scarring. Caused by destruction of hair follicles and replacement with scar tissue as a result of inflammation, trauma, fibrosis, or unknown causes; examples include lichen planopilaris and discoid lupus erythematosus.
  • Exogen Phase of the hair Follicle Cycle where hair shaft is shed from the follicle.
  • Follicle cycle Hair growth in each follicle occurs in a cycle that includes the following phases: anagen (growth phase), catagen (involuting/regressing stage), telogen (the quiescent phase), exogen (shedding phase), and re-entry into anagen.
  • Integumental Pertaining to the integumentary system, which comprises the skin (epidermis, dermis, hypodermis (or subcutanea)) and all cells contained therein regardless of origin, and its appendages (including, e.g., hair and nails).
  • Kenogen Latent phase of hair cycle after hair shaft has been shed and growth is suspended in follicle.
  • Telogen effluvium Excessive shedding of hair caused by an increased proportion of follicles entering the telogen stage; common causes include drugs and fever.
  • Terminal hair Large, usually pigmented hairs on scalp and body.
  • Vellus hair Very short, nonpigmented hairs (e.g., those found diffusely over nonbeard area of face and bald scalp as a result of miniaturization of terminal hairs).
  • Figure 1 Types of human hair follicles.
  • Figure 2 Architecture of the skin.
  • Figure 3 Diagram of human hair follicle.
  • Figure 4 Cellular structure of the human hair bulb.
  • Formulation 35A' lithium chloride emulsion cream; see Table 2 is plotted over time, in hours (x-axis).
  • Figure 7 Release of Li ions through dermis (y-axis) from Formulation BX (lithium chloride gel; see Table 2) is plotted over time, in hours (x-axis).
  • Cadaver skin was dermabraded, with a standard dermabrader to remove the stratum corneum and epidermis prior to administration of the lithium compound.
  • Figure 8 Release of Li ions through dermis (y-axis) from Formulation BV-001- 003A (lithium chloride hydrogel; see Table 2) is plotted over time, in hours (x-axis).
  • Cadaver skin was dermabraded with a standard dermabrader to remove the stratum corneum and epidermis prior to administration of the lithium compound.
  • FIG. 9 Release of Li ions through dermis (y-axis) from Formulation 28A (lithium chloride topical dispersion cream; see Table 2) is plotted over time, in hours (x-axis).
  • Cadaver skin was dermabraded, with a standard dermabrader to remove the stratum corneum and epidermis prior to administration of the lithium compound.
  • FIG. 12 Skin lithium concentrations calculated in mM, as a function of increasing doses of a formulation of lithium chloride dissolved in isotonic saline in mg/kg administered subcutaneously to mice dermabraded prior to dosing. "Peak samples" were taken 1 h post last dosing. [00132] Figure 13. Comparison of Peak lithium concentrations in plasma and skin upon subcutaneous administration of a formulation of lithium chloride dissolved in isotonic saline following DA.
  • FIG. 14 Lithium concentrations calculated in mM, in total blood (red blood cells (RBC) + plasma), as a function of increasing doses of a formulation of lithium chloride dissolved in isotonic saline in mg/kg, administered subcutaneously to mice dermabraded prior to dosing.
  • B Skin lithium concentrations calculated in ⁇ g/kg, as a function of increasing doses in mg/kg. In the wounded groups, skin was dermabraded prior to administration of the formulation of lithium chloride dissolved in isotonic saline. Non-wounded comparisons are shown (square, diamond) with dermabrasion wounded groups (cross, triangle). * It is noted that in this experiment, dermabrasion was accomplished using a microdermabrasion device.
  • FIG. 15 Skin lithium concentrations calculated in mM, as a function of increasing doses of a formulation of lithium chloride dissolved in isotonic saline in mg/kg.
  • the lithium formulation was administered subcutaneously following full thickness excision (FTE) of skin. Dosing was started on the day of scab detachment (-11-14 days post-FTE). Lithium ion concentrations were measured by a validated bioanalytical ICP method (see Section 13.2 infra).
  • FIG. 16 Plasma lithium Concentrations calculated in mM, as a function of increasing doses of a formulation of lithium chloride dissolved in isotonic saline in mg/kg.
  • the lithium formulation was administered subcutaneously following FTE. Dosing was started on the day of scab detachment ( ⁇ 11-14 days post-FTE). Lithium concentrations were measured by a validated bioanalytical ICP method (see Section 13.2 infra).
  • Figure 17 Comparison of Peak lithium concentrations in plasma and skin upon subcutaneous administration of a formulation of lithium chloride dissolved in isotonic saline following FTE.
  • FIG. 1 Pharmacokinetic analysis of lithium concentrations in skin and plasma with once daily topical dosing of 8% lithium chloride or 8% lithium gluconate hydrogel ("lithium gluconate") following FTE. Lithium ion concentrations were measured by
  • ICP/MS/MS using a validated method (see Section 13.2 infra).
  • Arrows indicate peak levels of Lithium ion in skin one hour post dosing.
  • N 2 per time point— error bars denote range [00138]
  • Figure 19 Pharmacokinetic analysis of lithium concentrations in skin and plasma with twice daily topical dosing of lithium gluconate, 1%; lithium gluconate, 8%; and lithium gluconate, 16% following DA.
  • FIG. 20 Topical lithium 8% increases the proportion of mature neogenic hair follicles in healed FTE wounds, based on histologic examination.
  • A Diagrams of selected stages of hair follicle development.
  • B Percentage of stageable neogenic hair follicles at stage 5 or greater following administration of topical lithium gluconate hydrogel ("lithium gluconate”), 1%, 8%, or 16% or lithium chloride hydrogel (“LiCl”), 8%. Numbers in the bars indicate the number of mice per group that were used for quantitation. Ratios above the bars indicate the number of NHF (neogenic hair follicles) > stage 5 divided by the total number of stageable NHF.
  • lithium gluconate hydrogel 1%, 8%, or 16%
  • LiCl lithium chloride hydrogel
  • Topical lithium 8% increases both the number and maturation of neogenic hair follicles in FTE wounds, as measured following administration of topical lithium gluconate hydrogel ("lithium gluconate”), 1%, 8%, or 16% or lithium chloride hydrogel (“LiCl”), 8%.
  • lithium gluconate hydrogel 1%, 8%, or 16%
  • LiCl lithium chloride hydrogel
  • Topical lithium 8% increases shaft thickness of regenerated hair follicles following DA. Tissues analyzed following administration of topical lithium gluconate hydrogel ("lithium gluconate”), 1%, 8%, or 16% or lithium chloride hydrogel (“LiCl”), 8%.
  • lithium gluconate hydrogel 1%, 8%, or 16%
  • LiCl lithium chloride hydrogel
  • FIG. 25 Topical lithium gluconate 8% results in a 16% increase thickness of regenerated hair shafts following DA.
  • LiCl lithium chloride hydrogel
  • Graph on right side shows simultaneous 90% confidence intervals, corrected for 4 comparisons by the Treatment Groups Bonferroni method.
  • FIG. 26 Healed FTE wounds treated with topical LiCl 8% have increased numbers of neogenic hair follicles, as assessed by in vivo scanning laser microscopy, imaging the wounded area approximately 60-80 ⁇ beneath the skin surface.
  • FIG. 28 Topical LiCl 8% increases the total number of neogenic hair follicles (also referred to as "HF") per FTE wound by 3-fold, based on histology of tissue sections.
  • Left graph: Median + first and third quartiles shown. Numbers above columns total number of neogenic hair follicles ("NHF”) combined from individual mice (parentheses indicate number of NHF that could not be staged).
  • NHF total number of neogenic hair follicles
  • FIG. 32 Topical LiCl 8% does not affect density of regenerated hair follicles following DA. Median + first and third quartiles shown. Right graph: Hodges-Lehman estimate of median difference. Simultaneous 90% confidence intervals, corrected for 4 comparisons by the Bonferroni method.
  • FIG 35 Complexed Lithium Gluconate encapsulated within biodegradable poly (D,L-lactide-co-glycolide) ("PLG”) microspheres.
  • PLG biodegradable poly
  • FIG. 37 Synthetic Biodegradable Matrices from PLA/PLG Blends. A:
  • any compound or composition that can release a lithium ion (also referred to herein as lithium cation, Li+, or ionized lithium) is suitable for use in the compositions and methods.
  • Such compounds include but are not limited to a pharmaceutically acceptable prodrug, salt or solvate ⁇ e.g., a hydrate) of lithium (sometimes referred to herein as "lithium compounds").
  • the lithium compounds can be formulated with a pharmaceutically acceptable vehicle, carrier, diluent, or excipient, or a mixture thereof.
  • lithium- polymer complexes can be utilized to developed various sustained release lithium matrices.
  • lithium is best known as a mood stabilizing drug, primarily in the treatment of bipolar disorder, for which lithium carbonate (L1 2 CO 3 ), sold under several trade names, is the most commonly used.
  • Other commonly used lithium salts include lithium citrate (L1 3 C 6 H 5 O 7 ), lithium sulfate (L1 2 SO 4 ), lithium aspartate, and lithium orotate.
  • a lithium formulation well-suited for use in the methods disclosed herein is lithium gluconate, for example, a topical ointment of 8% lithium gluconate (LithiodermTM), is approved for the treatment of seborrheic dermatitis.
  • lithium succinate for example, an ointment comprising 8% lithium succinate, which is also used to treat seborrheic dermatitis.
  • the lithium formulation is an ointment comprising 8% lithium succinate and 0.05% zinc sulfate (marketed in the U.K. as Efalith). See, e.g., Efalith Multicenter Trial Group, 1992, J Am Acad Dermatol 26:452-457, which is incorporated by reference herein in its entirety.
  • Examples of lithium succinate formulations and other lithium formulations for use in the intermittent lithium treatments or pulse lithium treatment described herein are also described in U.S. Patent No. 5,594,031, issued January 14, 1997, which is incorporated herein by reference in its entirety. 5.1.1 LITHIUM SALTS
  • any pharmaceutically acceptable lithium salt may be used as a source of lithium ions in the intermittent lithium treatments or a pulse lithium treatment. It will be understood by one of ordinary skill in the art that pharmaceutically acceptable lithium salts are preferred. See, e.g., Berge et al, J. Pharm. Sci. 1977, 66: 1-19; Stahl & Wermuth, eds., 2002, Handbook of Pharmaceutical Salts, Properties, and Use, Zurich, Switzerland: Wiley -VCH and VHCA; Remington 's Pharmaceutical Sciences, 1990, 18 th eds., Easton, PA: Mack Publishing;
  • the compositions used for intermittent lithium treatment or a pulse lithium treatment comprise mixtures of one or more lithium salts.
  • a mixture of a fast-dissolving lithium salt can be mixed with a slow dissolving lithium salt proportionately to achieve the release profile.
  • the lithium salts do not comprise lithium chloride.
  • the lithium salt can be the salt form of anionic amino acids or poly(amino) acids. Examples of these are glutamic acid, aspartic acid, polyglutamic acid, polyaspartic acid.
  • lithium salts of the acids set forth above, applicants do not mean only the lithium salts prepared directly from the specifically recited acids. In contrast, applicants mean to encompass the lithium salts of the acids made by any method known to one of ordinary skill in the art, including but not limited to acid-base chemistry and cation-exchange chemistry.
  • lithium salts of anionic drugs that positively affect hair growth can be administered.
  • a large anion or multianionic polymer such as polyacrylic acid can be complexed with lithium, then complexed with a cationic compound, such as finasteride, to achieve a slow release formulation of both lithium ion and finasteride.
  • a lithium complex with a polyanion can be complexed further with the amines of minoxidil, at pHs greater than 5.
  • Lithium compounds for use in the methods provided herein may contain an acidic or basic moiety, which may also be provided as a pharmaceutically acceptable salt. See, Berge et al, J. Pharm. Sci. 1977, 66: 1-19; Stahl & Wermuth, eds., 2002, Handbook of Pharmaceutical Salts, Properties, and Use Zurich, Switzerland: Wiley-VCH and VHCA.
  • the lithium salts are organic lithium salts.
  • Organic lithium salts for use in these embodiments include lithium 2,2-dichloroacetate, lithium salts of acylated amino acids ⁇ e.g., lithium -acetylcysteinate or lithium N-stearoylcysteinate), a lithium salt of poly(lactic acid), a lithium salt of a polysaccharides or derivative thereof, lithium acetylsalicylate, lithium adipate, lithium hyaluronate and derivatives thereof, lithium polyacrylate and derivatives thereof, lithium chondroitin sulfate and derivatives thereof, lithium stearate, lithium linoleate, lithium oleate, lithium taurocholate, lithium cholate, lithium glycocholate, lithium deoxycholate, lithium alginate and derivatives thereof, lithium ascorbate, lithium L-aspartate, lithium benzenesulfonate, lithium benzoate, lithium
  • the organic lithium salt for use in these embodiments is lithium (S)-2- alkylthio-2-phenylacetate or lithium (R)-2-alkylthio-2-phenylacetate ⁇ e.g., wherein the alkyl is C2-C22 straight chain alkyl, preferably C8-16). See, e.g., International Patent Application Publication No. WO 2009/019385, published February 12, 2009, which is incorporated herein by reference in its entirety.
  • the organic lithium salts used for intermittent lithium treatment or a pulse lithium treatment comprise the lithium salts of acetic acid, 2,2- dichloroacetic acid, acetylsalicylic acid, acylated amino acids, adipic acid, hyaluronic acid and derivatives thereof, polyacrylic acid and derivatives thereof, chondroitin sulfate and derivatives thereof, poly(lactic acid-co-glycolic acid), poly(lactic acid), poly(glycolic acid), pegylated lactic acid, stearic acid, linoleic acid, oleic acid, taurocholic acid, cholic acid, glycocholic acid, deoxycholic acid, alginic acid and derivatives thereof, anionic derivatives of polysaccharides, poly(sebacic anhydride)s and derivatives thereof, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid
  • organic lithium salts for use in these embodiments is the lithium salt of (S)-2-alkylthio-2-phenylacetic acid or the lithium salt of (R)-2-alkylthio-2-phenylacetic acid ⁇ e.g., wherein the alkyl is C2- C22 straight chain alkyl, preferably C8-16). See, e.g., International Patent Application Publication No. WO 2009/019385, published February 12, 2009, which is incorporated herein by reference in its entirety.
  • the organic lithium salt can be modified to create sustained release lithium salts. Due to the size of the lithium ion, it is possible that the residence time of ion at the treatment site will be short. In efforts to generate sustained release lithium salts, the hydrophobicity of the salt can be enhanced and made "lipid-like," to, for example, lower the rate of ionization of the salt into lithium ions. For example, lithium chloride has a much faster rate of ionizing into lithium ions, than lithium stearate or lithium orotate.
  • the lithium salt can be that of a cholesterol derivative, or a long chain fatty acids or alcohols. Lipid complexed lithium salts of size less than 10 microns can also be effectively targeted to the hair follicles and "tethered" to the sebaceous glands, by hydrophobic-hydrophobic interactions.
  • the organic lithium salt can be in the form of complexes with anionic compounds or anionic poly(amino acids) and other polymers.
  • the complexes can be neutral, wherein all of the negative charges of the complexation agent are balanced by equimolar concentrations of Li ions.
  • the complexes can be negatively charged, with Lithium ions bound to an anionic polymer.
  • the complexes can be in the form of nano-complexes, or micro-complexes, small enough to be targeted to the hair follicles. If the complexes are targeted to the dermis, the charged nature of the complexes will "tether" the complexes to the positively charged collagen.
  • This mode of tethering holds the Li ions at the site of delivery, thereby hindering fast in-vivo clearance.
  • negatively charged polymers that can be used in this application are poly(acrylates) and its copolymers and derivatives thereof, hyaluronic acid and its derivatives, alginate and its derivatives, etc.
  • the anionic lithium complexes formed as described above can be further complexed with a cationic polymer such as chitosan, or polyethylimine form cell-permeable delivery systems.
  • the salt can be that of a fatty acid, e.g. , lithium stearate, thereby promoting absorption through skin tissues and extraction into the lipid compartments of the skin.
  • the lithium salt of sebacic acid can be administered to the skin for higher absorption and targeting into structures of the skin, such as hair follicles.
  • the lithium salts are inorganic lithium salts.
  • Inorganic lithium salts for use in these embodiments include halide salts, such as lithium bromide, lithium chloride, lithium fluoride, or lithium iodide.
  • the inorganic lithium salt is lithium fluoride.
  • the inorganic lithium salt is lithium iodide.
  • the lithium salts do not comprise lithium chloride.
  • Other inorganic lithium salts for use in these embodiments include lithium borate, lithium nitrate, lithium perchlorate, lithium phosphate, or lithium sulfate.
  • the inorganic lithium salts used for intermittent lithium treatment or a pulse lithium treatment comprise the lithium salts of boric acid, hydrobromic acid, hydrochloric acid, hydrofluoric acid, hydroiodic acid, nitric acid, perchloric acid, phosphoric acid, or sulfuric acid.
  • the lithium compounds used for intermittent lithium treatment or a pulse lithium treatment may be formulated with a pharmaceutically acceptable carrier (also referred to as a pharmaceutically acceptable excipients), i.e., a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, an encapsulating material, or a complexation agent.
  • a pharmaceutically acceptable carrier also referred to as a pharmaceutically acceptable excipients
  • each component is "pharmaceutically acceptable” in the sense of being chemically compatible with the other ingredients of a pharmaceutical formulation, and biocompatible, when in contact with the biological tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • Suitable excipients are well known to those skilled in the art, and non-limiting examples of suitable excipients are provided herein. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art, including, but not limited to, the method of administration. For example, forms for topical administration such as a cream may contain excipients not suited for use in transdermal or intravenous administration. The suitability of a particular excipient depends on the specific active ingredients in the dosage form.
  • Exemplary, non-limiting, pharmaceutically acceptable carriers for use in the lithium formulations described herein are the cosmetically acceptable vehicles provided in
  • the lithium compounds suitable for use in intermittent lithium treatments or a pulse lithium treatment may be formulated to include an appropriate aqueous vehicle, including, but not limited to, water, saline, physiological saline or buffered saline (e.g., phosphate buffered saline (PBS)), sodium chloride for injection, Ringers for injection, isotonic dextrose for injection, sterile water for injection, dextrose lactated Ringers for injection, sodium bicarbonate, or albumin for injection.
  • PBS phosphate buffered saline
  • Suitable non-aqueous vehicles include, but are not limited to, fixed oils of vegetable origin, castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenated soybean oil, and medium-chain triglycerides of coconut oil, lanolin oil, lanolin alcohol, linoleic acid, linolenic acid and palm seed oil.
  • Suitable water- miscible vehicles include, but are not limited to, ethanol, wool alcohol, 1,3-butanediol, liquid polyethylene glycol (e.g., polyethylene glycol 300 and polyethylene glycol 400), propylene glycol, glycerin, N-methyl-2-pyrrolidone ( ⁇ ), N,N-dimethylacetamide (DMA), and dimethyl sulfoxide (DMSO).
  • the water-miscible vehicle is not DMSO.
  • the lithium compounds for use in the methods disclosed herein may also be formulated with one or more of the following additional agents.
  • Suitable antimicrobial agents or preservatives include, but are not limited to, alkyl esters of p-hydroxybenzoic acid, hydantoins derivatives, propionate salts, phenols, cresols, mercurials, phenyoxyethanol, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride (e.g. , benzethonium chloride), butyl, methyl- and propyl-parabens, sorbic acid, and any of a variety of quarternary ammonium compounds.
  • Suitable isotonic agents include, but are not limited to, sodium chloride, glycerin, and dextrose.
  • Suitable buffering agents include, but are not limited to, phosphate, glutamate and citrate.
  • Suitable antioxidants are those as described herein, including ascorbate, bisulfite and sodium metabisulfite.
  • Suitable local anesthetics include, but are not limited to, procaine
  • suspending and dispersing agents include but are not limited to sodium carboxymethylcelluose (CMC), hydroxypropyl methylcellulose (HPMC), polyvinyl alcohol (PVA), and polyvinylpyrrolidone (PVP).
  • CMC carboxymethylcelluose
  • HPMC hydroxypropyl methylcellulose
  • PVA polyvinyl alcohol
  • PVP polyvinylpyrrolidone
  • Suitable emulsifying agents include but are not limited to, including polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate 80, and triethanolamine oleate.
  • Suitable sequestering or chelating agents include, but are not limited to, EDTA.
  • Suitable pH adjusting agents include, but are not limited to, sodium hydroxide, hydrochloric acid, citric acid, and lactic acid.
  • Suitable complexing agents include, but are not limited to, cyclodextrins, including a-cyclodextrin, ⁇ -cyclodextrin, hydroxypropyl-P-cyclodextrin, sulfobutylether- ⁇ - cyclodextrin, and sulfobutylether 7-P-cyclodextrin (CAPTISOL ® , CyDex, Lenexa, KS).
  • cyclodextrins including a-cyclodextrin, ⁇ -cyclodextrin, hydroxypropyl-P-cyclodextrin, sulfobutylether- ⁇ - cyclodextrin, and sulfobutylether 7-P-cyclodextrin (CAPTISOL ® , CyDex, Lenexa, KS).
  • Soothing preparations may contain sodium bicarbonate (baking soda), and coal tar based products.
  • Formulations may also optionally contain a sunscreen or other skin protectant, or a waterproofing agent.
  • a product for application to skin may additionally be formulated so that it has easy rinsing, minimal skin/eye irritation, no damage to existing skin or hair, has a thick and/or creamy feel, pleasant fragrance, low toxicity, and good biodegradability.
  • lithium gluconate - for example, 8% lithium gluconate (LithiodermTM), which is approved for the treatment of seborrheic dermatitis (see, e.g., Dreno and Moyse, 2002, Eur J Dermatol 12:549-552; Dreno et al., 2007, Ann Dermatol Venereol 134:347-351 (abstract); and Ballanger et al, 2008, Arch Dermatol Res 300:215-223, each of which is incorporated by reference herein in its entirety); 8% lithium succinate (see, e.g., Langtry et al, 1996, Clinical and Experimental Dermatology 22:216-219; and Cuelenaere et al, 1992, Dermatology 184: 194-197, each of which is incorporated by reference herein in its entirety); or 8% lithium succinate with 0.05% zinc
  • a preparation of lithium or lithium salt comprises an anionic polymer (such as, e.g., crosslinked polyacrylic acid), which may form a gel.
  • an anionic polymer such as, e.g., crosslinked polyacrylic acid
  • a preparation provided in the examples of Sections 16-19 below may be used.
  • modified release refers to a dosage form in which the rate or place of release of the lithium or other active ingredient(s) is different from that of an immediate dosage form when administered by the same route.
  • Modified release dosage forms include, but are not limited to, delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated- and fast-, targeted-, programmed-release, and gastric retention dosage forms.
  • compositions in modified release dosage forms can be prepared using a variety of modified release devices and methods known to those skilled in the art, including, but not limited to, matrix controlled release devices, osmotic controlled release devices, multiparticulate controlled release devices, ion-exchange resins, enteric coatings, multilayered coatings, microspheres, liposomes, and combinations thereof.
  • the release rate of the active ingredient(s) can also be modified by varying the particle sizes and polymorphism of the active ingredient(s).
  • the controlled release is achieved by using an adjuvant that causes a depot effect, i.e., that causes an active agent or antigen to be released slowly, leading to prolonged exposure to a target cell or tissue (e.g., cells of the follicle, or, in the case of
  • modified release examples include those described in International Patent Application Publication No. WO 2008/1 15961, published September 25, 2008, which is incorporated herein by reference in its entirety.
  • modified release examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598, 123; 4,008,719; 5,674,533; 5,059,595;
  • the modified release dosage form can be fabricated using a matrix controlled release device known to those skilled in the art. See, Takada et al, 1999, in Encyclopedia of Controlled Drug Delivery, Mathiowitz E, ed., Vol. 2, Wiley.
  • the modified release dosage form is formulated using an erodible matrix device, which is water-swellable, erodible, or soluble polymers, including, but not limited to, synthetic polymers, and naturally occurring polymers and derivatives, such as polysaccharides and proteins.
  • Materials useful in forming an erodible matrix include, but are not limited to, chitin, chitosan, dextran, and pullulan; gum agar, gum arabic, gum karaya, locust bean gum, gum tragacanth, carrageenans, gum ghatti, guar gum, xanthan gum, and scleroglucan; starches, such as dextrin and maltodextrin; hydrophilic colloids, such as pectin; phosphatides, such as lecithin; alginates; propylene glycol alginate; gelatin; collagen;
  • cellulosics such as ethyl cellulose (EC), methylethyl cellulose (MEC), carboxymethyl cellulose (CMC), CMEC, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), cellulose acetate (CA), cellulose propionate (CP), cellulose butyrate (CB), cellulose acetate butyrate (CAB), CAP, CAT, hydroxypropyl methyl cellulose (HPMC), HPMCP, HPMCAS, hydroxypropyl methyl cellulose acetate trimellitate (HPMCAT), and ethyl hydroxyethyl cellulose (EHEC); polyvinyl pyrrolidone; polyvinyl alcohol; polyvinyl acetate; glycerol fatty acid esters; polyacrylamide; polyacrylic acid; copolymers of ethacrylic acid or methacrylic acid (EUDRAGIT ® , Rohm America, Inc., Piscataway, NJ);
  • degradable lactic acid-glycolic acid copolymers poly-D-(-)-3-hydroxybutyric acid; and other acrylic acid derivatives, such as homopolymers and copolymers of butylmethacrylate, methyl methacrylate, ethyl methacrylate, ethylacrylate, (2-dimethylaminoethyl)methacrylate, and (trimethylaminoethyl)methacrylate chloride.
  • the compositions are formulated with a non-erodible matrix device.
  • the active ingredient(s) is dissolved or dispersed in an inert matrix and is released primarily by diffusion through the inert matrix once administered.
  • Materials suitable for use as a non-erodible matrix device include, but are not limited to, insoluble plastics, such as polyethylene, polypropylene, polyisoprene, polyisobutylene, polybutadiene,
  • the desired release kinetics can be controlled, for example, via the polymer type employed, the polymer viscosity, the particle sizes of the polymer and/or the active ingredient(s), the ratio of the active ingredient(s) versus the polymer, and other excipients or carriers in the compositions.
  • the modified release dosage forms can be prepared by methods known to those skilled in the art, including direct compression, dry or wet granulation followed by compression, and melt-granulation followed by compression.
  • the modified release dosage form can be fabricated using an osmotic controlled release device, including, but not limited to, one-chamber system, two-chamber system, asymmetric membrane technology (AMT), and extruding core system (ECS).
  • AMT asymmetric membrane technology
  • ECS extruding core system
  • such devices have at least two components: (a) a core which contains an active ingredient; and (b) a semipermeable membrane with at least one delivery port, which encapsulates the core.
  • the semipermeable membrane controls the influx of water to the core from an aqueous environment of use so as to cause drug release by extrusion through the delivery port(s).
  • the core of the osmotic device optionally includes an osmotic agent, which creates a driving force for transport of water from the environment of use into the core of the device.
  • an osmotic agent which creates a driving force for transport of water from the environment of use into the core of the device.
  • osmotic agents water- swellable hydrophilic polymers, which are also referred to as "osmopolymers" and
  • hydrogels Suitable water-swellable hydrophilic polymers as osmotic agents include, but are not limited to, hydrophilic vinyl and acrylic polymers, polysaccharides such as calcium alginate, polyethylene oxide (PEO), polyethylene glycol (PEG), polypropylene glycol (PPG), poly(2-hydroxyethyl methacrylate), poly(acrylic) acid, poly(methacrylic) acid,
  • hydrophilic vinyl and acrylic polymers polysaccharides such as calcium alginate, polyethylene oxide (PEO), polyethylene glycol (PEG), polypropylene glycol (PPG), poly(2-hydroxyethyl methacrylate), poly(acrylic) acid, poly(methacrylic) acid,
  • polyvinylpyrrolidone PVP
  • crosslinked PVP polyvinyl alcohol
  • PVA polyvinyl alcohol
  • PVA/PVP copolymers PVA/PVP copolymers with hydrophobic monomers such as methyl methacrylate and vinyl acetate, hydrophilic polyurethanes containing large PEO blocks, sodium croscarmellose, carrageenan, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), carboxymethyl cellulose (CMC) and carboxyethyl, cellulose (CEC), sodium alginate, polycarbophil, gelatin, xanthan gum, and sodium starch glycolate.
  • HEC hydroxyethyl cellulose
  • HPMC hydroxypropyl methyl cellulose
  • CMC carboxymethyl cellulose
  • CEC carboxyethyl
  • sodium alginate sodium carbcarbophil
  • gelatin xanthan gum
  • the other class of osmotic agents is osmogens, which are capable of imbibing water to affect an osmotic pressure gradient across the barrier of the surrounding coating.
  • Suitable osmogens include, but are not limited to, inorganic salts, such as magnesium sulfate, magnesium chloride, calcium chloride, sodium chloride, lithium chloride, potassium sulfate, potassium phosphates, sodium carbonate, sodium sulfite, lithium sulfate, potassium chloride, and sodium sulfate; sugars, such as dextrose, fructose, glucose, inositol, lactose, maltose, mannitol, raffinose, sorbitol, sucrose, trehalose, and xylitol; organic acids, such as ascorbic acid, benzoic acid, fumaric acid, citric acid, maleic acid, sebacic acid, sorbic acid, adipic acid, edetic
  • Osmotic agents of different dissolution rates can be employed to influence how rapidly the active ingredient(s) is initially delivered from the dosage form.
  • amorphous sugars such as MA OGEMTM EZ (SPI Pharma, Lewes, DE) can be used to provide faster delivery during the first couple of hours to promptly produce the desired therapeutic effect, and gradually and continually release of the remaining amount to maintain the desired level of therapeutic or prophylactic effect over an extended period of time.
  • the active ingredient(s) is released at such a rate to replace the amount of the active ingredient metabolized and excreted.
  • the core can also include a wide variety of other excipients and carriers as described herein to enhance the performance of the dosage form or to promote stability or processing.
  • Materials useful in forming the semipermeable membrane include various grades of acrylics, vinyls, ethers, polyamides, polyesters, and cellulosic derivatives that are water- permeable and water-insoluble at physiologically relevant pHs, or are susceptible to being rendered water-insoluble by chemical alteration, such as crosslinking.
  • Suitable polymers useful in forming the coating include plasticized, unplasticized, and reinforced cellulose acetate (CA), cellulose diacetate, cellulose triacetate, CA propionate, cellulose nitrate, cellulose acetate butyrate (CAB), CA ethyl carbamate, CAP, CA methyl carbamate, CA succinate, cellulose acetate trimellitate (CAT), CA dimethylaminoacetate, CA ethyl carbonate, CA chloroacetate, CA ethyl oxalate, CA methyl sulfonate, CA butyl sulfonate, CA p-toluene sulfonate, agar acetate, amylose triacetate, beta glucan acetate, beta glucan triacetate, acetaldehyde dimethyl acetate, triacetate of locust bean gum, hydroxylated ethylene-vinylacetate, EC, PEG, PPG, PEG/PPG copo
  • a semipermeable membrane can also be a hydrophobic microporous membrane, wherein the pores are substantially filled with a gas and are not wetted by the aqueous medium but are permeable to water vapor, as disclosed in U.S. Pat. No. 5,798, 1 19.
  • Such hydrophobic but water- vapor permeable membrane are typically composed of hydrophobic polymers such as polyalkenes, polyethylene, polypropylene, polytetrafluoroethylene, polyacrylic acid derivatives, polyethers, polysulfones, polyethersulfones, polystyrenes, polyvinyl halides, polyvinylidene fluoride, polyvinyl esters and ethers, natural waxes, and synthetic waxes.
  • hydrophobic polymers such as polyalkenes, polyethylene, polypropylene, polytetrafluoroethylene, polyacrylic acid derivatives, polyethers, polysulfones, polyethersulfones, polystyrenes, polyvinyl halides, polyvinylidene fluoride, polyvinyl esters and ethers, natural waxes, and synthetic waxes.
  • the delivery port(s) on the semipermeable membrane can be formed post-coating by mechanical or laser drilling. Delivery port(s) can also be formed in situ by erosion of a plug of water-soluble material or by rupture of a thinner portion of the membrane over an indentation in the core. In addition, delivery ports can be formed during coating process, as in the case of asymmetric membrane coatings of the type disclosed in U.S. Pat. Nos.
  • the total amount of the active ingredient(s) released and the release rate can substantially by modulated via the thickness and porosity of the semipermeable membrane, the composition of the core, and the number, size, and position of the delivery ports.
  • An osmotic controlled-release dosage form can further comprise additional conventional excipients or carriers as described herein to promote performance or processing of the formulation.
  • the osmotic controlled-release dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art. See Remington: The Science and Practice of Pharmacy, supra; Santus and Baker, J. Controlled Release 1995, 35, 1-21 ; Verma et al., Drug Development and Industrial Pharmacy 2000, 26, 695-708; and Verma et al., J. Controlled Release 2002, 79, 7-27.
  • compositions are formulated as AMT controlled- release dosage form, which comprises an asymmetric osmotic membrane that coats a core comprising the active ingredient(s) and other pharmaceutically acceptable excipients or carriers.
  • AMT controlled-release dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art, including direct compression, dry granulation, wet granulation, and a dip-coating method.
  • compositions are formulated as ESC controlled-release dosage form, which comprises an osmotic membrane that coats a core comprising the active ingredient(s), a hydroxylethyl cellulose, and other pharmaceutically acceptable excipients or carriers.
  • the lithium-containing compound can be loaded into a polymeric solution that consists of a water-soluble polymer that is a solution at room temperature (20-25°C) and below, but gels at physiological temperatures of 32-37°C.
  • the lithium-containing solution can be cooled to 2-8°C to impart a soothing effect, while being sprayed as a liquid spray on the tissue surface. Once sprayed on, the lithium- loaded solution will thicken into a gel, releasing the lithium-containing compound slowly over time.
  • the lithium-loaded solution can be injected as a liquid, to form an in situ depot within the tissue.
  • the lithium-loaded solution can be delivered as a solution, which can flow into orifices of the tissue, such as hair follicles and then, form a gel to release lithium for follicle-associated conditions, such as MPHL, folliculitis, or another condition described herein.
  • the temperature and time of gelation can be correlated to the concentration of the polymers and the length of the polymer blocks that constitute the polymers.
  • the a modified release dosage form can be fabricated as a multiparticulate controlled release device, which comprises a multiplicity of particles, granules, or pellets, ranging from about 10 ⁇ to about 3 mm, about 50 ⁇ to about 2.5 mm, or from about 100 ⁇ to about 1 mm in diameter.
  • Such multiparticulates can be made by the processes known to those skilled in the art, including microfluidization, membrane-controlled emulsification, oil-in-water, water-oil-water and oil-in oil emulsification and homogenization processes, complex coacervation, wet-and dry-granulation, extrusion/spheronization, roller-compaction, melt-congealing, and by spray-coating seed cores.
  • microfluidization membrane-controlled emulsification
  • compositions can be blended with the compositions to aid in processing and forming the multiparticulates.
  • the resulting particles can themselves constitute the multiparticulate device or can be coated by various film- forming materials, such as enteric polymers, water-swellable, and water-soluble polymers.
  • the multiparticulates can be further processed as a capsule or a tablet. 5.2.3 TARGETED DELIVERY
  • the lithium compounds for use herein may be formulated with a carrier that delivers the lithium to the site of action, for example, a follicle in a particular tissue. Such targeted delivery may be preferable in formulations for systemic administration, in order to reduce side effects associated with lithium therapy and/or ensure that the lithium reaches only follicles of particular tissues.
  • the carrier may be an aptamer targeted to a particular protein or cell type in the follicle, an antibody or antigen-binding fragment thereof, a virus, virus-like particle, virosome, liposome, micelle, microsphere, nanoparticle, or any other suitable compound.
  • compositions for use in the methods provided herein can also be formulated to be targeted to a particular tissue, follicle, or other area of the body of the subject to be treated, including liposome-, resealed erythrocyte-, and antibody-based delivery systems. Examples include, but are not limited to, those disclosed in U.S. Pat. Nos. 5,709,874; 5,759,542;
  • targeting is accomplished by the attachment of specific targeting moieties to the delivery systems containing the drug.
  • Targeting moieties can be in the form of antibodies, aptamers or small molecules that bind to specific proteins expressed in specific tissues.
  • Specific or guided targeting can "channel" the drug only to the specific tissue type, thus minimizing distribution to all tissues. This concept is especially useful if the drug causes side effects.
  • microspheres and nanospheres have been utilized, to deliver drugs into the hair follicle. Entry into the hair follicle is governed by the size of the drug-containing spheres, with microspheres of size 0.5-0.7 microns of ideal size for entry.
  • the surface of the microspheres can be functionalized with moieties that bind to specific surfaces in the follicular orifice to "retain" them at the site.
  • These moieties can be non-specific, such as hydrophobic coatings, or cationic coatings, in order to be bioadhesive to cells within the follicle.
  • the moieties can be specific and targeted to certain proteins that are expressed specifically on specific cell membranes. For example, proteins over-expressed on the follicular lymphoma cell surfaces can be targeted by delivery systems that have antibodies or aptamers designed to bind to these proteins.
  • the surface of the delivery systems can also be functionalized with cell-penetrating moieties such as cell-permeable peptides, positively charged polymers that bind to anionic cell surfaces.
  • the intermittent lithium treatments or a pulse lithium treatment described herein may be delivered locally to any part of the subject in which wound healing or scar revision is desired, including, e.g., the head (e.g., the scalp, cheek, chin, lips, ears, nose, eyelid or eyebrow), neck, abdomen, chest, breast, back, arms, armpits, stomach, genital area, buttocks, legs, hands, or feet of a subject.
  • the intermittent lithium treatment or a pulse lithium treatment is applied to wounded or scarred skin.
  • the intermittent lithium treatment or a pulse lithium treatment is applied before the skin is wounded or scarred.
  • Such local delivery of the intermittent lithium treatment or a pulse lithium treatment can be achieved by topical administration, transdermal, intradermal, subcutaneous (depot effect), or by intramuscular, intravenous and oral routes of delivery in formulations for targeting systemically delivered lithium to desired follicles. Such modes of delivery are discussed supra.
  • enhancement of wound healing or scar revision in wounded or otherwise integumentally perturbed skin is accomplished by a lithium treatment described herein in combination with a pre-designed biomaterial dressing that may serve as a substrate to encourage a step-wise attachment of keratinocytes and epithelial cells to it, such that formation of an organized extra-cellular matrix (ECM) is enhanced in order to promote wound healing.
  • ECM organized extra-cellular matrix
  • the scaffold for use in combination with lithium treatment may be comprised of a mesh of a biocompatible, bioabsorbable material that cells recognize and attach to, preferably with ease.
  • these materials can be collagen type I/III, hyaluronic acid, chitosan, alginates, or combinations and derivatives thereof or any other such material described herein or known in the art.
  • the mesh scaffold may be neutral, or charged. If the mesh is positively charged, it may permit cells (which are negatively charged) to adhere to it more effectively. If the mesh scaffold is negatively charged, it may contain signaling moieties that the cells will recognize and attach to. For example, polymers such as hyaluronic acid are present already in skin, and thus a mesh comprised of this material is thought to be compatible with cells.
  • the scaffold is pre-fabricated with a fine microstructure that is of the dimension of cells, for example, red blood cells that will initially diffuse throughout the scaffold, or epithelial cells and keratinocytes from surrounding tissue.
  • epitophelial tongue can move with greater ease and organization by crawling on the scaffold mesh.
  • the mesh scaffold has an "open-cell” structure, with the pores inter-connected, much like an open-celled foam.
  • the open, interconnecting nature of the scaffold may allow free diffusion of oxygen and cells, so that optimal organized wound healing can occur.
  • the mesh scaffold has the capacity to hydrate and remain hydrated throughout the wound healing period. This is useful because, without being bound by any theory, drying out of the wound results in a impermeable granular structure that the keratinocytes cannot "crawl upon.”
  • the mesh scaffold has moieties that act as molecular signals to the cells, for example, to aid their proliferation. These moieties include, but are not limited to, peptidoglycans and RGD integrin recognition sequences that encourage cell attachment and subsequent proliferation.
  • the mesh scaffold has incorporated within it one or more active agents, for example, a small molecule, or a nucleic acid, or a protein.
  • the additional active agent is a protein, such as noggin or WNT, or is a nucleic acid that encodes noggin or WNT.
  • a small molecule is incorporated into the scaffold, such as, e.g. , a GSK inhibitor, BMP inhibitor, or PPAR antagonist.
  • the compound incorporated in the mesh scaffold is a compound considered for use in the combination therapies described herein, for example, in Section 5.4, especially Sections 5.4.2 to 5.4.4.
  • the scaffold may incorporate superoxide dismutase, a free radical quenching molecule that functions in the reduction of inflammation.
  • compounds are included in the mesh scaffold that alter the kinetics of wound healing, for example, that slow wound healing. Such compounds are known in the art and described elsewhere herein.
  • Other compounds that may be incorporated in the mesh scaffold include growth factors that aid in cell proliferation and tissue regeneration. In some embodiments, the compounds aid in hair follicle migration or hair follicle neogenesis in the wound site.
  • the lithium compound itself is incorporated within the mesh scaffold.
  • the lithium compound is incorporated within one or more layers of a multilayered mesh scaffold.
  • the mesh scaffold contains the lithium compound in alternating layers, which may achieve a pulsatile delivery of lithium.
  • the lithium compound in incorporated in microspheres in the scaffold, enabling a controlled release of lithium from the scaffold.
  • the mesh scaffold can be fibrin gels that additionally contain lithium.
  • a fibrin network is the first scaffold that a cell encounters as it performs its role in healing wounds due to trauma or other insults to tissue.
  • the fibrin network (the "scab") assemble rapidly by a modified polycondensation reaction from fibrinogen, an abundant constituent of blood plasma, as soon as the protease thrombin is activated in the clotting cascade— the result is a three-dimensional network of branching fibers, What is envisioned is a fibrin delivery matrix containing lithium, fibrinogen and thrombin, that "gels" in-situ.
  • One issue that is encountered is the ability of lithium to diffuse through the fibrin "scab” - making the drug part of the scab solves this issue.
  • the mesh scaffold is a synthetic biodegradable dressing and lithium delivery system that also acts as a "sponge" and absorbs the exudates/bloods from a wound.
  • These exudates intercalating with the synthetic scaffold contain an abundance of fibrinogen, thrombin, fibronectin, cell adhesion proteins, growth factors and hyaluronic acid, all of which create an integrated structure that is an attractive matrix for cell attachment /differentiation and delivery of lithium.
  • the release rate of lithium can be modulated by varying the composition of polymers that comprise the synthetic scaffold, or sponge.
  • a synthetic scaffold fabricated out of poly(lactide)-co-(glycolide) (PLG) and poly(lactide) (PLA) can be developed to have varied release profiles of lithium. Changing the ratio of PLA to PLG will change the release profile of the lithium from the scaffold.
  • Other polymers that can utilized to generate synthetic scaffolds are chitosan, carregenan, alginate, poly(vinyl alcohol), poly(ethylene oxide) (PEO), poly(ethylene oxide)-co-poly(propylene oxide)-co-poly(ethylene oxide) (PEO-PPO-PEO), poly(acrylates) and poly(vinyl pyrrolidone) (PVP).
  • the rate of lithium release from the formulation can be controlled, so that it takes anywhere from 2 hours to 30 days for most (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 100%) of the lithium ion to be released.
  • most of the lithium is released from the formulation within 2 hours, within 4 hours, within 8 hours, within 16 hours, within 24 hours, within 36 hours, within 48 hours, within 3 days, within 5 days, within 7 days, within 10 days, within 14 days, within 30 days, or within 2 months or more.
  • the mesh scaffold releases the aforementioned compounds in a timed release manner, acting as a controlled release formulation such as described in Section 5.2.2 above.
  • the compounds may be bound to the mesh scaffold, and are then released at a sustained release manner as a result of de-binding kinetics from the mesh.
  • the compound may be bound to a polymer, which is then incorporated to the mesh scaffold, and which may allow the compound to diffuse from the mesh at a slow rate, resulting in sustained release.
  • the mesh scaffold is extruded as a gel, with certain components of the gel precipitating out to form a mesh in situ.
  • the in situ mesh can be sprayed on the wounded or otherwise perturbed surface, such as tissue that has been extensively burned. A large area can be covered in this manner.
  • the mesh scaffold is pre-fabricated as a dressing or a wrap, to cover large areas of wounded tissue.
  • the mesh scaffold can be cut to size to fit the size of the wound to present a compatible surface for favorable movement of the epithelial tongue.
  • the scaffold is prepared by melt spinning, electrospinning, micromachining, weaving, or other methods known in the art in which open cell foams are fabricated.
  • the mesh scaffold can be fabricated by these methods, with the optional incorporation of additional compound(s) (which are optionally sterilized), then sterilized by gentle ethylene oxide sterilization.
  • the additional compounds are sterilized, and then added to the sterile mesh scaffold.
  • a combinatorial strategy that uses a biodegradable scaffold combined with administration of a lithium formulation described herein (alone or in combination with another treatment, such as described in Section 5.4, especially Sections 5.4.2 to 5.4.4) is applied, which may result in the in situ generation of embryonic stem cells or recruitment of cells required for wound healing following wounding.
  • This approach may be used together with a form of integumental perturbation described in Section 5.4.1 ⁇ e.g.
  • dermabrasion accomplished by a standard dermabrader or a laser
  • deep full-thickness excision accomplished by a bulk ablative laser
  • integumental perturbation by acute wounds, chronic wounds, or wounds generated for the purpose of scar revision.
  • integumental perturbation in combination with a scaffold that administers drug results in the in situ generation of stem cells or recruitment of other cells required for the wound healing process and may facilitate more effective wound healing with little or no scarring.
  • the scaffold is biodegradable. Placement of a 3-dimensional biodegradable scaffold in the wound assists the attachment, growth and differentiation of cells.
  • tissue repair has been by autologous cell/tissue transplantation— however, autografts are associated with donor site morbidity and limited availability.
  • An alternative is allografts, but these are susceptible to immune responses and also carry the risk of disease transfer.
  • tissue engineering has emerged as an interdisciplinary field that makes use of biomaterials, cells and factors either alone, or in combination to restore tissues. The tissue engineering strategy generally involves isolation of healthy cells from a patient, followed by their expansion in vitro.
  • a fibrin network is the natural network that forms rapidly due to a polycondensation reaction from fibrinogen, an abundant constituent of blood plasma, as soon as the protease thrombin is activated in the clotting cascade. The fibrin clot then forms a three-dimensional network for cells to attach, for re-epithelialization.
  • the biodegradability of the scaffold is modulated.
  • the biodegradability of the scaffold should be matched to the formation of the new epithelium due to wound healing.
  • One skilled in the art would know how to measure whether a synthetic matrix is biodegradable.
  • biodegradability can be measured ex vivo in implants or using rats or another animal model, by histological and HPLC analysis.
  • biodegradability by hydrolysis can be assessed.
  • the scaffold structure of choice is incubated in phosphate buffered saline, pH 7.4 and 37 °C.
  • the incubation buffer includes enzymes. The scaffolds are weighed prior to incubation.
  • the scaffolds are retrieved two-at-a-time at predetermined time points and dried in a vacuum oven.
  • the scaffolds are weighed at each time point and a plot of weight versus time is generated to develop the rate of biodegradability.
  • the biodegradability of the scaffold matrix is modulated to coincide with the healing process, and can be modulated by changing the composition of polymers utilized to fabricate the mesh. For example, a percentage of polyethylene glycol (PEG) can be included in a composition with PLG (e.g., described in the example in Section 19) to increase
  • Biodegradable synthetic matrices can be created to mimic the extra-cellular micro- environment for the enhanced cellular attachment necessary for tissue regeneration.
  • cell-recognition motifs such as RGD peptides may be incorporated to encourage cells to attach themselves to the scaffold.
  • RGD peptides may be incorporated to encourage cells to attach themselves to the scaffold.
  • One skilled in the art would know how to measure whether the biodegradable synthetic matrix has biomimetic properties. For example, in one embodiment, the biomimetic nature of the scaffold is judged on the basis of the content of the mesh and resultant intercalating fibrin.
  • the properties of the synthetic scaffold are dependent upon the three-dimensional geometry, matching of the modulus of the matrix with the tissue type and the porosity. It has been shown that the differentiation process can be modulated if the modulus of the tissue type is matched with the modulus of the scaffold.
  • the modulus of the scaffold is matched with the modulus of the tissue type.
  • the compressive modulus of a scaffold or hydrogel can be measured by a standard Instron instrument ⁇ e.g., using the TA Instruments DMA Q800).
  • the micro-environment created by the cells is optimally highly biocompatible to the cells present at the site, namely keratinocytes and stem cells derived from the dermal papilla.
  • this can be accomplished through the use of hydrophilic components that can absorb water.
  • hydrophobic components such as petrolatum is likely to be occlusive and prevent rapid cell proliferation.
  • the scaffold is incubated with human foreskin fibroblasts (HFF) in vitro and the scaffold is considered to be biocompatible if the cells maintain their shape and attach appropriately.
  • HFF human foreskin fibroblasts
  • the biodegradable scaffold is permeable to water, nutrients, oxygen and growth factors, enabling easy exchange of nutrients between tissues and cells (see, e.g., ASTM D39857). In some embodiments, a non-occlusive, non-permeable barrier is avoided.
  • the scaffold is used to "fill" a deep wound, as is common in a deep burn, to provide a matrix for the cells to attach, grow and differentiate - existence of the scaffold will likely minimize the scar formation normally observed in deep, large-area wounds.
  • a loose, dry, highly porous network or scaffold or mesh is placed in the bleeding site of the wound to gently absorb the blood and the cell adhesion proteins released at the site, as a result of wounding.
  • This will result in creation of a highly rich environment that consists of a combination of a 3 -dimensional scaffold combined with fibrinogen and thrombin, which will ultimately result in a highly biocompatible hydrogel suitable for cell attachment and growth.
  • inclusion of blood components and cell adhesion proteins into the network is critical for establishment of the ECM
  • extracellular matrix necessary to form continuous tissue in-growth, particularly in the case of large-area and deep wounds.
  • a dry scaffold has the added advantage of absorbing the blood at the wound site.
  • a person's own blood components can be used to create a combined synthetic/natural ECM.
  • the scaffold has an added advantage of serving as a blood absorbing gauze.
  • the scaffold has cell-recognition motifs, such as RGD peptides, to recruit cells to the site and attachment, thereof. Once attached, cells will proliferate. Without being bound by any theory, it is hypothesized that the primary attachment of cells to the scaffold is a critical step to prevent premature cell death.
  • a dry, sterile biodegradable scaffold is placed onto the freshly formed wound.
  • the properties of the scaffold will be such that it will transform into an adherent hydrogel upon water absorption.
  • Methods that may be employed to fabricate the scaffold are known in the art, and include electrospinning, micromachining, and others. Nano-fiber meshes fabricated by electrospinning, hydrogel imprint technologies have been utilized to create three-dimensional microstructures that match the supramolecular architecture of the tissue type. In situ forming scaffolds are also contemplated.
  • the active agents are administered using an active agent-containing spray-on hydrogel.
  • the active agent after placement of the biodegradable scaffold, the active agent is sprayed on the tissue.
  • the active agent (or combination of active agents, e.g., lithium and another stem cell signaling agent) may be incorporated into a spray-on hydrogel that will be sprayed on as a liquid, but which transforms into a hydrogel after it is sprayed on the tissue. This will be especially useful if the area of the wound is large and uniform coverage is needed.
  • the active agent-containing spray-on hydrogel is applied on the wound site, forming a cross-linked hydrogel that releases active agent over the time period of healing or a shorter or longer time period, as necessary.
  • the active agent will either be incorporated in microencapsulates or nano-encapsulates and suspended into the pre-hydrogel solution.
  • the active agent can also be dissolved into the pre-hydrogel solution.
  • the "pre-hydrogel” solution is defined as the solution that will be sprayed on the tissue and which also contains the active agent.
  • the active agent is contained within microspheres that can be positively charged to rapidly bind themselves to the negatively charged collagen present in the dermis. Binding the microspheres to the dermis renders the active agent-releasing moiety immobile at the site.
  • the wound may be covered with a breathable, non-occlusive spray-on hydrogel to cover the wound from infection during healing.
  • the intermittent lithium treatments or a pulse lithium treatment can be provided by administration of the lithium compound (or combination treatments, discussed in Section 5.4 infra) in forms suitable for topical ⁇ e.g., applied directly to the skin, transdermal, or intradermal), subcutaneous, intramuscular, intravenous or by other parenteral means, oral administration, sublingual administration, or bucchal administration.
  • topical ⁇ e.g. , applied directly to the skin, transdermal, or intradermal) administration is accomplished with the use of a mechanical device, such as, e.g., an iontophoretic device.
  • the lithium compounds (or combination treatment) can also be formulated as modified release dosage forms, including delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated-, fast-, targeted-, programmed-release, and gastric retention dosage forms.
  • These dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art (see, Rathbone et al, eds., 2008, Remington: The Science and Practice of Pharmacy, supra; Modified-Release Drug Delivery Technology, 2nd ed., New York, NY: Marcel Dekker, Inc.).
  • the intermittent lithium treatments or a pulse lithium treatment can be administered by a health care practitioner or by the subject. In some embodiments, the subject administers the intermittent lithium treatments or a pulse lithium treatment to him or herself.
  • topical administration is to the skin, either to the skin surface, transdermally, or intradermally. Topical administration can be with or without occlusion with a bandage or other type of dressing. In some embodiments, topical administration is to orifices or mucosa, or conjunctival, intracorneal, intraocular, ophthalmic, auricular, nasal, vaginal, urethral, respiratory, and rectal administration. The formulation used for topical administration can be designed to retain the lithium in the skin or to deliver a dose of lithium systematically. In some embodiments, topical administration of a lithium compound is combined with another treatment described herein, such as, but not limited to, a technique of integumental perturbation described in Section 5.4.1 infra.
  • Dosage forms that are suitable for topical administration for preferably local but also possible systemic effect, include emulsions, solutions, suspensions, creams, gels, hydrogels, ointments, dusting powders, dressings, elixirs, lotions, suspensions, tinctures, pastes, powders, crystals, foams, films, aerosols, irrigations, sprays, suppositories, sticks, bars, ointments, sutures, bandages, wound dressings, microdermabrasion or dermabrasion particles, drops, and transdermal or dermal patches.
  • the topical formulations can also comprise micro- and nano-sized capsules, liposomes, micelles, microspheres, microparticles, nanosystems, e.g., nanoparticles, nano-coacervates and mixtures thereof. See, e.g.,
  • the nano-sized delivery matrix is fabricated through a well-defined process, such as a process to produce lithium encapsulated in a polymer.
  • the lithium-releasing compound is spontaneously assembled in aqueous solutions, such as in liposomes and micelles.
  • the formulation for topical administration is a shampoo or other hair product, tanning product or sun protectant, skin lotion, or cosmetic.
  • the selected formulation will penetrate into the skin and reach the hair follicle.
  • the stratum corneum and/or epidermis have been or are removed by a method of integumental perturbation described herein (including by wounding or scar revision procedure, by laser, or by dermabrasion or microdermabrasion, which is a less vigorous form of dermabrasion), permitting application of the dosage form for topical administration directly into the exposed dermis.
  • the formulation for topical administration will be lipid-based, so that it will penetrate the stratum corneum.
  • the formulation for topical administration will contain a skin penetrant substance, such as, e.g., propylene glycol or transcutol.
  • a formulation in ointment form comprises one or more of the following ingredients: wool alcohol (acetylated lanolin alcohol), hard paraffin, white soft paraffin, liquid paraffin, and water. See, e.g., Langtry et al, supra.
  • the selected formulation is inconspicuous when applied to the skin, for example, is colorless, odorless, quickly-absorbing, etc.
  • the selected formulation is applied on the skin surface as a solution, which can crosslink into a hydrogel within a few minutes, thus creating a biocompatible dressing.
  • the hydrogel may be biodegradable.
  • the solution will absorb into the skin and crosslink into depots releasing drug.
  • the lithium ion will be used to crosslink the polymer, with release of the lithium ion controlled by the rate of degradation of the hydrogel.
  • Pharmaceutically acceptable carriers and excipients suitable for use in topical formulations include, but are not limited to, aqueous vehicles, water-miscible vehicles, nonaqueous vehicles, antimicrobial agents or preservatives against the growth of
  • microorganisms such as, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, penetration enhancers, cryoprotectants, lyoprotectants, thickening agents, and inert gases.
  • Forms for topical administration can also be in the form of ointments, creams, and gels.
  • Suitable ointment vehicles include oleaginous or hydrocarbon vehicles, including lard, benzoinated lard, olive oil, cottonseed oil, mineral oil and other oils, white petrolatum, paraffins; emulsifiable or absorption vehicles, such as hydrophilic petrolatum, hydroxystearin sulfate, and anhydrous lanolin; water-removable vehicles, such as hydrophilic ointment; water-soluble ointment vehicles, including polyethylene glycols of varying molecular weight; emulsion vehicles, either water- in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, including cetyl alcohol, glyceryl monostearate, lanolin, wool alcohol (acetylated lanolin alcohol), and stearic acid ⁇ see, Remington: The
  • Suitable cream base can be oil-in-water or water-in-oil.
  • Suitable cream vehicles may be water-washable, and contain an oil phase, an emulsifier, and an aqueous phase.
  • the oil phase is also called the "internal" phase, which is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol.
  • the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant.
  • the emulsifier in a cream formulation may be a nonionic, anionic, cationic, or amphoteric surfactant.
  • Gels are semisolid, suspension-type systems. Single-phase gels contain organic macromolecules distributed substantially uniformly throughout the liquid carrier. Suitable gelling agents include, but are not limited to, crosslinked acrylic acid polymers, such as carbomers, carboxypolyalkylenes, and CARBOPOL ® ; hydrophilic polymers, such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers, and polyvinylalcohol; cellulosic polymers, such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methylcellulose; gums, such as tragacanth and xanthan gum; sodium alginate; and gelatin.
  • dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing, and/or stirring.
  • lithium gluconate e.g., 8% lithium gluconate (LithiodermTM), approved for the treatment of seborrheic dermatitis ⁇ see, e.g., Dreno and Moyse, 2002, Eur J Dermatol 12:549-552; Dreno et al, 2007, Ann Dermatol Venereol 134:347-351 (abstract); and Ballanger et al, 2008, Arch Dermatol Res 300:215-223, each of which is incorporated by reference herein in its entirety); 8% lithium succinate (see, e.g., Langtry et al, 1996, Clinical and Experimental Dermatology 22:216-219; and Cuelenaere et al, 1992, Dermatology 184: 194-197, each of which is incorporated by reference herein in its entirety);
  • Each of these methods of topical administration may be used alone to administer lithium compounds or in combination with one or more other treatments as described in Section 5.4 infra.
  • topical administration is by electrical current, ultrasound, laser light, or mechanical disruption or integumental perturbation.
  • electroporation include electroporation, RF ablation, laserporation, laser ablation (fractional or non- fractional), non- ablative use of a laser, iontophoresis, phonophoresis, sonophoresis, ultrasound poration, or using a device that accomplishes skin abrasion, or microneedle or needle-free injection, such as topical spray or POWDERJECTTM (Chiron Corp., Emeryville, CA), BIOJECTTM (Bioject Medical Technologies Inc., Tualatin, OR), or JetPeelTM (from TavTech, Tel Aviv, Israel), which uses supersonically accelerated saline to remove epidermis.
  • POWDERJECTTM Chiron Corp., Emeryville, CA
  • BIOJECTTM Bioject Medical Technologies Inc., Tualatin, OR
  • JetPeelTM from TavTech, Tel Aviv, Israel
  • the device for topical administration of lithium compounds is an automatic injection device worn continuously but delivers lithium intermittently.
  • the device for topical administration of lithium compounds is an automatic injection device that is inconspicuous, for example, can be worn without undue discomfort under clothes, in the hair, under a hairpiece, etc.
  • a device for administration of the intermittent lithium treatment or a pulse lithium treatment delivers the lithium at a controlled depth in the skin so that it reaches hair follicles, but entry into the circulation is minimized.
  • the stratum corneum and epidermis is previously removed using a method of integumental perturbation (or by integumental perturbation as a result of wounding) described herein, and thus the required delivery pressures and velocities can be reduced. This reduction reduces the required complexity of the firing mechanisms.
  • a narrow firing stream is used, particularly to accomplish systemic delivery.
  • the particle injection system administers the lithium compound over a broad area of skin.
  • An exemplary particle delivery device compatible with broad-based skin delivery includes a low pressure / low velocity firing mechanism with a spray nozzle designed to deliver to a broad area.
  • a single-shot device that delivers to a 25 -cm 2 area could be fired or used multiple times on the scalp or other skin surface until the entire area is treated.
  • a dry particle spraying mechanism similar to an airbrush or miniature grit-blaster can be used to "paint" drug or drug particles onto the perturbed, wounded, or scarred area.
  • the stratum corneum and epidermis are already removed, e.g., by a method of integumental perturbation (e.g., wounding) described herein, and thus permits effective use of the mechanism using lowered pressure and velocity requirements to achieve dermal delivery.
  • the lithium compound (and/or additional drug) is present in an aqueous suspension, permitting use of standard aerosol spray can technology to deliver the lithium compound to the desired skin area.
  • dermabrasion e.g., using a mechanical device, including microdermabrasion devices that can be used to dermabrade, or alumina-, silica- or ice-based dermabrasion (as described by Webber, U.S. 6,764,493; U.S. 6,726,693; and U.S. 6,306, 119) is customized to include a drug particle delivery feature using methods readily known in the art.
  • the device fires ablation particles at the skin, it could also fire smaller drug particles that would simultaneously embed in the exposed dermis.
  • the device could switch over to firing drug particles once it is determined that adequate skin disruption has occurred. See, International Patent Application Publication No. WO 2009/061349, which is incorporated herein by reference in its entirety.
  • a standard dermabrasion device can be modified to incorporate any of the devices described above, e.g., a spraying/painting device.
  • a spray nozzle is located behind the dermabrasion wheel such that drug is sprayed into the dermis as it is exposed by the wheel.
  • the dermabrasion device via internal controls, could turn off the abrasion wheel once it is determined that adequate skin disruption has occurred, and switch on the drug spray to convert to drug painting mode.
  • a pulsed dye laser (585-595 nm) is combined with drug spraying either before or without skin perturbation, in conjunction with skin perturbation, or following skin perturbation.
  • a non- fractional C(3 ⁇ 4 or Erbium- YAG laser is combined with drug spraying either without or before skin disruption, in conjunction with skin disruption, or following skin disruption.
  • a fractional non-ablative laser e.g., an Erbium- YAG laser used at 1540-1550 nm
  • drug spraying either before or without skin perturbation, in conjunction with skin perturbation, or following skin perturbation.
  • a fractional ablative laser e.g., an Erbium-YAG laser used at 2940 nm or a CO 2 laser used at 10,600 nm
  • drug spraying either before or without skin perturbation, in conjunction with skin perturbation, or following skin perturbation.
  • fractional ablative laser treatment of the skin e.g., an Erbium-YAG laser used at 2940 nm or a C(3 ⁇ 4 laser used at 10,600 nm
  • lithium compound delivery e.g., lithium compound delivery.
  • a fractional laser could be combined with a precise delivery means such that as the laser forms a hole in the skin, the inkjet-like delivery component could fill that same hole with drug.
  • adequate integrated hardware and software controls are required such that the laser ablation and drug delivery are properly timed resulting in each newly formed hole being properly filled with drug.
  • fractional ablative laser treatment of the skin e.g., an Erbium-YAG laser used at 2940 nm or a CO 2 laser used at 10,600 nm
  • lithium compound delivery e.g., lithium compound delivery.
  • a non-ablative, fractional laser could be combined with a precise delivery means such that as the laser forms a hole in the skin, the inkjet-like delivery component could fill that same hole with drug.
  • adequate integrated hardware and software controls are required such that the laser treatment and drug delivery are properly timed resulting in each newly formed hole being properly filled with drug.
  • topical administration comprises administration of lithium- containing particles.
  • the particles can be delivered to the skin in combination with any of the means above and described elsewhere infra. Additionally, the particles can be designed for intermittent or pulse delivery of lithium. In one embodiment, particles with different release properties are be delivered simultaneously to achieve pulse delivery.
  • topical administration comprises administration of a lithium-containing formulation that is delivered through channels that are created by the use of needling or micro-needle technology.
  • the formulation can be, e.g., a liquid, a gel or a dry spray.
  • topical administration may be through delivery of a lithium- containing formulation through hollow needles.
  • topical administration comprises administration of a lithium-containing formulation that is delivered into the skin by an iontophoretic patch.
  • a patch can be developed in which the lithium-containing formulation is incorporated.
  • topical administration comprises administration of a lithium-containing formulation that is incorporated into micro-needle shaped biodegradable polymers.
  • the biodegradable microneedles penetrate the targeted skin tissue, and are optionally left in place to deliver the lithium ions in a sustained fashion over time.
  • Administration can be parenterally by injection, infusion, or implantation, for local or systemic administration.
  • Parenteral administration includes intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial, intravesical, and subcutaneous administration.
  • Compositions for parenteral administration can be formulated in any dosage forms that are suitable for parenteral administration, including solutions, suspensions, emulsions, micelles, liposomes, microspheres, nanosystems, and solid forms suitable for solutions or suspensions in liquid prior to injection.
  • compositions intended for parenteral administration can include one or more pharmaceutically acceptable carriers and excipients, including, but not limited to, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, antimicrobial agents or preservatives against the growth of microorganisms, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, cryoprotectants, lyoprotectants, thickening agents, pH adjusting agents, and inert gases.
  • aqueous vehicles water-miscible vehicles, non-aqueous vehicles, antimicrobial agents or preservatives against the growth of microorganisms, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents,
  • compositions for parenteral administration can be formulated as a suspension, solid, semi-solid, or thixotropic liquid, for administration as an implanted depot.
  • the compositions are dispersed in a solid inner matrix, which is surrounded by an outer polymeric membrane that is insoluble in body fluids but allows the active ingredient in the pharmaceutical compositions diffuse through.
  • Suitable inner matrixes include, but are not limited to, polymethylmethacrylate, polybutyl-methacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers, such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinyl alcohol, and cross-linked partially hydrolyzed polyvinyl acetate.
  • Suitable outer polymeric membranes include but are not limited to, polyethylene,
  • polypropylene ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride, vinyl chloride copolymers with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, and ethylene/vinyloxyethanol copolymer.
  • compositions comprising lithium compounds for oral
  • oral administration can be provided in solid, semisolid, or liquid dosage forms for oral administration.
  • oral administration also includes buccal, lingual, and sublingual administration.
  • Suitable oral dosage forms include, but are not limited to, tablets, fastmelts, chewable tablets, capsules, pills, strips, troches, lozenges, pastilles, cachets, pellets, medicated chewing gum, bulk powders, effervescent or non-effervescent powders or granules, oral mists, solutions, emulsions, suspensions, wafers, sprinkles, elixirs, and syrups.
  • the pharmaceutical compositions can contain one or more pharmaceutically acceptable carriers or excipients, including, but not limited to, binders, fillers, diluents, disintegrants, wetting agents, lubricants, glidants, coloring agents, dye-migration inhibitors, sweetening agents, flavoring agents, emulsifying agents, suspending and dispersing agents, preservatives, solvents, non-aqueous liquids, organic acids, and sources of carbon dioxide.
  • Compositions for oral administration can be also provided in the forms of liposomes, micelles, microspheres, or nanosystems. Micellar dosage forms can be prepared as described in U.S. Pat. No. 6,350,458.
  • oral formulations approved for treating mood disorders e.g., lithium carbonate (L1 2 CO 3 ), sold under several trade names, lithium citrate (L1 3 C 6 H 5 O 7 ), lithium sulfate (Li 2 S0 4 ), lithium aspartate, or lithium orotate, may be administered in accordance with the methods described herein.
  • lithium carbonate L1 2 CO 3
  • lithium citrate L1 3 C 6 H 5 O 7
  • lithium sulfate Li 2 S0 4
  • lithium aspartate lithium orotate
  • the intermittent and pulse lithium treatments may also be administered to skin- derived cells or skin tissue ex vivo.
  • an intermittent or pulse lithium treatment may be used to enhance the re-association of dissociated hair follicle cells into follicles and their growth and expansion in culture for their implantation into fresh wounds and scar revisions.
  • hair follicles promoted by intermittent or pulse lithium treatments are added to the wound before, at the time of, and/or subsequent to, either acute wounding or, more typically, during the wounding that is induced in scar revision.
  • hair follicles can be introduced to the wound by migration or de novo hair follicle neogenesis, or by transplanting one or more of the following skin elements: full skin (xeno-; autologous human), follicular units, dissociated cells (donor dominance; recipient effects), ex vivo-expanded skin and/or follicular units, or human skin equivalents in vivo (universal donors).
  • Engineered human skin, or human skin equivalents can also be used for hair follicle neogenesis and scar revision platforms.
  • Human skin equivalents can be grown and assembled in vitro, with the advantage that they can be grown to theoretically to any size/shape; can be comprised of different types of cells, including keratinocytes (hair follicle derived and non-hair follicle derived), dermal cells (hair follicle derived and non-hair follicle derived), other cell types (e.g. , mesenchymal stem cells); can contain cells that are genetically modified to include, e.g., markers or "inducible" signaling molecules; provide an unlimited and uniform source of human cells; from normal skin based on histology and marker studies; are generally devoid of skin appendages; and can be wounded and show similar wound healing events as in vivo.
  • keratinocytes hair follicle derived and non-hair follicle derived
  • dermal cells hair follicle derived and non-hair follicle derived
  • other cell types e.g. , mesenchymal stem cells
  • the lithium compound or formulation thereof can be administered topically, subcutaneously, orally, etc. Regardless of the route of administration used for lithium ion delivery, the dosing regimen should be adjusted to achieve peak concentrations of lithium in the target skin area of at least about 0.1 mM to 10 mM, and/or peak concentrations of lithium in the blood (serum or plasma samples) of at least about 1 mM (these values are sometimes referred to herein as the "target concentration").
  • ionized lithium is a monovalent cation
  • the peak concentration of lithium can be established by taking samples when peak concentrations are achieved and assaying them for lithium content using techniques well known to those skilled in the art (see, e.g., the examples of Sections 1 1 to 15 and the techniques described therein; see also Wood et al, 1986, Neuropharmacology 25: 1285-1288; and Smith, 1978, Acta Pharmacol et toxicol 43:51-54, each of which is incorporated herein by reference in its entirety).
  • samples can be taken when peak blood concentrations are typically achieved - for example, within 1 to 2 hours for standard release formulations, and 4-5 hours for sustained release formulations.
  • the peak concentration times for other formulations, including topical preparations, can be determined for the particular formulation used, and sampling can be adjusted accordingly.
  • the target concentration of lithium should be maintained in the skin and/or blood for at least 1 day; at least 2 days; at least 3 days; at least 5 days; at least 14 days; or at least 21 days; and, in certain embodiments, not more than 21 days.
  • This can be accomplished using, e.g., repeated applications of the lithium compound or a single application of a sustained release or extended release lithium formulation.
  • Either the single pulse protocol or the intermittent treatments can be used to achieve the target concentration of lithium for the shorter maintenance periods (i.e., for at least 1, 2 or 3 days). Maintenance periods longer than 3 days may require repeated application of the intermittent lithium treatments or the single pulse protocol.
  • topical administration of a lithium compound is preferred over oral or subcutaneous administration.
  • a topically administered lithium compound may achieve a higher concentration of lithium in skin than in the blood, thereby reducing the risk of toxicity associated with elevated blood levels of lithium.
  • a subcutaneously or orally administered lithium compound may be preferred in order to achieve a controlled release of lithium from the blood to the skin.
  • lithium doses should be adjusted on the basis of the blood concentration (serum or plasma) drawn (by convention) 12 or 24 hours after the last dose of the lithium compound; this trough blood concentration should be maintained below 2 mM Li+ and preferably, below about 1.5 mM Li+. In some embodiments, the steady state blood concentration of lithium should not exceed a maximum of 1.5 mM to 2 mM.
  • the relatively stable and characteristic pharmacokinetics of the lithium ion in individual patients makes it possible to predict dosage requirements for that individual based on the results of administration of a single test dose, followed by a skin and/or blood sample assay (plasma or serum) at the peak concentration time; followed by blood sample assays to monitor toxicity at the 12 hour or 24 hour trough concentration; and 24 hours later (when lithium is generally eliminated) which serves as the control value.
  • a skin and/or blood sample assay plasma or serum
  • blood sample assays to monitor toxicity at the 12 hour or 24 hour trough concentration; and 24 hours later (when lithium is generally eliminated) which serves as the control value.
  • a trough concentration of lithium in the skin of no less than 0.01 mM to 0.05 mM is preferred. In some embodiments, a trough concentration of lithium in the skin of 0.05 mM to 0.1 mM is preferred. In some embodiments, a trough concentration of lithium in the skin of less than 1 mM is preferred. In some embodiments, a trough concentration of lithium in the skin of less than 3 mM is preferred. In some embodiments, lithium concentrations at trough can be increased by twice daily dosing, or more frequent dosing. In such embodiments, topical administration of a lithium compound is preferred.
  • a pulsatile effect is achieved by the multiple dosing, but the trough concentrations do not decline as much as when once daily dosing is used.
  • a trough skin concentration of lithium is maintained at 0.25 mM or higher, for example from 0.25 mM to 0.5 mM or 0.5 mM to 0.75 mM.
  • the trough concentration is maintained at approximately 0.6 mM to 1.4 mM lithium.
  • a trough skin concentration is maintained at 1 mM to 3 mM lithium. In some such
  • the trough skin concentration is maintained at less than 0.5 mM, or less than 0.75 mM, or less than 1 mM, or less than 2 mM, or less than 3 mM of lithium.
  • an effective amount of a lithium compound is administered such that the target concentration of lithium ions in plasma or serum, as measured 30 minutes to 1 hour after the lithium treatment, is 0.10-0.20 ⁇ , 0.20-0.50 ⁇ , 0.50-1.0 ⁇ , 1.0-5.0 ⁇ , 5.0-10 ⁇ , 10-20 ⁇ , 20-50 ⁇ , 50-100 ⁇ , 100-500 ⁇ , 0.1- 0.5 mM, 0.5-1.0 mM, 1.0 mM-2.0 mM, 2.0-2.5 mM, 2.5-3.0 mM, 3.0-4.0 mM, 4.0 mM-5.0 mM, 5.0-7.0 mM, or 7.0 mM or greater.
  • an effective amount of lithium is administered such that the plasma or serum lithium ion concentration measured either 8 hours, 16 hours, 1 day, 1 week, 2 weeks, or 1 month after the lithium treatment, is 0.1 to 0.5 ⁇ , 0.1 to 1.0 ⁇ , 0.5 to 1.0 ⁇ , 0.5 to 1.5 ⁇ , 1 to 10 ⁇ , 10 to 50 ⁇ , 50 to 100 ⁇ , 100 to 150 ⁇ , 150 to 200 ⁇ , 250 to 300 ⁇ , 100 to 250 ⁇ , 100 to 500 ⁇ , 200 to 400 ⁇ , 500 to 1000 ⁇ ; or 1000 to less than 1500 ⁇ .
  • the plasma or serum lithium concentration reaches at least 1 ⁇ . In one embodiment, the plasma or serum lithium concentration reaches at least 100 ⁇ .
  • the plasma or serum lithium concentration reaches at least 1 mM. In one embodiment, the plasma or serum lithium concentration does not exceed 1 mM. In another embodiments, the plasma or serum concentration of lithium does not exceed 1.5 mM. Serum lithium concentration may be measured using any technique known in the art, such as described in Sampson et ah, 1992, Trace Elements in Medicine 9:7-8.
  • an amount of a lithium compound is administered such that the target concentration of lithium in the skin is 0.01 to 0.05 ⁇ , 0.05 to 0.1 ⁇ , 0.1 to 0.5 ⁇ , 0.1 to 1 ⁇ , 0.5 to 1.0 ⁇ , 1.0 to 1.5 ⁇ , 1 to 2.5 ⁇ , 1 to 5 ⁇ , 5 to 10 ⁇ , 10 to 50 ⁇ , 50 to 100 ⁇ , 100 to 150 ⁇ , 150 to 200 ⁇ , 250 to 300 ⁇ , 100 to 250 ⁇ , 100 to 500 ⁇ , 200 to 400 ⁇ , 500 to 1000 ⁇ , 1 to 10 mM, 1 to 5 mM, 5 to 10 mM, 10 to 100 mM, 100 to 200 mM, or 500 to 1000 mM.
  • the concentration of lithium achieved in the skin is greater than 0.1 mM. In some embodiments, the concentration of lithium achieved in the skin is greater than 1.0 mM. In some embodiments, the concentration of lithium achieved in the skin is greater than 1.5 mM. In one embodiment, the amount of lithium achieved in the skin is approximately 1 mM to 5 mM. In one embodiment, the amount of lithium achieved in the skin is approximately 5 mM to 10 mM. In one embodiment, the amount of lithium achieved in the skin is approximately 100 to 200 mM. In one embodiment, the amount of lithium achieved in the skin does not exceed 5 mM. In one embodiment, the amount of lithium achieved in the skin does not exceed 10 mM.
  • the amount of lithium achieved in the skin does not exceed 50 mM.
  • an amount of lithium is administered such that the concentration of lithium delivered to the stratum corneum is 0.1 to 0.5 mM, 0.5 to 1 mM, 1 to 10 mM, 10 to 100 mM, 100 to 200 mM, or 500 to 1000 mM.
  • the concentration of lithium delivered to the stratum corneum is greater than 1.5 mM.
  • the amount of lithium achieved in the stratum corneum is approximately 100 to 200 mM.
  • the amount of lithium achieved in the stratum corneum does not exceed 5 mM.
  • the amount of lithium achieved in the stratum corneum does not exceed 10 mM.
  • lithium concentrations in skin using techniques known in the art, for example, mass spectroscopy, e.g., inductively coupled plasma mass spectroscopy (ICP-MS).
  • mass spectroscopy e.g., inductively coupled plasma mass spectroscopy (ICP-MS).
  • ICP-MS inductively coupled plasma mass spectroscopy
  • concentration of lithium in skin can be measured using the method provided in the example of Section 13.2 below or equivalent methods.
  • the lithium concentration is measured in the hair shaft using techniques known in the art, e.g., Tsanaclis & Wicks, 2007, Forensic Science Intl. 176: 19-22, which is incorporated by reference herein in its entirety.
  • lithium can be applied topically, e.g., as a cream, gel, ointment, or other form for topical administration as described in Section 5.2 supra. Topical lithium may be administered to wounded or unwounded skin.
  • the lithium formulation for topical administration comprises lithium (or monovalent lithium salt) at a concentration of 50 niM, 75 niM, 100 niM, 125 niM, 150 niM, 175 niM, 200 niM, 250 niM, 300 niM, 350 niM, 400 niM, 450 niM, 500 niM, 550 niM, 600 niM, 650 niM, 700 niM, 750 niM, 800 niM, 900 niM, 1 M, 1.1 M, or 1.2 M, or more.
  • a monovalent lithium salt ⁇ e.g., lithium gluconate, lithium chloride, lithium stearate, lithium orotate, etc.
  • a divalent lithium salt ⁇ e.g., in some embodiments, lithium succinate, lithium carbonate
  • a trivalent lithium salt ⁇ e.g., in some embodiments, lithium citrate), refers to a salt form of lithium in which there are three lithium cations for each anion of the salt.
  • a lithium formulation comprising lithium (or monovalent lithium salt) at a concentration in the range of 50 mM to 200 mM is chosen for use in the embodiments described herein.
  • a lithium formulation comprising lithium (or monovalent lithium salt) at a concentration in the range of 200 mM to 400 mM is used.
  • a lithium formulation comprising lithium (or monovalent lithium salt) at a concentration in the range of 400 mM to 600 mM is used.
  • a lithium formulation comprising lithium (or monovalent lithium salt) at a concentration in the range of 600 mM to 800 mM is used.
  • concentration of lithium in a particular topical lithium formulation to deliver the intended dose of lithium will depend on the release properties of the lithium ion, the hydrophobicity of the lithium salt form, the partition coefficient of the lithium salt form, etc.
  • Lithium formulations comprising the foregoing lithium (or monovalent lithium salt) concentrations may be achieved using, for example, a formulation comprising, w/w, lithium ions at a concentration of 0.10% lithium, 0.15% lithium, 0.20% lithium, 0.25% lithium, 0.30% lithium, 0.35% lithium, 0.40% lithium, 0.45% lithium, 0.50% lithium, 0.55% lithium, 0.60% lithium, 0.65% lithium, 0.70% lithium, 0.75% lithium, 0.80% lithium, 0.85% lithium, 0.90% lithium, 0.95% lithium.
  • the form of lithium for topical administration comprises, w/w, 0.1% to 0.5% lithium ions, 0.2% to 0.5% lithium ions, 0.5% to 1% lithium ions, or more.
  • the amount of a salt form of lithium to generate a topical lithium formulation with one of the aforementioned concentrations of lithium ion is readily deducible by one of ordinary skill in the art, and depends upon several factors including, e.g. , the valency of the salt form, the stability of the salt form, the ability of the salt form to release the lithium ion, the hydrophobicity or hydrophilicity, etc.
  • Lithioderm (Labcatal) comprises 8% lithium gluconate, which corresponds to 0.275% lithium ion ⁇ i.e., 274.8 mg Li+/100 g gel).
  • a formulation of topical 8% lithium gluconate, w/w contains approximately 80 mg/ml lithium gluconate, which is approximately 400 mM lithium gluconate (and, thus, 400 mM lithium ion).
  • a formulation for topical administration comprises a salt form of lithium ⁇ e.g., lithium gluconate or other form described in Section 5.1 above) at a concentration, w/w, of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 16%, 18%, 20%, or more.
  • a salt form of lithium for topical administration comprises, w/w, 1% to 2% lithium salt ⁇ e.g., lithium gluconate or other form described in Section 5.1 above), 2% to 5% lithium salt, 5% to 10% lithium salt, 10% to 15% lithium salt, 15% to 20% lithium salt, 20% to 25% lithium salt, or 25% to 50% lithium salt.
  • the form of lithium for topical administration is 1% to 20% w/w lithium salt.
  • a topical formulation of lithium comprises l%-4% lithium gluconate (w/w). In some embodiments, a topical formulation of lithium comprises 4%-8% lithium gluconate (w/w). In some embodiments, a topical formulation of lithium comprises 8%-16% or more lithium gluconate (w/w). In some embodiments, a topical formulation of lithium comprises 0.2%-l%, or l%-5%, or more lithium chloride (w/w). In some embodiments, a topical formulation of lithium comprises 0.5%-2%, or 2%-4%, or 4%-8%, or 8%-16, or more lithium succinate (w/w).
  • a topical formulation of lithium comprises 0.5%-6%, 6%-12%, or 12%-25%, or more lithium stearate (w/w). In some embodiments, a topical formulation of lithium comprises l%-4%, 4%-8%, or 8%-16%, or more lithium orotate (w/w). In some embodiments, a topical formulation of lithium comprises 0.25%-0.75%, 0.75%-1.5%, or 1.5%-3%, or more lithium carbonate (w/w). In some embodiments, a topical formulation of lithium comprises 0.25%- 1.5%, 1.5%-3.0%, or 3%-6%, or more 8% lithium citrate (w/w).
  • a 50 kg patient is administered a single droplet - approximately 0.1 ml - of 8% (w/w) lithium gluconate at 3 sites, twice daily. This corresponds to approximately 8 mg lithium gluconate (0.274 mg Li+) per site, i.e., 0.16 mg/kg lithium gluconate (0.005 mg/kg Li+) per site. Over three sites twice daily, this corresponds to approximately 0.96 mg/kg lithium gluconate (0.033 mg/kg Li+) per day.
  • a patient ⁇ e.g., a 50 kg patient
  • a topical lithium formulation is administered once daily.
  • a topical lithium formulation is administered twice daily.
  • doses are administered 6 hours apart, or 7 hours apart, or 8 hours apart, or 9 hours apart, or 10 hours apart, or 11 hours apart, or 12 hours apart. In a particular embodiment, the doses are administered 7 to 8 hours apart.
  • an amount of lithium is administered such that the peak lithium concentration in skin is between 0.01 mM and 0.05 mM, 0.05 mM and 0.1 mM, 0.1 mM and 0.5 mM or between 0.5 mM and 10 mM, for example, between 0.1 and 0.5 mM, 0.5 mM and 1 mM, 1 mM and 2 mM, between 2 mM and 5 mM, 5 mM to 10 mM, or 10 mM to 50 mM.
  • the peak lithium concentration in blood may be one or more orders of magnitude lower than the peak concentration in skin (for example, 0.001 mM to 0.01 mM, 0.01 mM to 0.1 mM, or 0.1 mM to 0.5 mM, 0.5 mM to 1.0 mM, or 1.0 mM to 10 mM).
  • the steady state blood concentration of lithium should not exceed a maximum of 1.5 mM to 2 mM.
  • a formulation of lithium described herein (by non-limiting e.g., lithium gluconate, lithium chloride, lithium succinate, lithium carbonate, lithium citrate, lithium stearate, lithium orotate, etc.) is administered subcutaneous ly, to either wounded or unwounded skin.
  • the form of lithium for subcutaneous administration is administered at a dose comprising 0.001 mg lithium ion per kg of patient weight.
  • the dose is 0.001 mg/kg, 0.002 mg/kg, 0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg, 0.010 mg/ kg, 0.020 mg/kg, 0.025 mg/kg, 0.050 mg/kg, 0.075 mg/kg, 0.10 mg/kg, 0.15 mg/kg, 0.20 mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.40 mg/kg, 0.50 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg,
  • the dose does not exceed 50 mg/kg.
  • the lower ranges of dosages may be preferably used for bolus dosing.
  • the maximum dosage that may be administered at any one time may vary depending on the release kinetics of the lithium and the concentration of efficacy of the formulation.
  • concentration of a salt form of lithium required to generate a subcutaneously administered formulation that delivers lithium ions at one of the aforementioned dosages is readily deducible by one of ordinary skill in the art, and depends upon several factors including, e.g., the valency of the salt form, the stability of the salt form, the ability of the salt form to release the lithium ion, the hydrophobicity or hydrophilicity, etc.
  • a formulation comprising lithium gluconate may be subcutaneously administered at a dosage of approximately 10 mg lithium gluconate per kg of patient weight (mg/kg), 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 650 mg/kg, 700 mg/kg, 750 mg/kg, 800 mg/kg, 850 mg/kg, 900 mg/kg, 950 mg/kg, or 1000 mg/kg.
  • the formulation for subcutaneous administration contains a dose of 10 mg/kg to 50 mg/kg, 50 mg/kg to 100 mg/kg, 100 mg/kg to 200 mg/kg, 200 mg/kg to 400 mg/kg, 400 mg/kg to 600 mg/kg, or 100 mg/kg to 600 mg/kg of lithium gluconate.
  • the formulation for subcutaneous administration contains a dose in the range of 30 mg/kg to 150 mg/kg lithium gluconate.
  • the formulation for subcutaneous administration contains a dose in the range of about 30 mg/kg to 300 mg/kg lithium gluconate.
  • the dose for subcutaneous administration does not exceed 300 mg/kg lithium gluconate.
  • the dose for subcutaneous administration does not exceed 600 mg/kg lithium gluconate.
  • the lower ranges of dosages may be preferably used for bolus dosing.
  • the maximum dosage that may be administered at any one time may vary depending on the release kinetics of the lithium and the concentration of efficacy of the formulation.
  • the lithium formulation is administered subcutaneously once daily. In some embodiments, the lithium formulation is administered subcutaneously twice daily. In some embodiments of a twice daily treatment regimen, doses are administered 6 hours apart, or 7 hours apart, or 8 hours apart, or 9 hours apart, or 10 hours apart, or 11 hours apart, or 12 hours apart. In a particular embodiment, the doses are administered 7 to 8 hours apart.
  • an amount of lithium is administered such that the peak lithium concentration in skin is between 0.1 ⁇ and 0.2 ⁇ , 0.2 ⁇ and 0.5 ⁇ , 0.5 and 1 ⁇ , 1 ⁇ and 2 ⁇ , 2 ⁇ to 10 ⁇ , 10 ⁇ to 100 ⁇ , 100 ⁇ to 500 ⁇ , 500 ⁇ to 1000 ⁇ .
  • peak values will depend on the lithium release properties of the formulation, the hydrophobicity of the lithium salt form, the partition coefficient of the lithium salt form, etc.
  • the peak concentration in skin is 0.2 ⁇ to 1.5 ⁇ lithium.
  • the peak concentration in skin should not exceed 1 ⁇ or 1.5 ⁇ lithium. In some embodiments, the peak concentration in skin is 10 ⁇ to 100 ⁇ lithium. In some embodiments, the peak concentration in skin is 100 ⁇ to 1000 ⁇ lithium. In some such embodiments, the peak lithium concentration in blood may be several orders of magnitude higher, for example, 0.1 mM to 0.5 mM, or 0.5 mM to 1.1 mM, 1.1 to 1.5 mM, 1.5 mM to 5 mM, 5 mM to 10 mM, 10 mM to 50 mM, or 50 mM to 100 mM.
  • the steady state blood concentration of lithium should not exceed a maximum of 1.5 mM to 2 mM.
  • a formulation of lithium described herein (by non-limiting e.g., lithium gluconate, lithium chloride, lithium succinate, lithium carbonate, lithium citrate, lithium stearate, lithium orotate, etc.) is administered orally, for example, once daily, or twice daily as determined by the medical practitioner and in accordance with Section 5.3 above.
  • an oral formulation comprising of 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or more, but preferably less than 10 mM, of lithium ions (or monovalent lithium salt) is administered.
  • an oral formulation comprising lithium ions or a monovalent lithium salt in the range of 0.1 to 0.5 mM, 0.4 to 0.6 mM, 0.5 to 1 mM, 0.6 to 1.2 mM, or 1 to 1.5 mM, is administered.
  • Administration of the foregoing amounts of lithium may be achieved by oral administration of a lithium formulation at a dosage comprising 0.001 mg lithium ion per kg of patient weight.
  • the dose is 0.001 mg/kg, 0.002 mg/kg, 0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg, 0.010 mg/ kg, 0.020 mg/kg, 0.025 mg/kg, 0.050 mg/kg, 0.075 mg/kg, 0.10 mg/kg, 0.15 mg/kg, 0.20 mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.40 mg/kg, 0.50 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/
  • the dose does not exceed 50 mg/kg Li+.
  • the maximum dosage that may be administered at any one time may vary depending on the release kinetics of the lithium and the concentration of efficacy of the formulation.
  • concentration of a salt form of lithium required to generate an orally administered formulation that delivers lithium ions at one of the aforementioned dosages is readily deducible by one of ordinary skill in the art, and depends upon several factors including, e.g., the valency of the salt form, the stability of the salt form, the ability of the salt form to release the lithium ion, the hydrophobicity or hydrophilicity, etc.
  • a formulation comprising lithium carbonate which is a divalent lithium salt ⁇ e.g., trade names Eskalith CR, Eskalith, Lithobid
  • the oral formulation contains a dose of 2 mg/kg to 10 mg/kg, 10 mg/kg to 25 mg/kg, 25 mg/kg to 50 mg/kg, 50 mg/kg to 100 mg/kg, 100 mg/kg to 200 mg/kg, or 200 mg/kg to 500 mg/kg of lithium carbonate. In one embodiment, the oral formulation contains a dose in the range of 5 mg/kg to 100 mg/kg lithium carbonate. In one embodiment, the oral formulation contains a dose in the range of about 5 mg/kg to 50 mg/kg lithium carbonate. In one embodiment, the oral formulation contains a dose in the range of about 10 mg/kg to 100 mg/kg lithium carbonate. In one embodiment, the oral formulation contains a dose that does not exceed 300 mg/kg lithium carbonate.
  • the maximum dosage that may be administered at any one time may vary depending on the release kinetics of the lithium and the concentration of efficacy of the formulation.
  • an amount of lithium compound is administered such that the peak lithium concentration in skin is between 0.1 ⁇ and 0.2 ⁇ , 0.2 ⁇ and 0.5 ⁇ , 0.5 and 1 ⁇ , 1 ⁇ and 2 ⁇ , 2 ⁇ to 10 ⁇ , 10 ⁇ to 100 ⁇ , 100 ⁇ to 500 ⁇ , 500 ⁇ to 1000 ⁇ .
  • peak values will depend on the lithium release properties of the formulation, the hydrophobicity of the lithium salt form, the partition coefficient of the lithium salt form, etc.
  • the peak concentration in skin is 0.2 ⁇ to 1.5 ⁇ lithium.
  • the peak concentration in skin should not exceed 1 ⁇ or 1.5 ⁇ lithium. In some embodiments, the peak concentration in skin is 10 ⁇ to 100 ⁇ lithium. In some embodiments, the peak concentration in skin is 100 ⁇ to 1000 ⁇ lithium. In some such embodiments, the peak lithium concentration in blood may be several orders of magnitude higher, for example, 0.1 mM to 0.5 mM, or 0.5 mM to 1.1 mM, 1.1 to 1.5 mM, 1.5 mM to 5 mM, 5 mM to 10 mM, 10 mM to 50 mM, or 50 mM to 100 mM.
  • the steady state blood concentration of lithium should not exceed a maximum of 1.5 mM to 2 mM.
  • the pulse lithium treatment can be administered one time, or multiple times at intervals of time. It is understood that the precise dosage and duration of treatment may vary with the age, weight, and condition of the patient being treated, and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test or diagnostic data. It is further understood that for any particular individual, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations. For example, in the treatment of bipolar disorder, therapeutically useful amounts of lithium ( ⁇ 0.4 to 1.2 mM) are only slightly lower than toxic amounts (>1.5 mM), so the skilled practitioner knows that the blood levels of lithium must be carefully monitored during treatment to avoid toxicity.
  • a pulse lithium treatment is administered at the time of integumental perturbation. In some embodiments, a pulse lithium treatment is administered following integumental perturbation. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered before scab formation. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered during scab formation. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered periscab detachment.
  • a pulse lithium treatment in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered immediately after scab detachment. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered 1 hour after scab detachment. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered up to 6 hours after scab detachment.
  • the pulse lithium treatment in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered 6- 12 hours after scab detachment. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered 12-18 hours after scab detachment. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered 18- 24 hours after scab detachment.
  • the pulse lithium treatment in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered 1 day after scab detachment. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered 2 days after scab detachment. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered 3 days after scab detachment.
  • the pulse lithium treatment in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, is administered within 3 days, 5 days, 7 days, 10 days, 2 weeks, or 3 weeks after integumental perturbation.
  • the pulse lithium treatment is administered at the time of integumental perturbation and then maintained for 3 or 4 or 5 days thereafter (in some embodiments, a scab forms during this time). In some embodiments, a pulse lithium treatment is administered as soon as the scab falls of and maintained for 3 or 4 or 5 days. In some embodiments, the pulse lithium treatment is administered in order to modulate the neoepidermis that forms underneath the scab. In some such embodiments, the pulse lithium treatment is administered at the time of integumental perturbation and is maintained up to some time after scab falls off, for example, between 5 - 14 days following integumental perturbation.
  • the course of treatment with lithium is short, for example, limited to a few days just following scab detachment, or even continued only for as long as the scab is still attached.
  • the timing of integumental perturbation and lithium administration is preferably monitored and adjusted so that optimal results are achieved.
  • a pulse treatment is combined with a form of integumental perturbation that does not lead to formation of a scab.
  • the pulse lithium treatment is administered at the time of integumental perturbation.
  • a pulse lithium treatment is administered following integumental perturbation.
  • the pulse lithium treatment is administered following an integumental perturbation that does not lead to formation of a scab
  • the pulse lithium treatment is administered within 15 minutes of, or 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 10 days, 2 weeks, or 3 weeks after integumental perturbation.
  • the intermittent lithium treatment can be administered one time ⁇ e.g., using a controlled release formulation), or multiple times at intervals of time. It is understood that the precise dosage and duration of treatment may vary with the age, weight, and condition of the patient being treated, and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test or diagnostic data. It is further understood that for any particular individual, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations.
  • lithium can be administered daily ⁇ e.g., once, twice or three times daily) for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 7 days; and in some embodiments not more than 14 days.
  • Holidays can be interspersed for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 7 days; and in some embodiments not more than 14 days.
  • an intermittent lithium treatment is begun at the time of integumental perturbation. In some embodiments, an intermittent lithium treatment is begun following integumental perturbation. In one embodiment, in which an intermittent lithium treatment is begun following an integumental perturbation that leads to formation of a scab, the intermittent lithium treatment is begun before scab formation. In one embodiment, in which an intermittent lithium treatment is begun following an integumental perturbation that leads to formation of a scab, the intermittent lithium treatment is begun during scab formation. In one embodiment, in which an intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium in the intermittent lithium treatment is periscab detachment.
  • the first administration of lithium is immediately after scab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium is up to 6 hours after scab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium is 6-12 hours after scab detachment.
  • the first administration of lithium is 12-18 hours after scab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium is 18-24 hours after scab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium is 1 day after scab detachment.
  • the first administration of lithium is 2 days after scab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium is 3 days after scab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium is administered immediately after scab detachment, followed by another administration each day for several days to 1 week.
  • the pulse lithium treatment is begun within 3 days, 5 days, 7 days, 10 days, 2 weeks, or 3 weeks after integumental perturbation.
  • the intermittent lithium treatment is begun at the time of integumental perturbation and then administered daily (or twice daily) for 5 days thereafter (in some embodiments, a scab forms during this time). In some embodiments, the intermittent lithium treatment is begun as soon as the scab falls off, and administered daily for 5 days. In some embodiments, the intermittent lithium treatment is to modulate the neoepidermis that forms underneath the scab. In some such embodiments, the intermittent lithium treatment is begun at the time of integumental perturbation and is continued with daily dosing up to some time after scab falls off, for example, between 5 - 14 days following integumental perturbation.
  • the course of treatment with lithium is short, for example, limited to daily doses for a few days just following scab detachment, or even continued only for as long as the scab is still attached.
  • the timing of integumental perturbation and lithium administration is preferably monitored and adjusted so that optimal results are achieved.
  • an intermittent lithium treatment is combined with a form of integumental perturbation that does not lead to formation of a scab.
  • the intermittent lithium treatment is begun at the time of integumental perturbation.
  • an intermittent lithium treatment is begun following integumental perturbation.
  • the intermittent lithium treatment is begun within 15 minutes of, or 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 10 days, 2 weeks, or 3 weeks after integumental perturbation.
  • Intermittent lithium treatment or a pulse lithium treatment in combination with other methods enhances the effectiveness of these methods.
  • the effect that each drug offers could be an additive or synergistic improvement, or a combination of two different pharmacologically defined effects, to achieve the desired end result.
  • the combined modality of treatment could involve alternating treatment of each dosage form or concurrent or simultaneous treatment. Synergism occurs when the combination has an effect that is more than would be expected from merely the additive effect of each element in the combination, for example, if branched hair follicles or more hair follicles per pore were produced by the combination and not by either alone.
  • the intermittent lithium treatments or the pulse lithium treatment described herein may be in combination with any additional treatment(s) described or incorporated by reference herein or determined to be appropriate by the medical practitioner.
  • the amount of an additional treatment(s) will depend on the desired effect and the additional compound that is selected. Dosages and regimens for administering such additional treatment(s) are the dosages and regimens commonly in use, which can be easily determined by consulting, for example, product labels or physicians' guides, such as the Physicians' Desk Reference (“PDR”) (e.g., 63rd edition, 2009, Montvale, NJ: Physicians' Desk Reference).
  • PDR Physicians' Desk Reference
  • the combination treatment comprises lithium and an additional compound(s) formulated together.
  • the lithium in such formulations may be released concurrently with or separately from the additional compound(s), or may be released and/or delivered to the tissue site with different pharmacokinetics.
  • one or more of the compounds in the formulation undergoes controlled release, whereas one or more of the other compounds does not.
  • one or more of the compounds in the formulation undergoes sustained release whereas one or more of the other compounds undergoes delayed release.
  • the combination treatment comprises lithium and an additional compound(s) formulated separately.
  • the separate formulations may be administered concurrently, sequentially, or in alternating sequence.
  • the lithium compound may be administered sequentially, or concurrently with another compound to achieve the desired effect of improved wound healing or scar revision.
  • the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more treatments selected from, e.g., cell therapy (such as a stem cell), a formulation for gene therapy (such as, e.g., a virus, virus-like particle, virosome), an antibody or antigen-binding fragment thereof, an herb, a vitamin (e.g., a form of vitamin E, a vitamin A derivative, such as, e.g., all-trans retinoic acid (ATRA), a B vitamin, such as, e.g., inositol, panthenol, or biotin, or a vitamin D3 analog), a mineral, essential oils, an antioxidant or free radical scavenger, amino acids or amino acid derivatives, a shampoo ingredient (e.g.
  • cell therapy such as a stem cell
  • a formulation for gene therapy such as, e.g., a virus, virus-like particle, virosome
  • an antibody or antigen-binding fragment thereof an
  • ammonium chloride ammonium lauryl sulfate, glycol, sodium laureth sulfate, sodium lauryl sulfate, ketoconazole, zinc pyrithione, selenium sulfide, coal tar, a salicylate derivative, dimethicone, or plant extracts or oils
  • a conditioning agent e.g., a soap product, a moisturizer, a sunscreen, a waterproofing agent, a powder, talc, or silica, an oil-control agent, alpha-hydroxy acids, beta-hydroxy acids (e.g., salicylic acid), poly-hydroxy acids, benzoyl peroxide, antiperspirant ingredients, such as astringent salts (e.g., zinc salts, such as zinc pyrithione, inorganic or organic salts of aluminum, zirconium, zinc, and mixtures thereof, aluminum chloride, aluminum
  • octachlorohydrate aluminum zirconium octachlorohydrex GLY (abbreviation for glycine), aluminum zirconium pentachlorohydrate, aluminum zirconium pentachlorohydrex GLY, aluminum zirconium tetrachlorohydrate, aluminum zirconium trichlorohydrate, aluminum zirconium tetrachlorohydrate GLY, and aluminum zirconium trichlorohydrate GLY, potassium aluminum sulphate, (also known as alum (KAl(S0 4 ) 2 l2H20)), aluminum undecylenoyl collagen amino acid, sodium aluminum lactate+ aluminum sulphate
  • tridecyl carboxy alkylates cerulenin or a cerulenin analog, including pharmaceutically acceptable salts or solvates thereof, another fatty acid synthase inhibitor, such as triclosan or analogs thereof, a polyphenol extracted from green tea (EGCG), available from Sigma Corporation (St.
  • a massage agent e.g., an exfoliant, an anti-itch agent, a pro-inflammatory agent, an immunostimulant (e.g., interferon, cytokines, agonists or antagonists of various ligands, receptors and signal transduction molecules of the immune system, immunostimulatory nucleic acids, an adjuvant that stimulates the immune response and/or which causes a depot effect).
  • an immunostimulant e.g., interferon, cytokines, agonists or antagonists of various ligands, receptors and signal transduction molecules of the immune system, immunostimulatory nucleic acids, an adjuvant that stimulates the immune response and/or which causes a depot effect.
  • adjuvants and/or other stimulators of local cytokines are used in conjunction with the intermittent lithium treatment or pulse lithium treatment.
  • one rationale for administering adjuvants and/or other stimulators of local cytokines in conjunction with the intermittent lithium treatment or pulse lithium treatment is that the production of local cytokines may induce changes in the hair follicle cell cycle and recruit new follicle stem cells to follicles.
  • the combination ntreatment comprises lithium in ccombination with a cell cycle regulator, a hormonal agonist, a hormonal antagonist (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), an inhibitor of hormone biosynthesis and processing, a steroid (e.g., dexamethasone, retinoids, deltoids, betamethasone, Cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, hydrocortisone, mineralocorticoids, estrogen, testosterone, progestins), antigestagens (e.g., mifepristone, onapristone), an antiandrogen (e.g., cyproterone acetate), an antiestrogen, an antihistamine (e.g., mepyramine,
  • a hormonal antagonist e.g., flutamide
  • an anti-inflammatory e.g., corticosteroids (such as, e.g., Dermatop®), NTHEs, and COX-2 inhibitors, adrenocorticoids, beclomethasone, budesonide, flunisolide, fluticasone, triamcinolone, methylprednisolone, prednisolone, prednisone, hydrocortisone), an anesthetic (e.g., vocal anesthesia, lidocaine, bupivacaine, etidocaine, etc., with or without epinephrine or sodium bicarbonate) a retinoid (e.g., 13-cis-retinoic acid, adapalene, all-trans-retinoic acid, and etretinate), PMMA, Restylane, poly-L-lactic acid, collagen, hyaluronic acid, which may be present in microspheres, or other skin fillers,
  • an anti-inflammatory e.g.
  • aspirin, ibuprofen, diclofenac, and COX-2 inhibitors pain relievers, leukotreine antagonists (e.g., montelukast, methyl xanthines, zafirlukast, and zileuton), beta2-agonists (e.g.
  • hexylresorcinol methylbenzethonium chloride, cetrimide, chlorhexidine, chlorobutanol, chlorocresol, cresol, glycerin, imidurea, phenol, phenoxyethanol, phenylethylalcohol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, potassium sorbate, sodium benzoate, sodium proprionate, sorbic acid, and thiomersal (thimerosal)).
  • the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more narcotic analgesics, selected from the group of, e.g., alfentanil, benzylmorphine, codeine, codeine methyl bromide; codeine phosphate, codeine sulfate, desomorphine, dihydrocodeine,
  • one or more narcotic analgesics selected from the group of, e.g., alfentanil, benzylmorphine, codeine, codeine methyl bromide; codeine phosphate, codeine sulfate, desomorphine, dihydrocodeine,
  • hydromorphone methadone hydrochloride, morphine, morphine hydrochloride, morphine sulfate, nicomorphine, normethadone, normorphine, opium, oxycodone, oxymorphone, phenoperidine, and propiram.
  • the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more non-narcotic analgesics, selected from the group of, e.g., aceclofenac, acetaminophen, acetanilide, acetylsalicylsalicylic acid; aspirin, carbamazepine, dihydroxyaluminum acetylsalicylate, fenoprofen, fluproquazone, ibufenac, indomethacin, ketorolac, magnesium acetylsalicylate, morpholine salicylate, naproxen, phenacetin, phenyl salicylate, salacetamide, salicin, salicylamide, sodium salicylate, and tolfenamic acid.
  • Other pain treatments that may be used in combination with the lithium treatments described herein include nerve blocks or non-traditional pain medications, such as, e.g., Lyrica (pregabalin) or Neuront
  • the present invention is based, in part, on the appreciation that hair follicles play a role in wound healing.
  • Inducing the formation of new hair follicles in wounds, or enhancing the entry of hair follicles into wounds (for example, by transplanting hair follicles into wounds) harnesses their regenerative capacity and provides a transformational approach to scar revision and the management of wounds.
  • the approaches described herein permit scar revision under sterile and controlled conditions that recreates and harnesses the fetal skin's plastic and regenerative capacity.
  • Fractional laser treatment of scarred tissue creates areas of small micro-injuries with intact epidermis in- between, and the integumental perturbation of the laser activates hair follicle deposition into the injury sites, either by migration from the intact epidermis or by inducing hair follicle neogenesis in the wound.
  • laser- induced wounding of columns (the non- ablative coagulum is a preferred embodiment) triggers the regenerative capacity of the intervening normal skin stem cells.
  • This technique may have utility in, for example, revising small scars (to improve texture, pigmentation and other features).
  • one advantage of using combinations comprising integumental perturbation is that the perturbation provides a signal for hair follicle deposition and/or deposition of other adnexal structures into the wound site, e.g., by their migration and/or by generation of new hair follicles (hair follicle neogenesis) or adnexal structures.
  • hair follicle neogenesis new hair follicles
  • a wound heals by scarring may depend on the efficiency of hair follicle or other adnexal structure deposition into the wound. If these structures, e.g., hair follicles, are not timely deposited into the healing wound, the process will result in a scar.
  • wound healing without scarring may be effected by improving the efficiency of adnexal structure ⁇ e.g., hair follicles) deposition into the wound or by slowing wound healing in order to allow sufficient time for deposition of these structures into the wound site.
  • adnexal structure e.g., hair follicles
  • enhancement of wound healing or scar revision is accomplished by lithium treatment alone, for example, in acutely wounded skin or skin affected by a chronic non-healing wound, i.e., skin already subjected to integumental perturbation.
  • the lithium treatment is administered to skin that has been damaged and which no longer contains follicles.
  • the lithium treatment may restore follicle production in that area of skin.
  • an area of skin containing a wound that has not healed correctly, such as a scar ⁇ e.g., a keloid scar) is administered a lithium treatment in order to restore hair follicles and/or hair growth to that area of skin.
  • enhancement of wound healing or scar revision is accomplished by a combination of integumental perturbation and a pulse or intermittent lithium treatment.
  • the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with integumental perturbation or, optionally, also comprises another treatment known in the art or described herein.
  • Combinations comprising integumental perturbation are preferred for skin that is not already acutely wounded, since wounding itself is a form of integumental perturbation.
  • Integumental perturbation can be achieved by any means known in the art or described herein, such as, for example, using chemical or mechanical means.
  • integumental perturbation comprises disrupting the skin of the subject (for example, resulting in the induction of re-epithelialization of the skin of the subject).
  • a certain area of the epithelium is partially or wholly disrupted.
  • a certain area of both the epithelium and stratum corneum are partially or wholly disrupted.
  • Integumental perturbation can be used to induce, for example, a burn, excision, dermabrasion, full-thickness excision, or other form of abrasion or wound.
  • the combination of integumental perturbation and lithium treatment is administered to skin that has been damaged and which no longer contains hair follicles.
  • the combination of integumental perturbation and lithium treatment restores follicle production in that area of skin.
  • an area of skin containing a wound that has not healed correctly such as a scar ⁇ e.g. , a keloid scar
  • a combination treatment of integumental perturbation and lithium is administered in order to restore hair follicles and/or growth of hair to that area of skin.
  • Mechanical means of integumental perturbation include, for example, use of sandpaper, a felt wheel, ultrasound, supersonically accelerated mixture of saline and oxygen, tape-stripping, spiky patch, or peels.
  • Chemical means of integumental perturbation can be achieved, for example, using phenol, trichloroacetic acid, or ascorbic acid.
  • Electromagnetic means of integumental perturbation include, for example, use of a laser (e.g., using lasers, such as those that deliver ablative, non-ablative, fractional, non-fractional, superficial or deep treatment, and/or are CC based, or Erbium-YAG-based, etc.).
  • Integumental perturbation can also be achieved through, for example, the use of visible, infrared, ultraviolet, radio, or X-ray irradiation.
  • integumental perturbation is by light energy, such as described in Leavitt et al, 2009, Clin. Drug. Invest. 29:283-292.
  • Electrical or magnetic means of disruption of the epidermis can be achieved, for example, through the application of an electrical current, or through electroporation or RF ablation.
  • Electric or magnetic means can also include the induction of an electric or a magnetic field, or an electromagnetic field. For example, an electrical current can be induced in the skin by application of an alternating magnetic field.
  • a radiofrequency power source can be coupled to a conducting element, and the currents that are induced will heat the skin, resulting in an alteration or disruption of the skin. Integumental perturbation can also be achieved through surgery, for example, a biopsy, a skin transplant, skin graft, follicular unit extraction, hair transplant, cosmetic surgery, open- heart surgery, etc.
  • integumental perturbation is by laser treatment, as discussed below.
  • Exemplary laser treatments for integumental perturbation include Fraxel, laser abrasion, Erbium- YAG laser, Ultrapulse CO 2 fractional laser, Ultrapulse CO 2 ablative laser, Smooth Peel Full-ablation Erbium laser (Candela), as described, for example, in the examples of Section 8 below.
  • a laser treatment is chosen in which the integumental perturbation achieved most resembles that achieved by dermabrasion (for example, a dermabrasion method described herein).
  • integumental perturbation by laser treatment is by a fractional laser.
  • a fractional laser treatment is treatment with an Erbium- YAG laser at around 1540 nm or around 1550 nm (for example, using a Fraxel® laser (Solta Medical)). Treatment with an Erbium-YAG laser at 1540 or 1550 nm is typically non-ablative, and pinpoint bleeding typical of laser treatment is not observed since the stratum corneum is left in tact.
  • integumental perturbation by laser treatment is by a fractional laser, using, e.g. , a CO 2 laser at 10,600 nm. Treatment with a CO 2 laser at 10,600 nm is typically ablative, and typically leads to the appearance of pinpoint bleeding.
  • a standard CO 2 or Erbium- YAG laser can be used to create superficial and, optionally, broad based, integumental perturbation similar to dermabrasion (discussed below). Although the pinpoint bleeding clinical endpoint may not be achieved due to the coagulation properties of (particularly non-ablative) lasers, use of a laser has an advantage making it possible to select the specific depth of skin disruption to effectively remove the stratum corneum and epidermis, or portions thereof.
  • the laser treatment is ablative.
  • full ablation of tissue is generated by the targeting of tissue water at wavelengths of 10,600 nm by a CO 2 laser or 2940 nm by an Erbium- YAG laser.
  • the epidermis is removed entirely and the dermis receives thermal tissue damage.
  • the depth of tissue ablation may be a full ablation of the epidermis, or a partial ablation of the epidermis, with both modes causing adequate wounding to the skin to induce the inflammatory cascade requisite for regeneration.
  • the depth of ablation may extend partially into the dermis, to generate a deep wound.
  • a lithium composition described herein is delivered by a sustained release depot that is comprised of biocompatible, bioabsorbable polymers that are compatible to tissue.
  • the standard full thickness excision model is created using scissors or with a scalpel in animal models (see, also, the examples of Section 16 infra). This procedure, while contemplated for use herein, carries with it the risk of scarring.
  • various fractional laser modalities could be used to achieve a similarly deep disruption on a grid pattern.
  • a fractional laser can be use to "drill," for example, 1-mm diameter holes with a 1-mm hole spacing (the fractional laser can make holes of smaller dimensions). Although the skin is completely removed within the 1-mm hole, the surrounding intact skin prevents scarring and therefore the full thickness excision model is invoked within each hole.
  • the laser treatment is ablative and fractional.
  • fractional tissue ablation can be achieved using a CO2 laser at 10,600 nm or an Erbium- YAG laser at 2940 nm ⁇ e.g., the Lux 2940 laser, Pixel laser, or Pro fractional laser).
  • the lasing beam creates micro-columns of thermal injury into the skin, at depths up to 4 mm and vaporizes the tissue in the process.
  • Ablative treatment with a fractional laser leads to ablation of a fraction of the skin leaving intervening regions of normal skin intact to rapidly repopulate the epidermis. Approximately 15%— 25% of the skin is treated per session.
  • micro thermal zones can be varied to create a dense "grid" of injury columns surrounded by intact skin and viable cells.
  • the density of the grid on the treatment area plays an important role. The denser the grid, the more the thermal injury and the type of injury begins to approximate full ablation. Therefore, it is appreciated that there may be an "optimum" MTZ density that is appropriate for use in the methods disclosed herein.
  • a lithium composition described herein is delivered into the dermis immediately after wounding, or after initial re-epithelialization has occurred.
  • the mode of laser treatment is non-ablative, wherein the stratum corneum and the epidermis are intact after treatment, with the dermis selected for the deep thermal treatment required for the requisite injury to tissue.
  • This can be accomplished by cooling the epidermis during the laser treatment.
  • the depth of treatment may be 1 mm to 3 mm into the skin.
  • contact cooling such as a copper or sapphire tip.
  • Lasers that are non- ablative have emission wavelengths between 1000-1600 nm, with energy fluences that will cause thermal injury, but do not vaporize the tissue.
  • the non-ablative lasers can be bulk, wherein a single spot beam can be used to treat a homogenous section of tissue. In some embodiments, multiple treatments are required to achieve the desired effect.
  • a lithium composition described herein is delivered deep into the dermis in polymeric micro-depots and released in a sustained fashion.
  • Lasers that are non-ablative include the pulsed dye laser (vascular)(at, e.g., 585-595 nm), the 1064 Nd:YAG laser, or the Erbium- YAG laser at 1540 nm or 1550 nm ⁇ e.g., the Fraxel® laser).
  • the mode of laser treatment is fractional and non-ablative. Treatment with a fractional, non-ablative laser leads to perturbation of a fraction of the skin, leaving intervening regions of normal skin intact to rapidly repopulate the epidermis.
  • Approximately 15%— 25% of the skin is treated per session.
  • the skin barrier function is maintained, while deep thermal heating of dermis can occur.
  • zones of dermis and epidermis are coagulated and the stratum corneum is left essentially intact.
  • This process has been coined "fractional photothermolysis" and can be accomplished, e.g., using the Erbium-YAG laser with an emission at or around 1540 nm or 1550 nm.
  • a lithium composition described herein is delivered immediately after the tissue injury, deep into the dermis.
  • a combination of bulk and fractional ablation modes of tissue injury are used.
  • a combination treatment comprising use of a laser includes administration to the skin of a compound absorbing light at wavelengths between 1000-1600 nm for the purpose of efficient channeling of light to heat energy.
  • This method of channeling energy may cause micro-zones of thermal injury within the skin.
  • the compound may be delivered to the skin homogenously in the treatment zone, then subsequently irradiated with a non-ablative laser to efficiently capture the vibrational energy of the infrared beam. This method may result in evenly distributed and deep thermal injury, without causing tissue vaporization.
  • a combination treatment comprising use of a laser includes administration of a lithium compound formulation that is encapsulated in matrices that are highly hydrophilic and charged, for example, linked to the dermis by covalent or ionic bonding to prevent the matrices from being cleared by phagocytosis, as part of the wound healing process.
  • a combination treatment comprising use of a laser includes the step of placing a biocompatible, synthetic skin substitute on the newly created wound, especially if the wound is deep, covers large area, and is bulk ablated. This process can help minimize or prevent the rapid wound contraction that occurs after loss of a large area of tissue, frequently culminating in scar tissue formation and loss of skin function.
  • the biocompatible synthetic skin substitute is be impregnated with depots of a slow releasing lithium formulation described herein. This method of treatment may enable treating a large area in one session at the treatment clinic.
  • other molecules are also co-eluted at the site through the skin substitute, such as, e.g.
  • the skin substitute in the presence or absence of a lithium compound and/or other compounds described herein, may also be pre-cooled and applied to the wound to provide a feeling of comfort to the patient. This mode of lithium or other compound application may prevent the lithium or other compound from being cleared away from the wound site as the wound heals.
  • a fractional like hole pattern (similar to that achieved with a fractional laser or full thickness excision) is achieved with using an array of punch biopsy needles.
  • 1 -mm punch biopsies can be arranged with 1 -mm hole spacing.
  • the cored skin samples can be removed and, thus, an effect approximating the full thickness excision model is invoked within each hole.
  • microneedles e.g., 19 or 21 gauge needles
  • micro-coring needles could be used.
  • integumental perturbation is by dermabrasion (also referred to herein as "DA"), a well-established dermatological procedure that has been used for decades as a skin resurfacing technique (Grimes, 2005, Microdermabrasion. Dermatol Surg 31 : 1351-1354). While the popularity of mechanical DA has decreased in recent years with the advent of laser-based procedures, DA is still used for removing facial scars resulting from acne and other trauma. Small, portable mechanical dermabrasion equipment uses interchangeable diamond firaises operated at different rotation speeds to remove the epidermis and dermis to differing skin depths levels.
  • Dermabrasion may be carried out using any technique known in the art or as described herein, e.g., in the examples of Sections 9, 10 and 16 infra.
  • dermabrasion may be carried out using standard DA with aluminum oxide crystals using the Aseptico Econo-Dermabrader, Advance Microderm DX system, or M2-T system; standard DA with Bell Hand Engine with diamond fraize; etc.
  • DA is carried out using an abrasive wheel.
  • DA with an abrasive wheel is used in order to achieve pinpoint bleeding.
  • DA may be carried out using an abrasive wheel to achieve larger globules of bleeding and frayed collagen.
  • non-powered devices such as abrasive cloths can also be used to achieve the DA, with the optional achievement of the same endpoint(s).
  • DA is accomplished using a device typically used for microdermabrasion.
  • a microdermabrasion device is used to remove a greater depth and/or area of skin than is typical for microdermabrasion (also referred to herein as "MDA").
  • MDA microdermabrasion
  • the microdermabrasion device is used under sterile conditions.
  • DA is achieved by using a device typically used for microdermabrasion to the point where treatment is stopped upon the observation of pinpoint bleeding, which signals the removal of the stratum corneum and epidermis into the papillary dermis.
  • DA is achieved by using a device for
  • this extended use is reduced by using a microdermabrasion device with increased output pressure and increased abrasion particle size, which may accelerate the skin removal process.
  • DA is accomplished by removal of surface skin by particle bombardment (also referred to herein as "particle mediated dermabrasion" (“PMDA”)), for example, with alumina-, ice- or silica-based particles.
  • particle bombardment also referred to herein as "particle mediated dermabrasion” (“PMDA)
  • PMDA particle mediated dermabrasion
  • micron- sized particles are propelled toward the surface of the skin via short strokes of a handpiece, such as a particle gun, as known in the art.
  • the velocity of particles is controlled through positive or negative pressure.
  • the depth of skin removed by particle bombardment DA ⁇ e.g., PMDA) is a function of the volume of particles impacting the skin, the suction or positive pressure, the speed of movement of the handpiece, and the number of passes per area of the skin.
  • integumental perturbation by one or more of the aforementioned methods achieves removal of part or all of the epidermis. In some embodiments, integumental perturbation removes the entire epidermis. In some
  • integumental perturbation disrupts the papillary dermis.
  • integumental perturbation removes the papillary dermis.
  • integumental perturbation removes the reticular dermis.
  • the depth of integumental perturbation depends on the thickness of the skin at a particular treatment area. For example, the skin of the eyelid is significantly thinner than that of the scalp.
  • the occurrence of pinpoint bleeding indicates that the epidermis and portions of the dermis have been removed. Deeper penetration can results in much more bleeding, and the perturbation can go as deeps as the hypodermis.
  • integumental perturbation by one or more of the aforementioned methods is to a skin depth of 60 ⁇ . In some embodiments, integumental perturbation is to a skin depth of 60-100 ⁇ . In some embodiments, integumental perturbation is to a skin depth of 100 ⁇ . In some embodiments, integumental perturbation is to a skin depth of 150 ⁇ . In some embodiments, integumental perturbation is to a skin depth of 100-500 ⁇ . In some embodiments, integumental perturbation is to a skin depth of less than 500 ⁇ . In some embodiments, integumental perturbation is to a skin depth of 500- 1000 ⁇ .
  • integumental perturbation is to a skin depth of 1 mm or more. In some embodiments, integumental perturbation is to a skin depth of 1 mm to 3 mm. In some embodiments, integumental perturbation is to a skin depth of 1 mm to 5 mm.
  • Integumental perturbation such as occurs during wounding, produces in the affected skin tissue an increase in the number of hair follicle stem cells and in the plasticity of hair follicle cells.
  • the pulse or intermittent lithium treatments cause formation of new hair follicles or enhanced branching, division, or differentiation of existing hair follicles or hair follicle progenitors. Accordingly, and without being bound by any theory for how the invention works, integumental perturbation (or wounding) in combination with a pulse or intermittent lithium treatment provides an environment for the formation of a large number of follicles to enhance wound healing and, preferably, wound healing with reduced scarring.
  • integumental perturbation techniques described herein for example, laser techniques or surgical dissection of follicles, etc., lead to hair follicle transection in vivo. It is thought that at least 40% of follicles, after bisection, form two follicles (which would result if all the "bottoms” from the -30% of failed "tops” successfully yielded a hair fiber and in addition, if all the "tops” from the -30% of failed bottom bisected follicles successfully yielded a hair fiber.) Therefore, the percentage of bisected follicles that produce two new follicles is in the range of -40-70% (with the maximum -70% being the result of all bisections producing two new follicles and no bisections resulting in either a top or a bottom (but not both) producing a follicle). It is expected that this efficiency will be increased when pulsatile lithium is applied because it induces differentiation.
  • FSCs originate from one or more of the following: (i) existing follicles ("follicle derived follicle stem cells” or “FDFSC”) (see, e.g., Toscani et al, 2009, Dermatol Surg.
  • FDFSC follicle derived follicle stem cells
  • TDFSC tissue derived follicle stem cells
  • BMDFSC bone marrow derived follicle stem cells
  • FSCs generate new hair follicles that preserve the type of hair follicle that is typical for each location of skin or scalp.
  • FSCs from the coronal scalp of a male with MPHL typically generate atrophic follicles with vellus or club hairs.
  • FSCs from the occipital scalp of the same male typically generate follicles with terminal hair that are not subject to involution in response to DHT
  • FSCs responsible for follicle formation may be reprogrammed.
  • FSCs in the process of asymmetric division and subsequent differentiation are susceptible to signals (such as estrogen or testosterone) that alter the determinism of their differentiation program.
  • a pulse or intermittent lithium treatment in combination with integumental perturbation provides a window during which a third treatment that alters the follicle development program may be administered in order to significantly change the number and quality of follicles in a particular area of skin.
  • any treatment that enhances hair growth or, alternatively, that prevents hair growth or removes excessive hair, that is known in the art or yet to be developed is contemplated for use in such combination treatments.
  • treatments that promote hair growth include minoxidil, finasteride, bimatoprost (Latisse), CaCl 2 , or adenosine, or techniques of integumental perturbation such as by mechanical means, chemical means, electromagnetic means (e.g., using a laser such as one that delivers ablative, non-ablative, non-fractional, superficial, or deep treatment, and/or are CCVbased, or Erbium- YAG-based, etc.), irradiation, radio frequency (RF) ablation, or surgical procedures (e.g., hair transplantation, strip harvesting, follicular unit extraction (FUE), scalp reduction, etc.).
  • RF radio frequency
  • treatments that remove unwanted hair or prevent hair growth include, e.g., cytotoxic drugs, hair growth retardants, such as eflornithine HC1 (Vaniqa), 5-fluorouracil (5- FU) (e.g., Efudex 5% cream), or other epilation or depilation methods.
  • cytotoxic drugs such as eflornithine HC1 (Vaniqa), 5-fluorouracil (5- FU) (e.g., Efudex 5% cream), or other epilation or depilation methods.
  • hair growth retardants such as eflornithine HC1 (Vaniqa), 5-fluorouracil (5- FU) (e.g., Efudex 5% cream), or other epilation or depilation methods.
  • a third treatment may comprise treatment with an estrogen or testosterone modulator, such as those described in Poulos & Mirmirani, 2005, Expert Opin. Investig. Drugs 14: 177
  • the third treatment is administered simultaneously with integumental perturbation. In some embodiments, the third treatment is administered after integumental perturbation. In some embodiments, the third treatment is administered 1 day, 2 days, 3 days, 5 days, 7 days, 10 days, or 2 weeks after integumental perturbation. In one embodiment, the third treatment is administered at the time of integumental perturbation and then daily for 5 days thereafter (in some embodiments, a scab forms during this time). In some embodiments, the third treatment is administered daily for 5 days beginning as soon as the scab falls off. In some embodiments, the third treatment is administered in order to modulate the neoepidermis that forms underneath the scab.
  • the third treatment is administered at the time of integumental perturbation and up to some time after scab falls off, for example, between 5 - 14 days following integumental perturbation.
  • the course of treatment with the third treatment is short, for example, limited to a few days just following scab detachment, or even continued only for as long as the scab is still attached.
  • the timing of the integumental perturbation, lithium administration, and the third treatment is preferably monitored and adjusted so that optimal results are achieved.
  • This invention is based, in part, on the discovery that there is a correlation between the extent of wound contraction and the deposition of adnexal structures, such as new hair follicles, in wounded areas.
  • adnexal structures such as new hair follicles
  • intermittent and pulse lithium treatments may promote wound healing and scar revision by, at least in part, promoting the entry of hair follicles into the wound as it heals. This may occur by inducing the generation of new hair follicles and/or promoting migration of hair follicles into the wound site.
  • the intermittent and pulse lithium treatments described herein may (i) promote hair follicle neogenesis ⁇ e.g. , de novo formation of hair follicles from tissue or bone-marrow derived stem cells or disintegration of preexisting follicles into cells that mix together and reform the hair follicle); and/or (ii) promote branching ⁇ e.g., with the assistance of stem cells from dissociated hair follicles) and division of existing hair follicles.
  • hair follicle neogenesis e.g. , de novo formation of hair follicles from tissue or bone-marrow derived stem cells or disintegration of preexisting follicles into cells that mix together and reform the hair follicle
  • branching e.g., with the assistance of stem cells from dissociated hair follicles
  • a superficial wound is healed by the assistance of hair follicles remaining in the wound.
  • the hair follicles disintegrate and are reorganized and reformed by the presence of stem cells that enter the wound.
  • hair follicle neogenesis promotes wound healing.
  • the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more agents that increase the number of hair follicles or that counteract hair follicle cell senescence (also referred to herein as "anti-senescence agents"), for example, anti-oxidants such as glutathione, ascorbic acid, tocopherol, uric acid, or polyphenol antioxidants); inhibitors of reactive oxygen species (ROS) generation, such as superoxide dismutase inhibitors;
  • agents that increase the number of hair follicles or that counteract hair follicle cell senescence also referred to herein as "anti-senescence agents”
  • anti-oxidants such as glutathione, ascorbic acid, tocopherol, uric acid, or polyphenol antioxidants
  • ROS reactive oxygen species
  • ROS breakdown such as selenium
  • mTOR inhibitors such as rapamycin
  • sirtuins or activators thereof such as resveratrol, or other SIRT1, SIRT3 activators, or nicotinamide inhibitors.
  • the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more agents that induce an immune response or cause inflammation, such as, e.g., tetanus toxoid, topical non-specific irritants (anthralin), or sensitizers (squaric acid dibutyl ester [SADBE] and diphenyl cyclopropenone [DPCP]). While not intending to be bound by any theory, it is thought that by contacting these agents to the skin, lymphocytes and hair follicle stem cells may be recruited to skin.
  • agents that induce an immune response or cause inflammation such as, e.g., tetanus toxoid, topical non-specific irritants (anthralin), or sensitizers (squaric acid dibutyl ester [SADBE] and diphenyl cyclopropenone [DPCP]). While not intending to be bound by any theory, it is thought that by contacting these agents to the skin, lymphocytes
  • the combination treatment comprises a pulse or intermittent lithium treatment together with a cytokine thought to regulate the activity of Dermal Papillae, which is believed to be the target of androgen regulation of hair growth.
  • Interleukin-1 alpha decreases responses to androgen in cultured dermal papilla cells (Boivin et al, 2006, Exp Dermatol. 15:784-793).
  • TGF- ⁇ may mediate androgen-induced hair growth suppression, since in culture, human dermal papilla cells (DPCs) from androgenetic alopecia (AGA) subjects that transiently expressing androgen receptor were co-cultured with keratinocytes (KCs), and secreted TGF- ⁇ that inhibited KC growth (Inui et al., 2003, J Investig Dermatol Symp Proc. 8:69-71). Thus, a TGF-B1 inhibitor may be used in a combination treatment.
  • DPCs human dermal papilla cells
  • AGA androgenetic alopecia
  • KCs keratinocytes
  • TGF-B1 inhibitor may be used in a combination treatment.
  • Melatonin is a protein hormone secreted by the pineal gland that modulates hair growth, pigmentation and/or molting in many species. Human scalp hair follicles in anagen are important sites of extra-pineal melatonin synthesis. Melatonin may also regulate hair Follicle Cycle control, since it inhibits estrogen receptor-alpha expression (Fischer et al, 2008, Pineal Res. 44: 1-15). Melatonin and the other treatments described herein can be administered, for example, during the lithium treatment "holidays.” Alternatively, these treatments can be administered prior to or subsequent to a pulse lithium treatment.
  • the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with a chemical or mechanical (such as those discussed infra) treatment that induces an inflammatory process in the skin. While not intending to be bound by any theory, inducing inflammation in the site where hair growth is desired helps to recruit stem cells to the tissues that drive the formation of new follicles.
  • the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with an antiapoptotic compound.
  • the antiapoptotic compound is not a Wnt or a Wnt agonist.
  • the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more of stem cell therapy, hair cloning or hair plugs, follicular unit extraction, hair or skin transplantation, massage, a skin graft, or any surgical procedure aimed at skin or hair restoration.
  • the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with use of a laser device or other mode of accomplishing "photo-biostimulation" of the hair follicles.
  • a laser device or other mode of accomplishing "photo-biostimulation" of the hair follicles.
  • the Hairmax Lasercomb or the Leimo laser are non-limiting examples of devices that can be used in combination with the methods described herein.
  • intermittent lithium treatment or a pulse lithium treatment alone or in combination with other treatments described herein, synchronizes hair follicle cells in the cell cycle.
  • lithium is administered to arrest hair follicle cells in G2/M phase, which synchronizes them; then the lithium treatment is removed; and then their re-entry into the cell cycle and mitotic division is stimulated with other drugs (which leads to anagen follicles and an increased number of follicles).
  • the lithium treatment arrests hair follicle cells in late prophase or metaphase, which synchronizes them; the lithium treatment is removed; and then their re-entry into the cell cycle and mitotic division is stimulated with other drugs (which leads to anagen follicles and an increased number of follicles).
  • the lithium treatment arrests hair follicle stem cells in G2/M phase, which synchronizes them; then the lithium treatment is removed; and then their re-entry in to the cell cycle and mitotic division is stimulated with other drugs (which leads to anagen follicles and an increased number of follicles).
  • the lithium treatment arrests hair follicle stem cells in late prophase or metaphase, which synchronizes them; the lithium treatment is removed; and then their reentry into the cell cycle and mitotic division is stimulated with other drugs (which leads to anagen follicles, and an increased number of follicles).
  • intermittent lithium treatment or a pulse lithium treatment alone or in combination with the aforementioned combination treatments, synchronizes hair follicle cells in the Follicle Cycle.
  • the treatment regimen induces follicles to enter anagen.
  • the treatment regimen prevents follicles from entering catagen.
  • the treatment regimen induces follicles in telogen to enter exogen, or induces follicles in exogen to enter anagen.
  • a combination treatment comprises a pulse or intermittent lithium treatment in combination with another treatment that modulates wound healing, including any treatment described herein or known in the art to modulate wound healing.
  • the pulse or intermittent lithium treatment is administered in combination with a treatment that enhances one or more of the steps of wound healing discussed in Section 2.1.1 above, including any treatment described herein or known in the art to enhance wound healing.
  • enhancement of a step of wound healing or enhancement of wound healing is meant the hastening of healing, improvement of healing, or reduction of scarring, etc.
  • the pulse or intermittent lithium treatment is administered in combination with a wound dressing or skin replacement, such as, for example, gauze, calcium-alginates, impregnated gauzes, films, foams, hydrogels, hydrocolloids, adsorptive powders and pastes, silicone, mechanical vacuum, dermal matrix replacements, dermal living replacements, or skin living replacements, a collagen dressing, cadaveric skin, or other matrix useful to promote healing of the wound such as described herein or known in the art. See, e.g., Table 10.3 in Lorenz & Longaker, which is incorporated by reference herein in its entirety.
  • the pulse or intermittent lithium treatment is administered in combination with a pain reliever, antibiotic and antibacterial use or other anti-infectives (such as, e.g., tea tree oil), debridement, drainage of wound fluid, mechanical removal of bacteria, removal of devitalized tissue (such as, e.g., by surgery or maggot therapy), irrigation (e.g., by pulsed lavage), vacuum-assisted closure (otherwise referred to as negative pressure wound therapy), warming, oxygenation (e.g., using hyperbaric oxygen therapy), antioxidant therapy, revascularization therapy, moist wound healing, removing mechanical stress, use of elastase inhibitors, or adding cells or other materials to secrete or enhance levels of healing factors.
  • a pain reliever such as, e.g., tea tree oil
  • debridement such as, e.g., tea tree oil
  • debridement such as, e.g., tea tree oil
  • drainage of wound fluid such as, e.g., by surgery or maggot therapy
  • the pulse or intermittent lithium treatment is administered in combination with the upregulation of endogenous growth factors or exogenous application of growth factors, which may accelerate normal healing and improve healing efficacy.
  • growth factors include, but are not limited to, vascular endothelial growth factor (VEGF), insulin-like growth factor 1-2 (IGF), PDGF, transforming growth factor- ⁇ (TGF- ⁇ ), epidermal growth factor (EGF), EGF -receptor, members of the FGF family, and others described herein and listed in, e.g., Table 10.2 in Lorenz & Longaker, which is incorporated by reference herein in its entirety.
  • Such growth factors can be applied exogenously or may be applied by spreading onto the wound a gel of the patient's own platelets, implanting cultured keratinocytes into the wound, or treating the wound with artificial skin substitutes that have fibroblasts and keratinocytes in a matrix of collagen.
  • the pulse or intermittent lithium treatment is administered in combination with a treatment that reduces the time it takes for a wound to heal or that reduces the extent of the wound.
  • treatments include, for example, periodic rotation of the patient or wounded tissue or use of an air mattress, use of a lower pressure cast or relieving excessive suture tension, cleansing of the wound, debridement of tissue, particularly necrotic tissue, improvement of circulation and oxygen delivery to the tissue by, e.g., hyperbaric oxygen therapy or other oxygen administration, whirlpool therapy, ultrasound therapy, electrical stimulation, magnetic therapy have been utilized to aid the body in healing wounds coverage of wound with vascularized tissue, revascularization of the wounded tissue, treatment of circulatory obstruction or other treatment that improves circulation, treatment of ischemia, edema, or hypoxia, or improvement of the hematocrit (e.g., to at least 15%).
  • Treatment of tissue necrosis treatments or prevention of infection (e.g., with antibiotics such as povidone-iodine, chlorhexidine gluconate, hexachlorophene, or silver sulfadiazine and others described herein (particularly for burn wound care), irrigation (e.g., with saline), and/or debridement), improvement of nutrition ⁇ e.g., increasing intake of vitamins, e.g., vitamin A, C, Bl, B2, B5, or B6, or trace metals, such as, e.g., zinc and copper, amino acids such as arginine, glutamine, or Bromelain, Curcumin, etc.), herbal supplements (e.g., Aloe Vera, Centella), diabetes treatment (for example, to improve vascular conditions, or by administering glucose), skin graft, treatment with hormones (such as estrogen) or treatment with growth factors (e.g., epigallocate, hematoma, hematoma, hematoma, hematoma
  • the pulse or intermittent lithium treatment is administered in combination with a treatment that slows the natural adult wound healing process.
  • such combination treatments are used in the presence of a sterile wound dressing that obviates the need to heal the wound quickly (for example, in natural wound healing, the wound heals quickly in order to avoid infection).
  • the pulse or intermittent lithium treatment is administered in combination with a treatment that causes the postnatal wound healing process to resemble the fetal wound healing process. In some embodiments, this is accomplished by placing the wounded skin into a womb-like environment, for example, using a dressing and/or heat.
  • the pulse or intermittent lithium treatment is administered in combination with an agent that reduces or inhibits the inflammatory phase of wound healing, using, e.g. , an anti-inflammatory agent such as a NSAID or a topical glucocorticoids, an anti- androgen, or an antagonist of TNFa, TGF , NFkB, IL-1, IL-6, IL-8, IL-10, IL-18, or an antagonist of one or more other proinflammatory cytokines.
  • the pulse or intermittent lithium treatment is administered in combination with an agent that slows the wound healing process by extending the inflammatory phase, e.g., an androgen (see, e.g., Gilliver et al, 2007, Clin.
  • the treatment is administered in combination with an agent that suppresses the proliferative phase of wound healing, or the maturation and remodeling phase of wound healing.
  • the treatment is administered in combination with an agent that slows or interferes with fibrin deposition, clotting caused by fibrin, or fibrin-induced immunity.
  • the treatment is administered in combination with a treatment that inhibits the activity of fibrinogen.
  • the treatment is administered in combination with an agent that decreases the activity of myofibroblasts.
  • the treatment is administered in combination with a treatment that reduces collagen synthesis, deposition, or accumulation, for example, collagenases.
  • the treatment is administered in combination with a treatment that maintains the wound in an open state for a longer than normal period of time.
  • a treatment is administered in combination with rapamycin or corticosteroids.
  • a biocompatible, synthetic skin substitute is placed on the wound, especially if the wound is deep, covers large area, and is bulk ablated. This process can help minimize or prevent the rapid wound contraction that occurs after loss of a large area of tissue, frequently culminating in scar tissue formation and loss of skin function.
  • the biocompatible synthetic skin substitute is impregnated with depots of a slow releasing lithium formulation described herein. This method of treatment may enable treating a large area in one session at the treatment clinic.
  • other molecules are also co-eluted at the site through the skin substitute, such as, e.g., anesthetics and antibiotics, to prevent further pain and minimization of infection, or any other compound described herein.
  • the skin substitute in the presence or absence of a lithium compound and/or other compounds described herein, may also be pre-cooled and applied to the wound to provide a feeling of comfort to the patient. This mode of lithium or other compound application may prevent the lithium or other compound from being cleared away from the wound site as the wound heals.
  • a pulse or intermittent lithium treatment is administered in combination with a treatment that improves wound healing, in order to reduce the appearance or extent of scarring.
  • a pulse or intermittent lithium treatment is administered in combination with a treatment that improves the appearance and/or function of scarred skin, including any such treatment described herein or known in the art.
  • a pulse or intermittent lithium treatment is administered in combination with scar revision, such as by skin graft, serial expansion of surrounding skin, or laser treatment as described in Section 5.4 above.
  • a pulse or intermittent lithium treatment is administered in combination with re-excision with subsequent healing by primary intention, treatment with steroids (e.g., corticosteroid injection), silicone scar treatments (e.g.
  • porcine fillers or other cosmetic fillers e.g., inserted under atrophic scars
  • ribosomal 6 kinase (RSK) antagonists e.g., ribosomal 6 kinase (RSK) antagonists
  • RSK ribosomal 6 kinase
  • antagonists of pro-inflammatory cytokines such as TGF 2 or TNF
  • osteopontin antagonists the use of pressure garments, needling, dermabrasion, collagen injections, low-dose radiotherapy, or vitamins (e.g., vitamin E or vitamin C or its esters).
  • a pulse or intermittent lithium treatment is administered in combination with a treatment that reduces surgical scarring, e.g. , by placement of elective incisions parallel to the natural lines of skin tension (Langer's lines) or by applying sutures in a "zigzag" pattern.
  • the pulse or intermittent lithium treatment is administered in combination with a treatment of wounds that minimizes scarring, by, for example, administering physical therapy to a subject (e.g., range-of-motion exercises), reducing infection, reducing separation of wound edges, minimizing collagen synthesis, deposition, or accumulation or otherwise causing the process of healing by secondary intention to better resemble healing by primary intention.
  • intermittent lithium treatment or a pulse lithium treatment in combination with the aforementioned methods for enhancing scar revision or wound healing improves the effectiveness of these methods, making the treatment more effective, efficient, cost-effective, pain-free, and/or user friendly. For example, fewer treatments may be required.
  • one of the previously described wound healing or scar revision treatments on its own is not cosmetically satisfactory, does not adequately restore function of the skin, or the benefits are too short-lived.
  • the intermittent lithium treatment or a pulse lithium treatment can be administered prior to, concurrently with, or subsequent to the administration of a second (or third, or more) treatment.
  • the intermittent lithium treatment or a pulse lithium treatment is administered to a subject at reasonably the same time as the other treatment.
  • This method provides that the two administrations are performed within a time frame of less than one minute to about five minutes, or up to about sixty minutes from each other, for example, at the same doctor's visit.
  • the intermittent lithium treatment or a pulse lithium treatment and other treatment are administered at exactly the same time.
  • the intermittent lithium treatment or a pulse lithium treatment and the other treatment are administered in a sequence and within a time interval such that the intermittent lithium treatment or a pulse lithium treatment and the other treatment can act together to provide an increased benefit than if they were administered alone.
  • the intermittent lithium treatment or a pulse lithium treatment and other treatment are administered sufficiently close in time so as to provide the desired outcome.
  • Each can be administered simultaneously or separately, in any appropriate form and by any suitable route.
  • the intermittent lithium treatment or a pulse lithium treatment and the other treatment are administered by different routes of
  • each is administered by the same route of administration.
  • the intermittent lithium treatment or a pulse lithium treatment and the other treatment can be administered at the same or different sites of the subject's body.
  • the intermittent lithium treatment or a pulse lithium treatment and the other treatment may or may not be administered in admixture or at the same site of administration by the same route of administration.
  • the intermittent lithium treatment or a pulse lithium treatment and the other treatment are administered less than 1 hour apart, at about 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 1 1 hours apart, 11 hours to 12 hours apart, no more than 24 hours apart or no more than 48 hours apart.
  • the intermittent lithium treatment or a pulse lithium treatment and other treatment are administered 2 to 4 days apart, 4 to 6 days apart, 1 week a part, 1 to 2 weeks apart, 2 to 4 weeks apart, one month apart, 1 to 2 months apart, 2 to 3 months apart, 3 to 4 months apart, 6 months apart, or one year or more apart.
  • the intermittent lithium treatment or a pulse lithium treatment and the other treatment are administered in a time frame where both are still active. One skilled in the art would be able to determine such a time frame by determining the half life of each administered component.
  • the intermittent lithium treatment or a pulse lithium treatment and the other treatment are administered within the same patient visit. In one embodiment, the intermittent lithium treatment or a pulse lithium treatment is administered prior to the administration of the other treatment. In an alternate embodiment, the intermittent lithium treatment or a pulse lithium treatment is administered subsequent to the administration of the other treatment.
  • the intermittent lithium treatment or a pulse lithium treatment and the other treatment are cyclically administered to a subject.
  • Cycling treatment involves the administration of the intermittent lithium treatment or a pulse lithium treatment for a period of time, followed by the administration of the other treatment for a period of time and repeating this sequential administration.
  • the first treatment may be with the intermittent lithium treatment or a pulse lithium treatment or with the other treatment, depending on the subject's prior treatment history and the intended outcome.
  • cycling treatment can also reduce the development of resistance to one or more of the treatments, avoid or reduce the side effects of one of the treatments, and/or improve the efficacy of the treatment.
  • alternating administration of the intermittent lithium treatment or a pulse lithium treatment may be followed by the administration of another treatment (or vice versa) 1 year later, 6 months later, 3 months later, 1 month later, 3 weeks later, 2 weeks later, 1 week later, 4 to 6 days later, 2 to 4 days later, or 1 to 2 days later, wherein such a cycle may be repeated as many times as desired.
  • the intermittent lithium treatment or a pulse lithium treatment and the other treatment are alternately administered in a cycle of 3 weeks or less, once every two weeks, once every 10 days or once every week.
  • Such time frames can be extended or reduced depending on whether a controlled release formulation of either the lithium compound or the other treatment formulation is used, and/or depending on the progress of the treatment course.
  • an area of skin that was pre-treated with lithium is used as a source for transplanted follicles.
  • treatment with lithium at the wounds(s) from which transplanted tissue was obtained and/or the site of implantation is initiated for one week, and then discontinued and optionally followed by another treatment.
  • a candidate subject for intermittent lithium treatment i.e., alternating lithium treatment with "vacation/holiday” periods
  • a pulse lithium treatment for promoting hair growth is any subject at risk for, has, or has had a wound or scar.
  • the subject may be any subject, preferably a human subject, including male, female, intermediate/ambiguous ⁇ e.g., XO), and transsexual subjects.
  • a human subject including male, female, intermediate/ambiguous ⁇ e.g., XO), and transsexual subjects.
  • the subject is a Caucasian subject. In certain embodiments, the subject is an African subject or an African-American subject. In certain embodiments, the subject is a human adolescent. In certain embodiments, the subject is undergoing puberty. In certain embodiments, the subject is a young adult. In certain embodiment, the subject is a middle- aged adult. In certain embodiments, the subject is a premenopausal adult. In certain embodiments, the subject is undergoing menopause. In certain embodiments, the subject is postmenopausal. In certain embodiments, the subject is elderly.
  • the subject is a human of 1 year old or less, 2 years old or less, 2 years old, 5 years old, 5 to 10 years old, 10 to 15 years old, e.g., 12 years old, 15 to 20 years old, 20 to 25 years old, 25 to 30 years old, 30 years old or older, 30 to 35 years old, 35 years old or older, 35 to 40 years old, 40 years old or older, 40 to 45 years old, 45 to 50 years old, 50 years old or older, 50 to 55 years old, 55 to 60 years old, 60 years old or older, 60 to 65 years old, e.g., 65 years old, 65 to 70 years old, 70 to 75 years old, 75 to 80 years old, 80 to 85 years old, 85 to 90 years old, 90 to 95 years old or 95 years old or older.
  • the subject is a male 20 to 50 years old. In some embodiments, the subject is a male or female 12 to 40 years old. In some embodiments, the subject is not a female subject. In some embodiments, the subject is not pregnant or expecting to become pregnant. In some embodiments, the subject is not a pregnant female in the first trimester of pregnancy. In some embodiments, the subject is not breastfeeding.
  • the intermittent lithium treatment or a pulse lithium treatment is delivered to an area in which enhanced wound healing or scar revision is desired, for example, the scalp, face (e.g., the eyebrow, eyelashes, upper lip, lower lip, chin, cheeks, beard area, or mustache area) or neck, or another part of the body, such as, e.g., the chest, breasts, sternum, abdomen, arms, armpits (site of axillary hair), legs, hands, feet, or genitals.
  • a wounded or scarred part of the skin is treated.
  • the wounded or scarred part of the skin is a flexion surface or involves the extremities, breasts, sternum, face, or neck.
  • Wounds treatable by the methods described herein include, but are not limited to, any form of wound known in the art or to be discovered.
  • wounds treatable by the methods described herein include acute wounds (surgical and non-surgical), chronic or non-healing wounds, pressure sores (also referred to as decubitus ulcers or bed sores), pressure necrosis, lower extremity ulcers, radiation injury (such as, e.g., caused by radiation overdose), an erythema, skin abrasion, or a non-healing wound caused by wounding (e.g., a surgical incision) of irradiated skin.
  • the methods described herein are used to enhance healing of wounds caused by blisters, cutaneous trauma, and surgery, such as described in Mulvaney & Harrington, 1994, Chapter 7, "Cutaneous trauma and its treatment," in Textbook of Military Medicine: Military Dermatology, Office of the Surgeon General, Department of the Army, Virtual Naval Hospital Project, which is incorporated by reference herein in its entirety.
  • the methods described herein are used to enhance (e.g., hasten, improve, minimize scarring, etc.) healing of wounds by primary intention.
  • the methods described herein are used to enhance healing of wounds by secondary intention.
  • the methods described herein are used to enhance healing of wounds by tertiary intention.
  • the wound to be treated by the methods described herein has wound dehiscence, which is the premature "bursting" open of a wound along surgical suture.
  • the patient is at risk for wound dehiscence, based on one or more of the following risk factors: age, diabetes, obesity, poor knotting or grabbing of stitches, and trauma to the wound after surgery, or inadequate ability to form scars.
  • the methods described herein are used to treat a radiation scar, acne scar, curettage scar, spread scar, split-thickness scar, flap necrosis, scarring following infection, leg ulcer, burn scar, sternotomy scar, or as treatment to minimize scarring following curettage, following surgical excision, following follicular unit transplantation, or following Cesarean section, as exemplified in the examples of Section 7.
  • the methods described herein are used to enhance healing of transplanted skin at recipient sites ⁇ e.g., skin grafts or hair transplantation, such as long- term frontal hair scalp or eyebrow plugs), so that, for example, the skin blends in with the skin at the recipient site with regard to thickness, pigmentation, hair patterning, etc.
  • a scar that results from skin grafting where the graft edges join the host skin, common in battlefield wounds is treated by the methods described herein. In general any "flap" surgery or "free flap” graft will result in these scars.
  • the methods described herein are used to enhance healing of a split thickness skin graft.
  • the split-thickness donor skin tissue for grafting of wound sites is taken from the scalp, as described in Weyandt, et al, 2009, Dermatol. Surg. 35: 1873- 1879, which is incorporated herein by reference in its entirety.
  • lithium treatment may benefit this process by facilitating the "recipient dominance" phase (that temporally follows "donor dominance”). It is postulated that pulse or intermittent lithium treatment can make skin grafts (even pinch grafts) take on attributes of the recipient site by stimulating "local" tissue stem cells to form site-appropriate follicles.
  • Such an intervention can help not only autologous grafts, but also allogeneic grafts, fetal cell grafts (like placenta stem cell “bandaids”), and also stem cell grafts ⁇ ex vivo expanded
  • Scars treatable by the methods described herein include, but are not limited to, any form of scar known in the art or to be discovered.
  • Non-limiting examples of scars that can be revised or otherwise treated by the methods described herein include scars that form by secondary intention, atrophic scars, hypertrophic scars, keloid scars, hypopigmented scars, hyperpigmented scars, depressed scars (including ice-pick scars), and spread scars.
  • Scars form following a variety of causes including, e.g., cosmetic procedures and skin transplants are not really clinical categories of scars.
  • scars caused by a disease or disorder such as scarring (cicatricial) alopecia, scars caused by excessive wound healing, scars caused by joint contracture, or scars caused by burns or wounds.
  • the methods described herein may also be used to treat wounded skin, or skin that may become wounded, in order to prevent, minimize, or reduce scar formation.
  • the scar is caused by surgery, such as a open heart surgery, joint surgery, face lift, skin graft, or hair transplant, etc.
  • the subject for whom pulse or intermittent lithium treatment is intended is a patient who has scarring (cicatricial) alopecia, a condition of permanent hair loss in which the hair follicle is destroyed by inflammation and replaced with scar tissue.
  • scarring alopecia is moderate to severe.
  • the subject has wounding or scarring caused by, exacerbated by, or associated with medication, such as corticosteroid use, chemotherapy ⁇ e.g., anti-cancer therapy or cytotoxic drugs or other antiproliferative agents), thallium compounds, vitamins ⁇ e.g., vitamin A), retinoids, anti-viral therapy, or psychological therapy.
  • medication such as corticosteroid use, chemotherapy ⁇ e.g., anti-cancer therapy or cytotoxic drugs or other antiproliferative agents), thallium compounds, vitamins ⁇ e.g., vitamin A), retinoids, anti-viral therapy, or psychological therapy.
  • the subject has wounding or scarring caused by, exacerbated by, or associated with radiation (including therapeutic radiation treatment or radiation overdose), trauma (chronic or acute, mild or severe), physical trauma, endocrine dysfunction, surgery (including, for example, face lift, hair transplant, cosmetic surgery, and surgery of flexion surfaces, the extremities, breasts, sternum, and neck), sutures, x-ray atrophy, burning or other wound or injury, stress, aging, an inflammatory disease or condition (acute or chronic), an autoimmune disease or disorder, malnutrition (including, e.g., vitamin or trace metal deficiency, scurvy), anemia, diabetes, obesity, a circulatory disorder, such as, e.g., arterial or venous insufficiency, occlusive vascular disease, microvascular occlusive disease, vasoconstriction, hypovolemia, venous valvular disease, impaired oxygen delivery or tissue perfusion, caused by, e.g., ischemia, hypoxia, stroke,
  • radiation including
  • a human skin xenograft (without skin appendages) can be considered as similar to a scar, and can be wounded and then treated pharmacologically to induce hair follicles and/or monitor revision of the scar.
  • Xenografts can also be combined with inducible genetically modified cells to activate pathways know to form hair follicles.
  • the safety and efficacy of a pulse or intermittent lithium treatment is tested in a full thickness or a split thickness human skin xenograft ⁇ e.g., obtained surgically from scar revisions; from foreskin; or cadaveric), or may be tested in a three-dimensional organotypic human skin culture on SCID mice.
  • Success of a pulse or intermittent lithium treatment can be measured by:
  • any method known in the art may be used to evaluate the safety and efficacy of an intermittent lithium protocol or pulse lithium protocol, or of the combination treatments described in Section 5.4.
  • a human skin xenograft model is used.
  • an intermittent lithium treatment or pulse lithium treatment may be administered with a full thickness excision, laser, inflammatory stimulus, or dermabrasion procedure for integumental perturbation.
  • a synergistic effect of an intermittent lithium treatment or pulse lithium treatment on another treatment for enhancing wound healing or scar revision may be measured as an improvement over a control subject receiving only one of the two treatments (i.e., the intermittent lithium treatment or pulse lithium treatment alone or the second treatment alone).
  • Another animal model for use in evaluating treatment that may more closely mimic the biology of human skin and hair is a guinea pig model (see, Stenn & Paus, 2001, Physiol. Revs. 81 : 449-494).
  • the methods for evaluating treatment in animals described elsewhere in this section and in the example in Section 16 below may be applied to guinea pigs according to methods known in the art. See also, e.g., Kramer et al, 1990, Dermatol Monatsschr. 176:417-20; and Simon et al, 1987, Ann Plast Surg 19:519-23.
  • Other animal models that may be of use in evaluating the treatments described herein include pig, cat, or stumptailed macaque models.
  • success of a pulse or intermittent lithium treatment can be measured by:
  • hair follicle neogenesis or regeneration • increased proportion of hair follicles in anagen or decreased proportion of follicles in telogen
  • VSS Vancouver Scar Scale
  • the intermittent lithium treatment or pulse lithium treatment improves one of the foregoing measures by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more.
  • Such an improvement may be measured after 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or one year or longer after initiation of the intermittent lithium treatment or pulse lithium treatment.
  • a synergistic effect of an intermittent lithium treatment or pulse lithium treatment on another treatment described herein may be measured as an improvement over a control subject receiving only one of the two treatments (i.e., the intermittent lithium treatment or pulse lithium treatment alone or the second treatment alone).
  • Human skin equivalents can be grown and assembled in vitro, with the advantage that they can be grown to theoretically to any size/shape; can be comprised of different types of cells, including keratinocytes (hair follicle derived and non-hair follicle derived), dermal cells (hair follicle derived and non-hair follicle derived), other cell types ⁇ e.g. , mesenchymal stem cells); can contain cells that are genetically modified to include, e.g., markers or "inducible" signaling molecules; provide an unlimited and uniform source of human cells; from normal skin based on histology and marker studies; are generally devoid of skin appendages; and can be wounded and show similar wound healing events as in vivo.
  • keratinocytes hair follicle derived and non-hair follicle derived
  • dermal cells hair follicle derived and non-hair follicle derived
  • other cell types ⁇ e.g. , mesenchymal stem cells
  • the intermittent lithium treatments or pulse lithium treatments may facilitate wound healing and scar revision by:
  • epithelial stem cells located in the epidermis and in the hair follicle, giving rise to keratinocytes that re-epithelialize the wound (see, e.g., Lau et al, 2009, Exp. Dermatol. 18:921-933).
  • the invention is based, in part, on the principle that the lithium ion (Li+) is an inhibitor of the polyphosphoinositide cycle that can reversibly arrest cells in cell cycle.
  • the lithium ion has been shown to cause metaphase arrest that can be reversed by the addition of CaC12 or myo-inositol. (Wolniak, 1987, Eur. J. Cell Biol. 44: 286-293).
  • the lithium ion has also been shown to arrest cancer cell lines at certain stages of the cell cycle (see, e.g., Wang JS, 2008, World J. Gastroenterol. 14:3982-3989).
  • the invention is based in part on the inventors' recognition that the lithium ion can be used in a pulse or intermittent treatment regimen to synchronize groups of hair follicle cells or hair follicle stem cells that are in various stages of cell cycle (cycling asynchronously).
  • the lithium ion may cause hair follicle stem cells to stop dividing and more readily differentiate into hair follicles and thereby improve wound healing. Restarting the cell cycle at the termination of a pulse lithium treatment, or during the "holidays" between intermittent lithium treatments should restart cell cycle synchronously.
  • the synchronization phenomenon can be described by analogy to traffic lights: periodically arresting the motion of individual cars generates synchronization because cars pile up behind stop lights. Similarly, by introducing a signal that periodically arrests cell division, synchronization is generated because when the "stop" signal is removed, cells initiate division at the same time.
  • Such synchronization of cell cycles in the hair follicle cells results in relative synchronization of hair follicle cycle stage in groups of follicles that otherwise have a stochastic distribution of stages of follicle cycle (asynchronous follicle cycle).
  • the Follicle Stem Cells that are thought to be involved can be derived from (1) other Follicle Stem Cells (e.g., from the bulge or crypt), (2) from other tissue stem cells, termed "pre-Follicle Stem Cells" (from the interfollicular skin), (3) from bone marrow- derived stem cells ("BMST", such as hematopoietic stem cells), (4) uncommitted epithelial progenitor cells; and/or (5) from mesenchymal stem cells such as hair follicle dermal sheath cells and adipocyte stem cells.
  • BMST bone marrow- derived stem cells
  • mesenchymal stem cells such as hair follicle dermal sheath cells and adipocyte stem cells.
  • BMST bone marrow derived stem cells
  • their differentiation into Follicle Stem Cells requires intact follicles, whose cells can play the role of "nurse cells” and provide appropriate signals to guide the differentiation of bone marrow derived stem cells into Follicle Stem Cells.
  • Integumental perturbation (by wounding, e.g., during scar revision, or by the induction of inflammation) (1) provides signals for Follicle Stem Cells to divide symmetrically to begin the process of forming new follicles; (2) mobilizes tissue stem cells ("pre-Follicle Stem Cells”) from interfollicular skin to
  • intermittent or pulse lithium treatment organizes the normally asynchronous state of human hair follicle cells in Cell Cycle and human hair follicles in Follicle Cycle into relatively more
  • This protocol is adapted from the IACUC VA protocol. Specifically, 4 week old male SCID mice are obtained from Charles River and allowed to acclimate for at least 1 week. In preparation for surgery, mice are anesthetized with ketamine (80 mg/kg)/xylazine (20 mg/kg) delivered i.p. in a volume ⁇ 100 ⁇ , and monitored by toe pinch to determine the surgical plane of anesthesia. Full thickness adult human skin (measuring approximately 1.5 cm x 2 cm; removed during surgical procedures from the CHTN, NDRI or cadaver scalp skin from ABS) is sutured into a full thickness skin excision site on the dorsal surface of the mouse. The grafts are bandaged and allowed to heal for at least 5 weeks.
  • mice are anesthetized with ketamine (80 mg/kg)/xylazine (20 mg/kg) delivered i.p. in a volume of ⁇ 100 ⁇ , and monitored by toe pinch to determine the surgical plane of anesthesia.
  • the epidermis of the human skin is removed using a microdermabrasion device to dermabrade as described above.
  • mice may be required to confirm and/or optimize these settings for human scalp xenografts. Additionally, some mice may be required to test the differences between full thickness and split thickness human scalp xenografts. Furthermore, reducing the overall thickness of the human skin may improve the "take" rate of the grafts, which is
  • mice receive vehicle alone or the lithium composition, delivered systemically or topically, or neither vehicle nor lithium composition, for 5 consecutive days, (the lithium composition chosen is the one determined to be most efficacious in the
  • C57BL/6J model with efficacy determined to be increased number and/or size of neogenic hair follicles.
  • One dose of the lithium composition is delivered, using the most efficacious dose as described above, systemically and, in a separate experiment, a dose is delivered topically. Additionally, histology and/or photography is performed daily (until the end of the experiment) following scab detachment in order to monitor hair follicle neogenesis.
  • An additional set of mice are treated with the lithium composition or vehicle or neither, with the exception that the xenografted mice are not wounded, in order to assess the effect of the lithium composition in the absence of wounding.
  • mice At approximately 2 weeks post-scab detachment, all mice are anesthetized with ketamine (80 mg/kg)/xylazine (20 mg/kg) delivered i.p. in a volume of ⁇ 100 ⁇ , and monitored by toe pinch to determine the surgical plane of anesthesia. Subsequently, they have a terminal blood draw (to detect drug in the plasma), and are euthanized. The wound is then removed, which is trisected with one-third taken for biochemistry, one third for determination of lithium levels in the skin using mass spectrometry, and one third for histology/immunohistochemistry.
  • mice are needed for the optimization of microdermabrasion settings and split thickness versus full thickness xenografts. Considering that the "take" rate of human skin xenografts is approximately 50%, the total number of mice to optimally receive human skin grafts is approximately 500.
  • a female human subject 75 years old, underwent treatment of a basal cell carcinoma of the nose five years prior to presentation.
  • the resulting scar is atrophic, hypo- pigmented and lacking normal pore pattern.
  • the scar is mechanically disrupted by excision, dermatome planing, dermabrasion, laser abrasion, or Fraxel, and treatment with topical lithium gluconate 8% is initiated.
  • Response to treatment is determined by measuring skin thickness, return of pigmentation and re-establishment of adnexal structures.
  • a male human subject 28 years old, presents with extensive, broad, shallow, acne scars.
  • the scars are atrophic, hypo-pigmented and lack normal pore pattern.
  • the scars are dermabraded using a moderate grit diamond fraise and treatment with 8% topical lithium is initiated.
  • Response to treatment is determined by measuring skin thickness of the scar, return of pigmentation and re-establishment of adnexal structures.
  • Response to treatment is determined by measuring skin thickness, return of pigmentation and re-establishment of adnexal structures.
  • a male human subject 75 years old, underwent excision of a large malignant melanoma of the forehead with subsequent reconstruction using a split-thickness skin graft. After one year, the resulting graft demonstrates depression, skin atrophy, hyper-pigmentation and loss of normal pore pattern.
  • the graft is mechanically disrupted by excision, dermabrasion, laser abrasion, or Fraxel, and treatment with 8% topical lithium gluconate is initiated.
  • Response to treatment is determined by measuring skin thickness, establishment of normal pigmentation and re-establishment of adnexal structures.
  • a female human subject undergoes excision of a large congenital nevus of the cheek. Due to the tension of closure, the incision line spreads and widens over time resulting in a scar that is atrophic, hypo-pigmented and lacking normal pore pattern. The scar is mechanically disrupted by excision, dermabrasion, laser abrasion, or Fraxel, and treatment with 8% topical lithium gluconate is initiated.
  • Response to treatment is determined by measuring skin thickness, return of pigmentation and re-establishment of adnexal structures.
  • a female human subject 65 years old, underwent a face-lift procedure.
  • a portion of the cheek flap subsequently became necrotic and healed by secondary intent.
  • the resulting scar was atrophic, hypo-pigmented and lacked normal pore pattern.
  • the scar is mechanically disrupted by excision, dermabrasion, laser abrasion, or Fraxel, and treatment with topical 8% lithium gluconate is initiated.
  • Response to treatment is determined by measuring skin thickness, return of pigmentation and re-establishment of adnexal structures.
  • a female human subject 37 years old, underwent a phenol chemical peel of her lips to decrease rhytides.
  • the patient developed a staphylococcal (herpetic) infection of the treated area.
  • the infected area healed with an atrophic, hypo- pigmented scar that lacked the normal pore pattern.
  • the scar is mechanically disrupted by excision, dermabrasion, laser abrasion, or Fraxel, and treatment with 8% topical lithium gluconate is initiated.
  • Response to treatment is determined by measuring skin thickness, return of pigmentation and re-establishment of adnexal structures.
  • a male human subject 70 years old, developed non-healing leg ulcers involving the tibial aspects of both legs.
  • the affected area was pre-treated with 8% topical lithium carbonate and subsequently grafted using small pinch grafts harvested from the thighs.
  • Response to treatment is determined by measuring skin thickness and re- establishment of adnexal structures over the grafted area following graft take.
  • a female human subject 40 years old, underwent split thickness grafting to reconstruct a facial defect following excision of a squamous cell carcinoma.
  • the graft donor site healed with a hypo-pigmented scar that lacked the normal pore pattern.
  • the scar is mechanically disrupted by excision, dermabrasion, laser abrasion, or Fraxel, and treatment with 8% topical lithium gluconate is initiated.
  • Response to treatment is determined by measuring skin thickness, return of pigmentation and re-establishment of adnexal structures.
  • a male human subject 30 years old, suffered a burn of the left cheek that healed with a contracted, hypo-pigmented scar that lacked normal pore pattern.
  • the scar is mechanically disrupted by excision, dermabrasion, laser abrasion, or Fraxel, and treatment with 8% topical lithium gluconate is initiated.
  • Response to treatment is determined by measuring skin thickness, return of pigmentation and re-establishment of adnexal structures.
  • a 76 year old fair skin male with a history of multiple basal cell carcinomas presents with 2 new pigmented nodular pearly lesions of 1 cm on his left (A) and right (B) scapula. Shave biopsies reveal both lesions to be nodular BCCs.
  • Lesion (A) is treated with aggressive 3 pass curettage then application of aluminum chloride.
  • Lesion (B) is treated with aggressive 3 pass curettage then application of aluminum chloride.
  • Lesion B is post-treated with topical 8% lithium gluconate daily for 5 days post-procedure (in addition to routine wound care).
  • the lesions are compared with respect to skin thickness, return of pigmentation, re-establishment of adnexal structures and global assessment the physician and patient's satisfaction regarding the cosmetic result of the 2 distinct lesions using a Visual Analogue Scale (VAS).
  • VAS Visual Analogue Scale
  • a 76 year old fair skin male with a history of multiple basal cell carcinomas presents with 2 biopsy proven BCC lesions of 1 cm on his left scapula (A) and 1 cm on his right scapula(B). Conservative surgical excision is performed on both lesions .
  • Lesion (A) is treated with then treated with routine wound care.
  • Lesion (B) is treated with pre-treated with 5 days topical 8% lithium gluconate daily prior to surgery followed by application for 5 days post-procedure (in addition to routine wound care).
  • the lesions are compared with respect to skin thickness, return of pigmentation, re-establishment of adnexal structures and global assessment the physician and patient's satisfaction regarding the cosmetic result of the 2 distinct lesions using a Visual Analogue Scale (VAS).
  • VAS Visual Analogue Scale
  • a 74 year old black male with history CAD undergoes CABG surgery requiring median sternotomy. Following the procedure the scar is found to measure 14 cm in diameter. Starting Day 1 thru 7 he applies topical 8% lithium gluconate to the 7 cm superior portion of the scar (in addition to routine wound care).
  • the superior and inferior aspects of the sternotomy scar are compared with respect to skin thickness, return of pigmentation, re-establishment of adnexal structures and global assessment the physician and patient's satisfaction regarding the cosmetic result of the 2 distinct lesions using a Visual Analogue Scale (VAS).
  • VAS Visual Analogue Scale
  • a 51 year old white male with history Androgenic Alopecia undergoes Hair Transplant with the "Strip Harvesting Method" with donor area located at the posterior scalp along the occipital protuberance.
  • Surgery requires a long scar on the posterior scalp measuring 28 cm.
  • Starting Day 1 thru 7 topical 8% lithium gluconate is applied only to the 14 cm “left” portion of the scar (in addition to routine wound care).
  • the right and left aspects of the scar are compared with respect to skin thickness, return of pigmentation, re-establishment of adnexal structures and global assessment the physician and patient's satisfaction regarding the cosmetic result of the 2 distinct lesions using a Visual Analogue Scale (VAS).
  • VAS Visual Analogue Scale
  • hair that is regenerated in the treated donor area may be used as a source of future, repeated hair transplants in accordance with the foregoing method.
  • a 51 year old white male with history Androgenic Alopecia undergoes Hair Transplant with the "Follicular Unit Extraction" with donor area located at the posterior scalp above and below the occipital protuberance.
  • punch graft were taken from the posterior scalp and left to heal with secondary intention.
  • Starting Day 1 thru 7 topical 8% lithium gluconate is applied only to the "left" portion of the donor area (in addition to routine wound care).
  • the right and left aspects of the scar are compared with respect to skin thickness, return of pigmentation, re-establishment of adnexal structures and global assessment the physician and patient's satisfaction regarding the cosmetic result of the 2 distinct lesions using a Visual Analogue Scale (VAS).
  • VAS Visual Analogue Scale
  • hair that is regenerated in the treated donor area may be used as a source of future, repeated hair transplants in accordance with the foregoing method.
  • VAS Visual Analogue Scale
  • a patient has a 5 cm scar resulting from surgery. Two to three months after surgery, the scar is treated with fractional laser. Half the scar is treated with lithium and the whole scar is treated with laser. In another variation, a patient has two surgical scars, one of which is treated with the lithium and laser combination and the other of which is treated with laser alone.
  • the subject is administered a fractional and non-ablative laser therapy using an Erbium- YAG laser with an emission at 1540-1550 nm (set to 50-70 J/cm 2 , treatment level of 8-10 (density of the "dots"), and 8 passes) and the subject is provided with a topical preparation of Lithium gluconate 8% gel (Lithioderm 8% gel) and instructed to apply the Lithium gluconate 8% gel to the treated area of the ear for one week. After one week, treatment with lithium gluconate is discontinued and he is evaluated after three weeks.
  • Lithium gluconate 8% gel Lithium gluconate 8% gel
  • the treatments may alternatively be accomplished by applying an ablative laser treatment in place of the non-ablative laser treatment.
  • the application of Lithium gluconate is sterile and, optionally, the treatment area is covered by a bandage.
  • ablative laser treatment may accomplished using an Erbium- YAG laser at 2940 nm or a CO 2 laser at 10,600 nm.
  • Lidocaine HCL 2% with Epinephrine 1 : 100,000 are injected to anesthetize the surface of the area to be treated.
  • An Ultrapulse (fractional mode) CO 2 laser is used to disrupt the epidermis and dermis to approximately 100 to 500 ⁇ in depth.
  • the Ultrapulse laser produces an effect that is similar to that of dermabrasion yet the disruption produced delivers a greater amount of energy deeper into the skin in a non-scaring fractional ablation.
  • the treated area is a 1.5 cm x 1.5 cm square.
  • the Ultrapulse is set to deliver up to 350 mJ, up to 52.5 Watts, using pattern size #8, density #4, and fill the square treatment site with up to 5 passes.
  • Lidocaine HCL 2% with Epinephrine 1 : 100,000 are injected to anesthetize the surface of the area to be treated.
  • An Ultrapulse CO 2 laser (ablative mode) is used to disrupt the epidermis and dermis to approximately 100 to 500 ⁇ in depth.
  • the Ultrapulse laser produces an effect that is similar to that of dermabrasion yet the disruption produced delivers a greater amount of energy deeper into the skin in a non-scaring ablation that resembles the dermabrasion.
  • the treated area is a 1.5 cm x 1.5 cm square.
  • Ultrapulse is set to deliver up to 500 mJ in 1 msec, 1 Watts, using a spot size of 3 mm at 2 Hz to fill the square treatment site, which may require up to 15 passes.
  • Lidocaine HCL 2% with Epinephrine 1 : 100,000 are injected to anesthetize the surface of the area to be treated.
  • the ablative erbium laser is set to deliver up to 5 Joules 240 msec in of energy at level 3 so that in up to 15 passes it will produce a disruption up to 500 ⁇ deep.
  • the treated area is a 1.5 cm x 1.5 cm square.
  • the dermabrasion treatments provided in the examples of Sections 7.1-1.7, 7.9, and 7.10 may alternatively be accomplished by applying one of the following dermabrasion treatments.
  • Lidocaine HCL 2% with Epinephrine 1 : 100,000 is injected to anesthetize the surface of the area to be treated.
  • Standard dermabrasion using the Aseptico Econo-Dermabrader from Tiemann and Company, is performed to a depth of approximately 150 ⁇ , that includes removal the entire epidermis and disruption of the papillary dermis (detectable by a shiny, whitish appearance) inducing the formation of small pools of blood in the treated area.
  • Each dermabraded area is a 1.5 cm x 1.5 cm square.
  • a Bell Hand dermabrasion device may be used.
  • Section 11-16 which present mouse studies using dermabrasion, and the protocols for use in humans in the examples of Sections 7, 9 and 10.
  • dermabrasion was carried out using a microdermabrasion device. While dermabrasion in humans may also be carried out using a microdermabrasion device, where sterile conditions are preferential, a dermabrasion device is preferably used.
  • the following example provides a protocol for demonstrating the importance of timing of lithium gluconate treatment for the optimization of follicular neogenesis and wound healing/scar revision after integumental perturbation.
  • patients are treated with a pulse lithium of 8% lithium gluconate (topical gel) in combination with dermabrasion.
  • patients may be treated with the intermittent lithium treatment or a pulse lithium treatment alone (as described infra), dermabrasion alone (or with a vehicle, e.g., petrolatum), or may not receive any treatment.
  • any patient population may be treated, preferred patients are Caucasian males 20-50 years of age. Patients for whom treatment may be contraindicated (particularly at the clinical trial stage) are those who are currently participating in or have participated in any clinical study with an investigational drug within the thirty (30) days immediately preceding treatment, with current or recent use ( ⁇ 1 y) of isotretinoin (Accutane), currently taking hormone therapy, or steroids or other immunomodulators or have taken these medications within the past thirty (30) days (inhaled steroids are acceptable), currently using Rogaine or Propecia or used them in the past forty- five (45) days, immune compromised or undergoing therapy to treat an immune disorder, have a clinically significant medical condition that may interfere with the protocol described herein, have other active skin diseases (such as actinic keratosis or psoriasis) or skin infections (bacterial, fungal or viral, esp.
  • other active skin diseases such as actinic keratosis or psoriasis
  • skin infections bacterial, fungal or viral
  • HSV infection in the area to be treated, have a history of keloids or hypertrophic scarring, hypersensitivity to lidocaine, poor wound healing, diabetes, or coagulopathy, undergoing current drug or alcohol abuse, psychiatric dysfunction, or other factors that would limit compliance, have sunburned skin, or who are currently taking anti-platelet agents other than aspirin.
  • Dermabrasion using alumina particles is performed on Day 0. Dermabrasion is performed to a depth of approximately 100 ⁇ , which includes removal of the entire epidermis and disruption of the papillary dermis (detectable by a shiny, whitish appearance) inducing the formation of small pinpoints of blood in the treated area. Dermabrasion is performed in two sites of the skin. The area is then allowed to heal without manipulation. A 4 mm punch biopsy is performed on days 1 1 and 14, and the presence of new hair follicles is examined in these subjects based on histological assessment. A third biopsy is optionally performed on Day 14 on an untreated area 1 cm away from the treated area to serve as histologic control.
  • the protocol may be amended in accordance with the findings. For example, if dermabrasion causes presence of neogenic hairs in a 4 mm punch biopsy in, for example, at least three of the first 15 patients, then additional patients will be treated.
  • the procedure begins with shaving/clipping of the existing hair in the area to be treated followed by a thorough cleaning with antiseptic cleansing agent.
  • Numbing agents such as lidocaine HCL 2% and Epinephrine 1 : 100,000, are injected to anesthetize the surface to be treated.
  • Standard dermabrasion is performed to a depth of approximately 100 ⁇ , which includes removal the entire epidermis and disruption of the papillary dermis
  • Each dermabraded area is approximately a 1.5 cm x 1.5 cm square.
  • Suitable dermabrasion devices are the ASEPTICO ECONO-DERMABRADER from
  • sterilized sandpaper may be used for dermabrasion.
  • Adhesive ocular shields are worn by the patient during the procedure to avoid complications due to aluminum crystals entering the eye (chemosis, photofobia, punctuate keratitis) and the doctor should wear safety goggles.
  • the dermabrasion tool is carefully maneuvered over the area to carefully remove layers of skin until the desired level is reached. The procedure usually takes only a few minutes.
  • Pre-dermabrasion patients should be asked to: not wear contact lenses during the procedure, discontinue use of over the counter exfoliation products such as Retinol, Glycolic or other hydroxy acids, Salicylic acid, Beta hydroxyl acids 3 days prior to treatment, discontinue use of retinoids 30 days prior to treatment, not receive Botox or collagen injections for 2 weeks prior to treatment.
  • over the counter exfoliation products such as Retinol, Glycolic or other hydroxy acids, Salicylic acid, Beta hydroxyl acids 3 days prior to treatment, discontinue use of retinoids 30 days prior to treatment, not receive Botox or collagen injections for 2 weeks prior to treatment.
  • the treated skin will be red, swollen and tender, and the wound should be cared for as follows until new skin starts to grow; this usually takes 7-10 days: 1) Keep the area clean and dry for today.
  • the area should either be covered with Vaseline and bandaged after or covered with duoderm or a similar covering. Alternatively, it may be preferable to not cover, bandage, or otherwise manipulate the treated area; 2) Avoid touching the area when washing hair; 3) Pat the area dry. Do not cover, bandage, or otherwise manipulate the treated area.
  • the treated are may itch as the new skin grows and may be slightly swollen, sensitive, and bright pink for several weeks after dermabrasion.
  • the procedure begins with thoroughly cleaning the area to be biopsied with antiseptic cleansing agent.
  • Lidocaine HCL 2% and Epinephrine 1 : 100,000 (approximately 0.5 cc to each site) are injected to anesthetize the site that will be biopsied.
  • 4 mm punch biopsy is performed. Biopsied site is closed with 2 4.0 Ethilon sutures. Vaseline and band-aid are applied. Tissue samples are stored in formalin for histological analysis.
  • This example provides a protocol for characterizing and comparing the percutaneous absorption pharmacokinetics of four formulations containing a lithium salt, in human cadaver skin, using the in vitro skin finite dose model.
  • This model is a well- established tool for the study of percutaneous absorption and the determination of the pharmacokinetics of topically applied drugs.
  • the model uses human cadaver skin mounted in specially designed diffusion chambers allowing the skin to be maintained at a temperature and humidity that match typical in vivo conditions.
  • a dose (e.g., 0.1 gram) of formulation is applied to the top of the partial thickness skin or dermis and drug absorption is measured by monitoring its rate of appearance in the reservoir solution bathing the other surface of the skin.
  • compositions of the formulations are provided in Table 2 below.
  • the formulations were tested initially for stability in solution at 4 °C, 25 °C and 40 °C. All formulations were stable solutions or emulsions at the temperatures tested.
  • the excipients selected for the formulations were based on levels approved for topical drug formulations and each excipient was selected for its viscosity-enhancing properties or its ability to enhance permeation through tissues. Methylparaben was added to the formulations for its preservative activity.
  • hydrogel Carbopol 980 1.5%
  • Dermal tissue was prepared by heating the full thickness skin at 40 °C for 20 minutes in de- ionized water and removing the epidermis using sterile forceps. All cells were mounted in a diffusion apparatus in which the dermal bathing solution was stirred magnetically at approximately 600 RPM and its skin surface temperature maintained at 32.0° ⁇ 1.0 °C.
  • PBS Human Receptor Solution
  • the formulation BX is a neutral hydrogel, with its gel-like consistency produced by the presence of high molecular weight hydroxyethyl cellulose (HEC).
  • HEC high molecular weight hydroxyethyl cellulose
  • the diffusion of lithium through the hydrogel and through the dermis is slower than 35A', with 80% released in approximately 8 hours.
  • Use of an anionic hydrogel (formulation BV-001-003A) slowed down release even further, with 80% released in 12 hours. It is possible that complexation of lithium ions with the anionic polymer Carbopol 980 slows down the release of lithium ions from the hydrogel.
  • This example provides an assessment of the rate of permeation and residence time of lithium ions provided in various formulations in an in vivo mouse model developed for follicle neogenesis. Based on the data, appropriate formulations are selected for an in vivo mouse experiment to assess neogenesis. Formulations that have an adequate rate of permeation through the dermis and longest residence time are selected as formulations to enroll in an in vivo model for neogenesis. It is postulated that lithium ions can induce differentiation of stem cells into neogenic hair follicles.
  • Formulations selected in this experiment were : 35A', 35BX and BV-001-003A with their respective compositions as shown in Table 2 supra.
  • mice 24 C57/BL 6 mice were enrolled in each group. There were 6 groups in total, with 3 groups enrolled for dermabrasion (DA) and 3 for FTE treated skin. A different formulation was enrolled in each of the three groups for DA and FTE.
  • DA dermabrasion
  • FTE FTE
  • Dosing for the DA groups was started at day 0, immediately after debriding the mouse skin with dermabrasion, and continued to day 5. Scab formation on the wound occurs approximately at day 1 and thus the formulations are delivered on top of scabbed wounds.
  • Dosing for the FTE groups was started at approximately day 7, or when the scab detached from the wound. The formulations were delivered to the re-epithelialized skin for five days.
  • Each wound was dosed with a formulation volume of 0.1 ml, or 0.1 g since the density of each formulation was determined to be approximately 1 g/ml. Dosing was accomplished with a 100 microliter Wiretrol device. Post-dosing, the wound was covered with a non-stick Tegaderm bandage.
  • Blood levels were an order of magnitude lower than in skin, possibly because the formulation used, in which the Li ion is complexed with CarboPol 980 to form a polymer, enhances its residence in the skin, in contrast to Li ion in, for example, saline, which is expected to be highly water soluble.
  • Dermabrasion by any other means such as full-thickness or partial-thickness excision, micro-needle roller perturbation, laser fractional, non-fractional or ablative, are alternate means of integumental perturbation, prior to administration of lithium.
  • the skin and the corresponding plasma concentrations of Li ions were determined following subcutaneous administration of lithium chloride at increasing dose concentrations.
  • This protocol can also be adapted to determine follicular neogenesis as a function of increasing dose concentrations of lithium.
  • mice were treated with either DA or FTE or unwounded (see Table 3 below), and dosed subcutaneously with 0.1 ml of a formulation containing increasing concentrations of lithium chloride in isotonic saline.
  • DA mice received 42 mg/kg, 127 mg/kg, or 381 mg/kg subcutaneously, twice daily for 4 days, and one dose on the 5 th day.
  • Lithium treatment of FTE mice started the day of scab detachment (at day 10-11). FTE mice received 64 mg/kg, 150 mg/kg, or 240 mg/kg subcutaneously, twice daily for 4 days, and one dose on the 5 th day.
  • mice were sacrificed, and the entire wound area of skin was analyzed for Li concentration and blood was drawn and centrifuged into red blood cells (RBC) and plasma. Then, at the 21 st day, a section of skin was biopsied and analyzed for Li concentration, along with the corresponding plasma and RBC concentrations.
  • RBC red blood cells
  • mice that received FTE treatment as a mode of wounding were dosed on the day of scab detachment (e.g., on day 10-15 post-wounding) with a single dose of lithium chloride for 5 days.
  • the mice were sacrificed and the wound was biopsied and analyzed for Li concentration.
  • blood was drawn and centrifuged into RBC and plasma and assayed for lithium levels.
  • Lithium concentrations were measured by the validated bioanalytical ICP method provided below.
  • LOQ for Li in the assay 50 mM.
  • This method was developed to quantify lithium in murine skin, plasma and pellet of red blood cells (RBC).
  • RBC red blood cells
  • Known quantities of lithium were added to matrices collected from control mice that were not exposed to any lithium as part of preclinical testing. Processing of samples is described below and involved digestion with hot nitric acid to reduce interference by organic matter and to convert particulate-associated metals to a form that could be measured by ICP/MS. Acid digests were cooled and then filtered prior to injection into the ICP /MS.
  • the calibration process involved using lithium standards that were dissolved in purified water.
  • the primary lithium standard was diluted to make a set of lithium working standards.
  • One set of these working standards was used to generate the calibration curve.
  • a separate set was used to prepare the QC samples described in the section entitled "Accuracy.”
  • the method was validated for all three murine matrices.
  • the final calibration curve covered the concentration range from 0.05-50 ⁇ g/L.
  • Igepal-70 is a non-ionic detergent and was added to dissociate erythrocyte membranes.
  • the solution was triturated with a pipettor and then transferred to a microwave vessel. The mass of transferred solution was measured before adding known amounts of lithium working standard and 1.0 mL concentrated nitric acid. Mixture was heated in a microwave for approximately fifteen minutes. After digestion was complete, the digest was cooled and purified water was added to achieve a total volume of 25 mL. Once the solution was mixed and passed through a 0.45- ⁇ nylon filter, it was ready for injection into the ICP/MS.
  • LLOQ Precision at Lower Limit of Quantitation
  • Precision was measured as inter-injection variability, by analyzing six separate injections of the same QC sample, removed from the same vial. Analyte concentration for this test was near the mid-point of the calibration range. Results are presented in Table 8. In terms of %CV, precision was less than 1% for all matrices.
  • Precision was based on six replicate injections of the QC standard, the concentration of which corresponds to the mid-point of the calibration range.
  • the %CV for plasma was 0.642 for skin 0.631 and for RBC 0.540. The results are presented in Table 8.
  • Li concentrations in skin and blood increased in a dose-related fashion.
  • the concentrations of Li in RBC were negligible (data not shown).
  • Li concentrations in skin at trough were 0.0001-0.0009 mM Li.
  • Li concentrations in blood at peak (1 hr post dosing on day 5) were between 0.695 mM - 1.059 mM Li (see Figures 16 and 17), and trough concentrations in blood were 0.02-0.09 mM Li.
  • the data show that when lithium chloride is delivered subcutaneously, lithium ions extract to the skin and the plasma in a linear dose-related fashion, although plasma concentrations were many-fold higher than in skin.
  • the lithium concentration in skin at 5 days correlates in a linear dose-related fashion, with no difference observed between wounded skin and non-wounded skin (see Figure 14B).
  • the skin samples were obtained 1 hour post-dosing, in other words at "peak" skin concentrations.
  • the data demonstrate that lithium does distribute to the skin, where it may play a role in stem cell modulation toward differentiation into de novo hair follicles.
  • the purpose of the experiment in this example was to evaluate the absorption of Li ions into the skin and blood compartments in an in vivo mouse model developed for follicle neogenesis, with once/day topical administration of two lithium formulations, a Lithium Gluconate Hydrogel and a Lithium Chloride Hydrogel (see Table 10 below).
  • This example provides a protocol for and assessment of the rate of permeation and residence time of lithium ions provided in the formulations, which can be adapted for an in vivo experiment to assess HF neogenesis.
  • Hydrogel "HC730”; also referred to Hydrogel, "BV-001-003A”; also
  • lithium gluconate referred to herein as “lithium chloride”
  • CarboPol 980 CarboPol 980
  • mice Twenty-four (24) C57/BL 6 mice were enrolled in each group. There were 4 groups in total, with 2 groups enrolled for DA and 2 for FTE treatment. Dosing for the DA groups was started at day 1 , immediately after debriding the mouse skin with dermabrasion, and continued once daily to day 4 (i.e., single dose administered at Oh, 24 h, 48 h, 72 h). A thick scab forms on the wound after day 1 , and thus the formulations are delivered prior to scabbing of the wounds.
  • Dosing for the FTE groups was started at approximately day 10-15 post-FTE wound, when the scab detached from the wound (this is referred to herein as "Day 1"), and continued to day 4 post-scab detachment (i.e., single dose administered at Oh, 24 h, 48 h, 72 h post scab detachment).

Abstract

The invention relates to intermittent lithium treatments, or a single pulse lithium treatment for scar revision and wound healing in human subjects. Uses of compositions containing compounds that liberate lithium ions are described, including adjuvants and devices for administration. The intermittent treatment protocol involves multiple courses of lithium treatment interrupted by lithium treatment "holidays". For the single pulse protocol, a dose of lithium is administered over a short period of time. The lithium treatment(s) can be used in combination with other treatments for scar revision, wound healing, and hair follicle neogenesis. Such combination treatments may involve mechanical or physical treatments that modulate scar revision or wound healing, or that cause integumental perturbation, and/or chemical treatments that modulate wound healing, scar revision, or hair follicle neogenesis or that cause integumental perturbation or immune stimulation for the treatment of wounds or revision of scars.

Description

INTERMITTENT AND PULSE LITHIUM TREATMENTS
FOR SCAR REVISION AND WOUND HEALING
[0001] This application claims priority to U.S. provisional application Serial No.
61/241,857, filed September 1 1, 2009, U.S. provisional application Serial No. 61/330,250, filed April 30, 2010, U.S. provisional application Serial No. 61/356,534, filed June 18, 2010, and U.S. provisional application Serial No. 61/356,531, filed June 18, 2010, the entire contents of each of which is incorporated herein by reference in its entirety.
1. INTRODUCTION
[0002] The invention relates to intermittent lithium treatments, or a single pulse lithium treatment for scar revision and wound healing in human subjects. Uses of compositions containing compounds that liberate lithium ions are described, including adjuvants and devices for administration. The intermittent treatment protocol involves multiple courses of lithium treatment interrupted by lithium treatment "holidays." For the single pulse protocol, a dose of lithium is administered over a short period of time. The lithium treatment(s) can be used in combination with other treatments for scar revision, wound healing, and hair follicle neogenesis. Such combination treatments may involve mechanical or physical treatments that modulate scar revision or wound healing, or that cause integumental perturbation {e.g. such as laser, surgical treatments, including skin graft or full thickness wounding, or dermabrasion, dermatome planing, etc.); and/or chemical treatments that modulate wound healing, scar revision, or hair follicle neogenesis or that cause integumental perturbation or immune stimulation {e.g., such as adjuvants, antigens, cytokines, growth factors, etc.) for the treatment of wounds or revision of scars. The combination treatment(s) may be administered concurrently with, or during the "holidays" between, cycles of intermittent lithium treatments; or concurrently with, or before and/or after the pulse lithium treatment.
2. BACKGROUND
2.1 WOUND HEALING AND SCAR FORMATION
2.1.1 PHASES OF WOUND HEALING
[0003] Wound healing, or wound repair, is an intricate process in which the skin (or some other organ) repairs itself after injury. In normal skin, the epidermis (outermost layer) and dermis (inner or deeper layer) exist in a steady-state equilibrium, forming a protective barrier against the external environment. Once the protective barrier is broken, the physiologic process of wound healing is immediately set in motion. The classic model of wound healing is divided into three or four sequential, yet overlapping, phases: (1) hemostasis (not considered a phase by some authors); (2) inflammation; (3) proliferation; and (4) maturation and remodeling.
2.1.1.1 HEMOSTASTS
[0004] When tissue is wounded, blood platelets (thrombocytes) aggregate at the injury site to form a fibrin clot. This clot acts to control active bleeding (hemostasis). Fibrin and fibronectin crosslink to form a plug that traps proteins and particles and prevent further blood loss. This fibrin-fibronectin plug, also called the extracellular matrix, is also the main structural support for the wound until collagen is deposited. Migratory cells use this plug as a matrix to crawl across, and platelets adhere to it and secrete factors. The clot is eventually lysed and replaced with granulation tissue and then later with collagen.
2.1.1.2 TNFT ; A MM A TORY PHASE
[0005] The contact of blood with collagen during wounding triggers platelets to begin secreting inflammatory factors, extracellular matrix proteins, cytokines, and growth factors. The proinflammatory factors released by platelets, like serotonin, bradykinin, prostaglandins, prostacyclins, thromboxane, and histamine, increase cell proliferation and migration to the area, cause blood vessels to initially constrict to prevent further blood loss, and then cause blood vessels to become dilated and porous. Vasoconstriction lasts five to ten minutes and is followed by vasodilation, a widening of blood vessels, which peaks at about 20 minutes post- wounding. The main factor involved in vasodilation is histamine, which also causes blood vessels to become porous, allowing the tissue to become edematous. Increased porosity of blood vessels facilitates the entry of inflammatory cells into the wound site from the bloodstream.
[0006] Within an hour of wounding, polymorphonuclear neutrophils (PMNs) arrive at the wound site and become the predominant cells in the wound for the first two days after the injury occurs, with especially high numbers on the second day. They are attracted to the site by fibronectin, growth factors, and substances such as kinins. Neutrophils phagocytose debris and bacteria, kill bacteria by releasing free radicals, and cleanse the wound by secreting proteases that break down damaged tissue. Neutrophils usually undergo apoptosis once they have completed their tasks and are engulfed and degraded by macrophages. Other leukocytes to enter the wound area include helper T cells, which secrete cytokines to cause more T cells to divide and to increase inflammation and enhance vasodilation and vessel permeability. T cells also increase the activity of macrophages. Gamma-delta T cells are enriched in epidermis and become activated upon wounding, secreting growth factors that induce keratinocyte proliferation (Havran and Jameson, 2010).
[0007] Macrophages are essential to wound healing. They replace PMNs as the predominant cells in the wound by two days after injury. Attracted to the wound site by growth factors released by platelets and other cells, monocytes from the bloodstream enter the area through blood vessel walls. Numbers of monocytes in the wound peak one to one and a half days after the injury occurs. Once they are in the wound site, monocytes mature into macrophages. The main role of macrophages is to phagocytose bacteria and damaged tissue, and they also debride damaged tissue by releasing proteases. Macrophages also produce factors that induce and speed angiogenesis; secrete a number of factors such as growth factors and other cytokines, especially during the third and fourth post-wounding days, which attract cells involved in the proliferative stage of healing; and stimulate cells that re-epithelialize the wound, create granulation tissue, and lay down a new extracellular matrix.
[0008] As inflammation dies down, fewer inflammatory factors are secreted, existing ones are broken down, and numbers of neutrophils and macrophages are reduced at the wound site. Because inflammation plays a role in fighting infection, clearing debris and inducing the proliferative phase, it is a necessary part of healing. However, inflammation can lead to tissue damage if it lasts too long. Thus the reduction of inflammation is frequently a goal in therapeutic settings. Inflammation lasts as long as there is debris in the wound. Thus the presence of dirt or other objects can extend the inflammatory phase for too long, leading to a chronic wound.
2.1.1.3 PROT FERATTVE PHASE
[0009] About two or three days after wounding, fibroblasts begin to enter the wound site, marking the onset of the proliferative phase (also called the reconstruction phase) even before the inflammatory phase has ended. The proliferative phase is characterized by the overlapping steps of angiogenesis, collagen deposition, granulation tissue formation, epithelialization, and wound contraction.
Angiogenesis
[0010] In angiogenesis (neovascularization), new blood vessels are formed by vascular endothelial cells. Because the activity of fibroblasts and epithelial cells requires oxygen and nutrients, angiogenesis is also imperative for other stages in wound healing. Endothelial growth and proliferation is stimulated by the presence of lactic acid in the wound and by hypoxia. Endothelial stem cells from uninjured blood vessels, attracted to the wound by fibronectin found on the fibrin scab and chemotactically by angiogenic factors released by other cells, migrate through the extracellular matrix (ECM) into the wound site to establish new blood vessels. The tissue in which angiogenesis has occurred typically looks red (is erythematous) due to the presence of capillaries. When macrophages and other growth factor-producing cells are no longer in a hypoxic, lactic acid- filled environment, they stop producing angiogenic factors. Thus, when tissue is adequately perfused, migration and proliferation of endothelial cells is reduced. Eventually blood vessels that are no longer needed die by apoptosis.
Granulation tissue formation and fibroplasia
[0011] Granulation tissue functions as rudimentary tissue, and begins to appear in the wound during the inflammatory phase, two to five days post wounding, and continues growing until the wound bed is covered. Granulation tissue consists of new blood vessels, fibroblasts, inflammatory cells, endothelial cells, myofibroblasts, and the components of a new, provisional ECM. The provisional ECM is different in composition from the ECM in normal tissue. Its components - fibronectin, hyaluronan, collagen, glycosaminoglycans, elastin, glycoproteins and proteoglycans - originate from fibroblasts. Later this provisional matrix is replaced with an ECM that more closely resembles that found in non-injured tissue.
[0012] Fibroblasts begin entering the wound site two to five days after wounding and their numbers peak at one to two weeks post-wounding. In the first two or three days after injury, fibroblasts mainly proliferate and migrate, while later, they are the main cells that lay down the collagen matrix in the wound site. Fibroblasts from normal tissue migrate into the wound area from its margins. Initially fibroblasts use the fibrin scab formed in the inflammatory phase to migrate, adhering to fibronectin. Fibroblasts then deposit ground substance into the wound bed, and later collagen, which they can adhere to for migration. Growth factors (e.g., PDGF, TGF-β) and fibronectin encourage proliferation, migration to the wound bed, and production of ECM molecules by fibroblasts. Hypoxia also contributes to fibroblast proliferation and secretion of growth factors (e.g. , growth factors that attract epithelial cells to the wound site), though too little oxygen inhibits proliferation and deposition of ECM components, which may lead to excessive, fibrotic scarring. Fibroplasia ends two to four weeks after wounding.
[0013] One of fibroblasts' most important duties is the production of collagen.
Fibroblasts begin secreting appreciable collagen by the second or third post-wounding day, and its deposition peaks at one to three weeks. Collagen deposition is important because it increases the strength of the wound; before it is laid down, the only thing holding the wound closed is the fibrin- fibronectin clot, which does not provide much resistance to traumatic injury. Also, cells involved in inflammation, angiogenesis, and connective tissue
construction attach to, grow and differentiate on the collagen matrix laid down by fibroblasts. Collagen production continues rapidly for two to four weeks, after which its destruction by collagenases matches its production and so its deposition levels off; this homeostasis signals the onset of the maturation phase. Granulation gradually ceases and fibroblasts decrease in number in the wound once their work is done. At the end of the granulation phase, fibroblasts begin to commit apoptosis, converting granulation tissue from an environment rich in cells to one that consists mainly of collagen.
Re-epithelialization
[0014] Formation of granulation tissue in an open wound allows re-epithelialization, in which epithelial cells proliferate and migrate across the new tissue to form a barrier between the wound and the environment. Basal keratinocytes from the wound edges, and appendages such as hair follicles, sweat glands and sebaceous (oil) glands, are the main cells responsible for re-epithelialization. They are believed to advance in a sheet across the wound site and proliferate at its edges, ceasing movement when they meet in the middle.
[0015] Keratinocytes migrate without first proliferating. Migration of keratinocytes over the wound site is stimulated by lack of contact inhibition and by chemicals such as nitric oxide. Migration can begin as early as a few hours after wounding, although the time of onset of migration is variable. Epithelial cells require viable tissue to migrate across, so if the wound is deep it must first be filled with granulation tissue. Cells on the wound margins proliferate on the second and third day post-wounding in order to provide more cells for migration.
[0016] If the basement membrane is not breached, epithelial cells are replaced within three days by division and upward migration of cells in the stratum basale in the same fashion that occurs in uninjured skin. However, if the basement membrane is ruined at the wound site, re-epithelialization must occur from the wound margins and from skin appendages such as hair follicles and sweat and oil glands that enter the dermis. If the wound is very deep, skin appendages may also be ruined and their migration is believed to only occur from wound edges.
[0017] Epithelial cells climb over one another in order to migrate. This growing sheet of epithelial cells is often called the epithelial tongue. Basal and suprabasal cells become mobilized and both migrate and get "pushed" into the wound site (Usui et al, 2005). The more quickly this movement occurs, the less of a scar there will be.
[0018] Fibrin, collagen, and fibronectin in the ECM may further signal cells to divide and migrate. Like fibroblasts, migrating keratinocytes use the fibronectin cross-linked with fibrin that was deposited in inflammation as an attachment site to crawl across. As keratinocytes migrate, they move over granulation tissue but underneath the scab (if one was formed), separating it from the underlying tissue. Epithelial cells have the ability to phagocytose debris such as dead tissue and bacterial matter that would otherwise obstruct their path. Because they must dissolve any scab that forms, keratinocyte migration is best enhanced by a moist environment, since a dry one leads to formation of a bigger, tougher scab. To make their way along the tissue, keratinocytes must dissolve the clot, debris, and parts of the ECM in order to get through. They secrete plasminogen activator, which activates plasminogen, turning it into plasmin to dissolve the scab. Cells can only migrate over living tissue, so they must secrete collagenases and proteases to dissolve damaged parts of the ECM in their way, particularly at the front of the migrating sheet. Keratinocytes also dissolve the basement membrane, using instead the new ECM laid down by fibroblasts to crawl across.
[0019] As keratinocytes continue migrating, new epithelial cells form at the wound edges to replace them and to provide more cells for the advancing sheet. Proliferation behind migrating keratinocytes normally begins a few days after wounding and occurs at a rate that is 17 times higher in this stage of epithelialization than in normal tissues. Until the entire wound area is resurfaced, the only epithelial cells believed to proliferate are at the wound edges. Growth factors cause cells to proliferate at the wound edges. Keratinocytes themselves also produce and secrete factors, including growth factors and basement membrane proteins, which aid both in re-epithelialization and in other phases of healing. Growth factors are also important for the innate immune defense of skin wounds by stimulation of the production of antimicrobial peptides in keratinocytes.
[0020] Keratinocytes continue migrating across the wound bed until cells from either side meet in the middle, at which point contact inhibition causes them to stop migrating. When they have finished migrating, the keratinocytes secrete proteins that form the new basement membrane and become anchored once again to the basement membrane. Basal cells begin to divide and differentiate in the same manner as they do in normal skin to re-establish the strata found in re-epithelialized skin.
Contraction [0021] Around a week after wounding, fibroblasts differentiate into myofibroblasts and the wound begins to contract. In full thickness wounds, contraction peaks at 5 to 15 days post wounding. Contraction can last for several weeks and continues even after the wound is completely re-epithelialized. If contraction continues for too long, it can lead to
disfigurement and loss of function.
[0022] Contraction reduces the size of the wound. A large wound can become 40%-80% smaller after contraction. Wounds can contract at a speed of up to 0.75 mm per day, depending on how loose the tissue in the wounded area is. Contraction usually occurs along an "axis of contraction," which allows for greater organization and alignment of cells with collagen. At first, contraction occurs without myofibroblast involvement. Later, fibroblasts, stimulated by growth factors, differentiate into myofibroblasts. Myofibroblasts, which are similar to smooth muscle cells, are responsible for contraction. Attracted by fibronectin and growth factors, they move along fibronectin linked to fibrin in the provisional ECM in order to reach the wound edges, where they form connections to the ECM and to each other. Also, at an adhesion called the fibronexus, actin in the myofibroblast is linked across the cell membrane to molecules in the extracellular matrix like fibronectin and collagen.
Myofibroblasts have many such adhesions, which allow them to pull the ECM when they contract, reducing the wound size. In this part of contraction, closure occurs more quickly than in the first, myofibroblast-independent part. As the actin in myofibroblasts contracts, the wound edges are pulled together. Fibroblasts lay down collagen to reinforce the wound as myofibroblasts contract. The contraction stage ends as myofibroblasts stop contracting and commit apoptosis. The breakdown of the provisional matrix leads to a decrease in hyaluronic acid and an increase in chondroitin sulfate, which gradually triggers fibroblasts to stop migrating and proliferating. These events signal the onset of the maturation stage of wound healing.
2.1.1.4 MATTJRATTON AND REMODELING PHASE
[0023] When the levels of collagen production and degradation equalize, the maturation and remodeling phase of tissue repair is said to have begun. The maturation phase can last for a year or longer, depending on the size of the wound and whether it was initially closed or left open. During maturation, type III collagen, which is prevalent during proliferation, is gradually degraded and the stronger type I collagen is laid down in its place. Originally disorganized collagen fibers are rearranged, cross-linked, and aligned along tension lines. As the phase progresses, the tensile strength of the wound increases, with the strength approaching 50% that of normal tissue by three months after injury and ultimately becoming as much as 80% as strong as normal tissue. Since metabolic activity at the wound site is reduced, the scar loses its red appearance as blood vessels that are no longer needed are removed by apoptosis.
[0024] For reviews on wound healing, see Lorenz & Longaker, 2003, Chapter 7 in Surgery: Basic Science and Clinical Evidence, pp. 77-88.
2.1.2 HEALING BY PRIMARY. SECONDARY. AND
TERTIARY INTENTION
[0025] The phases of wound healing normally progress in a predictable, timely manner; if they do not, healing may progress inappropriately to either a chronic wound, such as a venous ulcer, or pathological scarring such as a keloid scar and other forms of scarring discussed in Section 2.1.3.
2.1.2.1 PRIMARY INTENTION
[0026] When wound edges are directly next to one another, and there is little tissue loss, wounds may heal by primary intention. Such wounds may be referred to as "closed wounds." These wounds are usually surgically closed in layers along tissue planes by a physician. In primary intention, a linear scar results at the intersection of the approximated tissues.
Scarring is often minimal, but can be variable depending on the size and location of the wound, the tension on tissue and other factors. Most surgical wounds are sutured closed, so they heal by primary intention. In primary intention, wound closure is usually performed with sutures, staples, or an adhesive. Other examples of wounds that heal by primary intention are well repaired lacerations, well reduced bone fractures, and wounds that heal after flap surgery (the edges of which tend to appear scarred).
2.1.2.2 SECONDARY INTENTION
[0027] Healing by secondary intention occurs when the extent of skin separation or skin tissue removed is too great for the edges of the wound to be placed in proximity (e.g., by bandages or sutures). Such wounds may be referred to as "open wounds." For example, wounds formed by blast injury, shrapnel (e.g., from improvised explosive devices ("IEDs")), blunt trauma, dental wounds (e.g., gingivectomy, gingivoplasty, tooth extraction sockets), poorly reduced fractures, and third degree burns heal by secondary intention. Healing by secondary intention follows the same basic steps as wounds that heal by primary intention, i.e., inflammation, proliferation, and remodeling, but each sequence may take much longer, especially the proliferative phase. Since the wound edges are not approximated, epithelial cell migration much occur over a longer distance, and before epiboly can occur, a provisional matrix must be present. Thus, in healing by secondary intention, there is much more granulation tissue formation and contraction, which carries a greater risk of scarring. The secondary intention healing process can be slow due to the presence of drainage from infection, and the surgeon may pack the wound with a gauze or use a drainage system.
Wound care must be performed daily to encourage wound debris removal to allow for granulation tissue formation. Depending on the size and location of the wound, placement of a partial or full-thickness skin graft may be considered if no infection is present and the area is of sufficient size that healing may not be complete for at least 2 or 3 weeks. Infection and inflammation of the wound can dysregulate repair and transform the wound into a clinically non-healing wound.
2.1.2.3 TERTTARY TNTENTTON
[0028] In wound healing by tertiary intention (delayed primary closure), the wound is initially cleaned, debrided, and observed, and typically 4 or 5 days elapse before closure. The wound is purposely left open. Examples include healing of wounds by use of tissue grafts.
2.1.3 SCAR FORMATION
[0029] A major component of wound healing in humans is scar formation. A scar ("cicatrix"; plural, "cicatrices") is an area of fibrous tissue that forms as part of the healing process to replace normal skin after injury. A hallmark of scars is altered extracellular matrix, notably a reduction of elastin fibers (De Vries et al, 1995). Scars result from damage to the dermis, and with the exception of very minor lesions, every wound results in some degree of scarring. Scars generally form in proportion to the extent of damage.
[0030] Adult mammalian skin repairs wounds in a process that results in scars, whereas fetal skin heals by a regenerative process that results in complete restoration of normal skin. Scarring is the consequence of the adult wound repair mechanism that focuses on hemostasis and rapid epidermal closure (to avoid infection) in lieu of cosmetic or functional outcomes - regeneration of the original tissue architecture is sacrificed. For example, during wound repair, adult skin undergoes epithelialization without adnexal structures such as hair follicles and sweat glands. This leads to alopecia, desiccation, and thermal dysregulation of the affected tissue. See, e.g., Fathke et al, 2006, BMC Cell Biol. 7:4. Scar tissue is also usually of inferior functional quality. A scar is a collagen-rich, elastin-poor dermal matrix with a simple stratified epithelial covering. Deposition of such a collagen-rich matrix in the neo- dermis is prone to contracture, loss in elasticity, and reduced tensile strength. Scars in the skin are also less resistant to ultraviolet radiation. For example, scars from skin transplants are typically dysfunctional, discolored, etc. Skin flaps and grafts are common methods of achieving rapid closure of large defect wounds. Not only do these methods tend to result in scarring at the donor site, but the sites of apposition of flap or graft edges to the wound edges can also result in linear scars.
[0031] Scars form differently based on the location of the injury on the body and the age of the person who was injured.
[0032] Recent research has implicated osteopontin in scarring.
http://jem.rupress.Org/cgi/content/abstract/205/l/43 "Molecular mechanisms linking wound inflammation and fibrosis: knockdown of osteopontin leads to rapid repair and reduced scarring"; BBC NEWS - Health - Gel "to speed up wound healing." Transforming Growth Factors (TGFs) are also believed to play a critical role in scar development, and current research is investigating the manipulation of these TGFs for drug development to prevent scarring from the emergency adult wound healing process. Another recent study implicated the protein Ribosomal s6 kinase (RSK) in the formation of scar tissue.
[0033] Scarring is likely to be extensive when wounds heal by secondary intention. In secondary intention, the wound heals by granulation, wherein epithelial cells grow over the wound from all sides of the normal skin, which results in a shiny layer of epithelial cells and fibrous tissue that is rich in collagen but does not contain underlying structures ("adnexal structures," including hair follicles). In addition to lacking adnexal structures, the scar also lacks the suppleness of normal skin. This type of scar can result in contractures when it occurs over the mouth or eyes or on the skin around joints, and can be disfiguring.
[0034] In addition to scars that form by secondary intention, there are numerous other types of scars that we distinguish, including atrophic scars, hypertrophic scars, keloid scars, hypopigmented scars, hyperpigmented scars, depressed scars (which, compared with atrophic scars, also have contour abnormality, while "atrophic" scars are implied to have only thinning), ice-pick scars (a type of depressed scar), spread scars (scars that widen due to tension over a time period, and which may become somewhat atrophic in the center), fineline scars, widespread (or stretched) scars, scar contractures, and other "intermediate" types of scars that are difficult to categorize. For more information on types of scars, see Bayat et al, 2003, BMJ. 326:88-92, the contents of which are incorporated herein by reference in its entirety.
2.1.3.1 ATROPHIC SCARS
[0035] Atrophic scars are characteristic of scars that are too thin, and typically form below the plane of the skin. Atrophic scars have a pitted appearance, and are caused when underlying structures supporting the skin, such as fat or muscle, are lost. This type of scarring is commonly associated with acne, but can also be caused by chickenpox, surgery or an accident. Acne scars and striae (scars from stretched skin) are exemplary of atrophic scars. Striae are caused when the skin is stretched rapidly (for instance during pregnancy, significant weight gain, or adolescent growth spurts), or when skin is put under tension during the healing process, usually near joints. This type of scar usually improves in appearance after a few years.
2.1.3.2 HYPERTROPHIC SCARS
[0036] Characteristic of scars that are too thick, and typically emerge above the plane of the skin. These often form from surgical incisions. Hypertrophic scars take the form of a red raised lump on the skin, but do not grow beyond the boundaries of the original wound, and they often improve in appearance after a few years.
2.1.3.3 KELOID SCARS
[0037] Like hypertrophic scars, keloid scars are raised above the surface of the skin, but by definition they grow beyond the boundaries of the original wound. Keloids can be viewed as a tumorous (although benign) growth. Keloid scars can occur on anyone, but they are most common in dark-skinned people. Keloid scars can be caused by surgery, an accident, by acne or, sometimes, from body piercings. In some people, keloid scars form spontaneously. Although they are primarily a cosmetic problem, keloid scars can be itchy or painful in some individuals. They tend to be most common on the shoulders, chest and earlobes.
2.2 HUMAN SKIN APPENDAGES AND THEIR ROLE IN WOUND HEALING
[0038] Human skin appendages, also referred to as "adnexal" structures, include hair and hair follicles, sebaceous glands (which secrete sebum onto hair follicle to oil the hair), eccrine and apocrine sweat glands, and nails. The skin of an adult human is essentially covered with hair follicles and contains approximately five million hair follicles, with approximately 100,000 - 150,000 covering the scalp. Only a minority of human hair follicles produce a hair fiber that can be appreciated visibly (a "terminal hair") and these specialized follicles are localized on specific regions of skin. The portions of human skin that lack visible hair contain, for the most part, hair follicles that produce "vellus hair" while certain other hair follicles may contain or produce no hair (see Figure 1).
[0039] Hair follicles, and particularly human hair follicles, are crypt structures comprised of distinct components, each comprised of several different specialized cells (see Figures 2 and 3). In addition to the cells and structures associated with making and anchoring the hair shaft, the vast majority of hair follicles contain units called sebaceous glands (which produce sebum), and a minority (follicles located in specialized areas of the skin) also contain apocrine glands (which produce sweat used primarily for olfactory cues). (Eccrine sweat glands are independent of hair follicles and produce sweat that functions in
thermoregulation). In addition to the hair shaft, the structures of the hair follicle include the follicular papilla (FP) and the germinative epithelium (GE) (together, the bulb). The FP is comprised of mesenchymal cells (and connective tissue). The other cells of the follicle are epithelial and include at least 8 cellular lineages including the outer root sheath (ORS), the companion layer (CL), the internal root sheath Henle's layer (He), internal root sheath Huxley's layer (Hu), the cuticle of the internal root sheath (Csth), the cuticle of the hair shaft (Csft), the cortex of the hair shaft, and the medulla of the shaft (Med). (Stenn & Paus, 2001, Physiol. Revs. 81 : 449-494.) (See also Figures 2-4.)
[0040] The importance of hair follicles to skin biology is now known not to be restricted to production of hair shafts and sebum. Rather, the hair follicle and other adnexal structures appear to be regenerative organs that play a central role in normal skin homeostasis and in response to wounding. Several lines of evidence suggest that hair follicles and other skin adnexal structures have the potential to provide skin with stem cells and other elements that are important for skin regeneration.
[0041] In non-human mammals, when the depth of a wound in mammalian skin approaches approximately one-half of the total skin thickness, the resultant repair process leads to a scar (Dunkin et al, 2007). This is in contrast to similar fetal wounds which heal by regeneration, a process that results in complete restoration of normal skin function (Buchanan et al, 2009). In several species (Buchanan et al., 2009), including primates (Lorenz et al, 1993), a prominent feature of complete skin regeneration in fetuses is the formation of hair follicles, a property that is lost in the perinatal period. The mechanism for the switch from regeneration to repair with age in mammals is not fully known, but involves cytokines and an immune response that promotes rapid epidermal closure, dermal fibroplasia and increased collagen deposition. During this reparative process, hair follicles do not form, thus contributing to the lack of adnexal structures in a scar. The abnormal structure of scars contributes to the their associated morbidity. Due to lack of eccrine glands, there is poor thermoregulation. Increased fibroplasia and collagen can lead to contracture and loss of mobility in affected areas of the body.
[0042] Several lines of evidence indicate that formation of hair follicles in a healing wound can reduce or even prevent scarring. In adult rodents, burn wounds heal faster if hair follicles are actively growing at the time of wounding (Zawacki & Jones, 1967). Direct evidence for the anti-scarring properties of hair follicles comes from full thickness excisional wounds in adult mice (such wounds normally form a scar) that heal with near-complete skin regeneration when dissociated hair follicle cells are implanted at the time of wounding (Prouty et al, 1996; Prouty et al, 1997). The regenerated skin contains hair follicles and normalized collagen and extracellular matrix. When hair follicles of mouse dorsal and tail skin (areas rich in hair follicles) are experimentally removed, such seen in EDAR mutant mice (Langton et al, 2008) and in photoepilated mouse skin (Huh et al, 2006), alterations in wound healing such as delayed re-epithelialization and dermal changes are observed.
[0043] It is also well known from surgical and medical experience that after comparable injury in humans, areas rich in hair follicles heal more rapidly and with less scarring compared to those relatively devoid of adnexal structures (Martinot et al, 1994; Jahoda & Reynolds, 2001). This is consistent with the hair follicle (both in mouse and human) being rich in Keratin 1-15 (K 1-15; K15)-positive stem cells and being a known central repository of skin regenerative capacity (Morris et al, 2004; Roh & Lyle, 2006). There is also evidence that connective tissue cells of the follicle play a role in dermal repair. For example, it has been observed that hair follicle keratinocytes contribute to epidermal closure, and hair follicle dermal sheath fibroblasts play central roles in dermal healing. See, Ito et al, 2005; Jahoda & Reynolds, 2001; Stenn & Paus, 2001, Physiol. Revs. 81 :449-494.
[0044] These data suggest that hair follicles participate in the regeneration of normal dermis and epidermis in healing wounds, thereby preventing or possibly reversing scarring or contributing to the formation of scars that are not disfiguring or dysfunctional.
[0045] The only skin tissue (aside from scar tissue) that normally lacks hair follicles is the glabrous skin on palmar and plantar aspects of hands and feet, respectively, and the lips and labia. Although human glabrous skin lacks hair follicles, it is rich in eccrine sweat glands. Wound healing studies in pig (Miller et al, 1998) have shown that sweat glands, by themselves, are capable of regenerating epidermis, which likely accounts for lack of scarring in glabrous skin wounds that spare the base of sweat glands. However, as with deep wounds to hairy skin, palmar (Barret at al, 2000) and plantar (Barret & Herndon, 2004) wounds can result in scarring, a sequelae related to increased depth of wound and delayed wound healing.
[0046] Until recently, it was believed that the only hair follicles involved in wound healing migrated into the wound from the surrounding epithelium. The general belief is that hair follicle formation occurs but once in a lifetime (in utero), so that a mammal, and particularly a human, is born with a fixed number of follicles, which does not normally increase thereafter. Despite early suggestions of the regenerative capacity of the adult mammalian skin to recreate the embryonic follicle, follicle neogenesis was never proven because of an incomplete understanding of the fundamental biology of the follicle and the lack of tools needed to demonstrate the occurrence of hair follicle neogenesis (see, Argyris et al, 1959, Dev. Biol. 1 : 269-80; Miller, 1973, J. Invest. Dermatol. 58: 1-9; and Kligman, 1959, Ann NY Acad Sci 83: 507-51 1); for example, hair follicles were not understood to be capable of neogenesis and bulge cells were not proven to be a source of stem cells for hair follicle neogenesis. Well-designed studies in the 1950s using full thickness skin excisions generated support both for and against hair follicle neogenesis. Following a 2.5 cm diameter full thickness skin excision on the rabbit dorsum, and placement of a barrier device to retard contracture, small neogenic hair follicles were observed histologically in the healed wound (Breedis, 1954). However, another experiment, using a very similar design in rabbits and a histological endpoint, was carried out specifically to address the issue of contracture and concluded that, despite the barrier device, the wound margins contracted and "dragged" preexisting hair follicles into the center of the wound (Straile, 1959). In human volunteers, following dermabrasion of the cheek to remove of 2 mm of skin (to depth of mid-dermis), neogenic hair follicles were identified histologically (Kligman & Strauss, 1956). However, without molecular markers to define neogenic hair follicles, these studies were only speculative of hair follicle neogenesis.
[0047] More recently, based on studies primarily in rodents, it has been proposed that wound healing in animals is associated with hair follicle neogenesis or reformation of hair follicles from dissociated parts. See, Stenn & Paus, 2001, Physiol. Revs. 81 :449-494. Prouty and colleagues showed that a full thickness excisional wound in mice, which normally forms a scar, heals with near-complete skin regeneration when dissociated hair follicles are implanted at the time of wounding. The regenerated skin contains hair follicles and normalized collagen and extracellular matrix. See Prouty et al, 1997, Lab. Invest. 76: 179- 189. Nielsen & Sun (U.S. Patent No. 5,767, 152) showed that application of the hair growth enhancer n-butyl cyanoacrylate to incisional and excisional wounds in mice stimulated hair follicle growth in the wound site in what would normally be a scar devoid of skin appendages, suggesting that hair follicle growth in wounds accelerates the process of wound remodeling. A series of murine experiments definitively showed that hair follicle-derived epithelial stem cell progenitors migrate out of the follicle and contribute to the re- epithelialization of injured skin {see, Morris et al, 2004, Nature Biotechnology 22:411-417; Ito et al, 2004, Differentiation 72:548-57; and Ito et al, 2005, Nature Medicine 11 : 1351- 1354).
[0048] Some of the molecular signals involved in the regulation of hair follicle regeneration have been investigated in murine systems. Based on experiments performed on murine cells in culture, Morgan implicated a role for Wnt signaling to the dermal papillae (DP) {e.g., resulting from β-catenin accumulation in the epidermis) in the coordination of hair follicle development (Kishimoto et al, 2000, Genes & Dev. 14: 1 181-1 185; see also U.S. Patent Application Publication No. US 2006/0134074, which postulates a role for Wnt produced in three-dimensional tissue cultures in vitro in promoting hair growth). Previous and subsequent reports were conflicting. One group, consistent with Morgan's theory, showed that continuous expression of stabilized β-catenin in the epidermis of transgenic mice resulted in hair follicle morphogenesis, but unfortunately led to the development of hair follicle tumors (Gat et al, 1998, Cell 95:605-614). Another group found that continuous activation of a hyperstabilized and highly amplified (12-21 copies) form of β-catenin in mice also resulted in hair follicle tumors, but that transient activation of this hyperstabilized and highly amplified form of β-catenin led to normal hair follicle patterning, although no hair growth was observed in mice harboring a single copy of the hyperstabilized form of β-catenin (Lo Celso et al, 2004, Development 131 : 1787-1799). These results are in contrast to the results of another study, in which a reduction of β-catenin signaling was found to cause hair follicles to develop into cysts in postnatal mice (Niemann et al, 2002, Development. 129:95- 109). Moreover, there are examples in which increased β-catenin was found to decrease hair growth and hair follicle formation. For example, one group showed that forced expression of β-catenin dependent Wnt 3a in murine skin decreased hair growth (Millar et al, 1999, Dev. Biol. 207: 133-149). In another study, continuous expression of β-catenin during
embryogenesis was found to induce placodes, but they became aborted and did not produce a hair shaft (Narhi et al.,. 2008, Development. 135: 1019-1028). In yet another study, increased β-catenin expression during embryogenesis was found to alter cell fate toward formation of hair follicles, but continued presence of β-catenin prevented further development of the hair follicles (Zhang et al, 2008, Development. 135:2161-2172). Finally, overexpression of Lef- 1 (a transcription factor in Wnt pathway) was found to lead to abnormal hair follicle growth, as reported in Zhou et al, 1995, Genes Dev. 9:700-713.
[0049] Despite this confusion about a role of β-catenin in hair growth, Morgan speculated that exogenous Wnt could extend the hair cycle and promote hair growth, and further, that "lithium chloride or similar small ions" that inhibit the activity of glycogen synthase kinase- 3β (Θ8Κ-3β) may induce Wnt signal transduction (through the accumulation of β-catenin), and thus, may be used to promote hair growth (US Patent 6,924,141 ; US Patent 7, 175,842; and US Patent Application Publication 2008/0286261). Morgan implicated a role for lithium chloride or similar small ions in hair growth promotion despite the fact that lithium had been shown to arrest mitosis (Wolniak, 1987, Eur. J. Cell Biol. 44: 286-293; and Wang, 2008, World J. Gastroenterol. 14:3982-3989) and cause pathological hair loss when systematically administered (see, e.g., Mercke et al, 2000, Ann. Clin. Psych. 12:35-42).
[0050] Fathke' s experiments tested Morgan's theory. In animal studies designed to explore the role of Wnt in tissue patterning following wounding, Fathke showed that prolonged activation of β-catenin dependent Wnt signaling (using lithium chloride as suggested by Morgan - in the case of Fathke, by continuous topical administration for two weeks until wound closure) during wound healing in mice resulted in generation of rudiments of hair follicles but did not result in the formation of hair follicles or growth of more hair (Fathke et al, 2006, BMC Cell Biol. 7:4). Fathke also noted that the epithelium that typically forms over the wound during wound healing was covered with cysts. Id. Fathke then turned the focus of their investigation to the -catenm-independent Wnts expressed in the skin - which are not activated by lithium chloride. Unfortunately, the prolonged expression of the β-catenin independent Wnt yielded the same results - mature hair follicles were not generated in the mice and large epithelial cysts formed in the wounds. Although Fathke interpreted their data as evidence for the restoration of tissue patterning in the adult mammalian wound epithelium - a feature not normally seen in adult cutaneous wound healing - they provided neither evidence of hair follicle neogenesis, nor the concomitant hair growth and enhanced wound healing that would be expected in connection with it.
[0051] The most compelling evidence for hair follicle neogenesis during wound healing to date comes from studies in mice by Cotsarelis and coworkers. As noted by Fathke, cutaneous repair in adult mammals following a full thickness wound is understood to result in scar tissue and the loss of the regenerative capability of the hair follicle. However, Cotsarelis showed, in mice, that following wound closure of large healed wounds created by full thickness excision (FTE) (1 cm2 square wounds), new hairs are formed at the center of the wound (Ito et al, 2007, Nature 447:316-321). Physically disrupting the skin and existing follicles was found to lead to follicle neogenesis. Here again the role of Wnt was examined. In these experiments, inhibition of Wnt signaling at the time of wounding decreased the number of new hairs formed in the healed wound. While increasing Wnt expression increased the number of new hairs formed in the healed wound, Cotsarelis noted that "the hair follicle dermal papilla of these mice contain -25-38% more cells compared with normal. It is possible that the larger number of dermal papilla cells contributes to the greater number of hair follicles that form after wounding" (Ito et al. 2007, Nature 447:316-321, full Methods, available at www.nature.com/nature). This suggests that these Wnt-overexpressing mice were already predisposed to growing more hair. Thus, Cotsarelis' recognition that "... to date there has been no evidence that extracellular Wnt ligands can promote actual hair follicle neogenesis in adult skin" (Ito et al. 2007, Nature 447:316-321, at p. 319) remains correct. Moreover, in both of Cotsarelis' experiments - the blockade of Wnt or Wnt overexpression - wound closure was normal (Ito et al. 2007, Nature 447:316-321, at p. 319).
[0052] Other studies that investigated the link (without consideration of hair follicles) between the Wnt pathway and wound healing found that activation of β-catenin by continuous application of lithium chloride actually interfered with wound healing. In one study, continuous administration of lithium chloride to activate β-catenin resulted in proliferation of dermal cells and the formation of larger wounds, suggesting that excessive activation of the Wnt pathway results extension of the proliferative phase of wound healing, consistent with Fathke's observations of cyst formation in the continuous presence of lithium chloride. See, Cheon et al, 2006, FASEB J. 20:692-701. Similarly, in another study, activation of Wnt by continuous lithium chloride treatment prevented keratinocyte migration (ostensibly due to overactivation of the preceding proliferative phase), which is needed for the re-epithelialization phase of wound healing, resulting in the formation of chronic wounds. Stojadinovic et al., 2005, Am. J. Pathol. 167:59-69. The results in Stojadinovic are consistent with those seen in Wolf e/ al. (2000, J. Eur. Acad. Dermatol. Venereol. 14:97-99), in which lithium carbonate was found to induce a psoriatic {i.e., chronically wounded) state in human skin explants.
[0053] Thus, while Wnt signaling in hair follicle neogenesis and, in turn, wound healing has generated interest, its role in these processes is, at best, unclear. The evidence that β- catenin activation causes cyst formation, is abnormally activated in the epidermis of chronic wounds, and that its overactivation shifts the wound healing process from the re- epithelialization stage to the proliferative stage suggest that activating Wnt in the epidermis could also have a negative effect on wound healing. Continuous activation of Wnt, it seems, may actually be bad for wound healing.
[0054] It is also important to note that although the above-described studies implicate hair follicles, and perhaps hair follicle neogenesis, in wound healing, mice are a poor model for human wound healing and scar formation. Mice tend to heal most wounds rapidly, with little or no scarring. In humans, however, severe wounds and burns are usually associated with cutaneous repair that results in scar tissue, no hair follicles, and the loss of regenerative capability that hair follicles may provide {see, Fathke et al, 2006, BMC Cell Biol. 7:4). One reason for the difference in wound healing capability between humans and mouse may be that the biology of hair follicles in humans and mice differs in several significant respects. In the mouse, a thick fur coating is essential to healthy life (because hair plays roles in
thermoregulation and other functions). Mouse skin is covered with hair follicles that produce terminal hair (fur), whereas significant regions of human skin are covered with hair follicles that produce vellus hair, which is invisible. Mouse and other non-primate mammals have synchronous Follicle Cycles, whereas human follicles progress through the Follicle Cycle in an asynchronous fashion.
2.3 CURRENT TREATMENTS FOR WOUNDS AND
SCARS IN HUMAN SUBJECTS
2.3.1 WOUND TREATMENT
[0055] Acute treatment of wounds is generally focused on hemostasis and antimicrobial considerations. The treatment depends on the type, cause, and depth of the wound as well as whether other structures beyond the skin are involved. Treatment of acute lacerations involves examination, cleaning, and closing the wound. If the laceration occurred some time ago, it may be allowed to heal by secondary intention due to the high rate of infection associated with immediate closure. Minor wounds like bruises tend to heal on their own with skin discoloration that usually disappears within 1 -2 weeks. Abrasions usually require no active treatment except keeping the area clean with soap and water, although scarring may occur. Puncture wounds may be prone to infection depending on the depth of penetration. The entry of a puncture wound is usually left open to allow for bacteria or debris to be removed from the inside. [0056] Appropriate treatment of chronic wounds seeks to address the problems at the root of chronic wounds, including ischemia, bacterial load, and imbalance of proteases. Various methods exist to ameliorate the problems associated with wounds, including antibiotic and antibacterial use, debridement, irrigation, vacuum-assisted closure, warming, oxygenation, moist wound healing, removing mechanical stress, and adding cells or other materials to secrete or enhance levels of healing factors. However, many of the foregoing treatments are good temporary measures but are not concerned with, and thus not designed for, optimal wound recovery. The addition of exogenous hair follicle cells to acute wounds can result in regenerated skin, after healing, as long as the hair follicles formed. See, e.g., Prouty et al, 1997, Lab. Invest. 76: 179-189; and Prouty et al, 1996, Am. J. Pathol. 148: 1871-1885.
2.3.1.1 CLEANING AND CLOSURE OF WOUNDS AND PREVENTING AND TREATING TNFECTTON
[0057] For simple lacerations cleaning can be accomplished using a number of different solutions including tap water, sterile saline solution, or antiseptic solution. Infection rates may be lower with the use of tap water in regions were water quality is high. Evidence for the effectiveness of any cleaning of a simple wound is, however, limited. Lacerations must be thoroughly cleaned and the edges trimmed. If the wounds are fresh and less than 12 hours old, they can be closed with sutures or staples. Any wound which is more than 24 hours old should be suspected to be contaminated and not closed completely. Only the deeper tissues can be approximated and the skin should be left open. If closure of a wound is decided upon a number of techniques can be used, such as bandages, a Cyanoacrylate glue, staples, and sutures. Absorbable sutures have the benefit over non-absorbable sutures of not requiring removal. They are often preferred in children.
[0058] Most clean open wounds do not require any antibiotics unless the wound is contaminated or the bacterial cultures are positive. Excess use of antibiotics may lead to resistance and side effects. To lower the bacterial count in wounds, therapists may use topical antibiotics, which kill bacteria and can also help by keeping the wound environment moist, which is important for speeding the healing of chronic wounds. Some researchers have experimented with the use of tea tree oil, an antibacterial agent which also has antiinflammatory effects. Disinfectants are usually contraindicated because they damage tissues and delay wound contraction. Further, they are rendered ineffective by organic matter in wounds like blood and exudate and are thus not useful in open wounds. [0059] A greater amount of exudate and necrotic tissue in a wound increases likelihood of infection by serving as a medium for bacterial growth away from the host's defenses. Since bacteria thrive on dead tissue, wounds are often surgically debrided to remove the devitalized tissue. Debridement and drainage of wound fluid are an especially important part of the treatment for diabetic ulcers, which may create the need for amputation if infection gets out of control. Mechanical removal of bacteria and devitalized tissue is also the idea behind wound irrigation, which is accomplished using pulsed lavage.
[0060] Removing necrotic or devitalized tissue is also the aim of maggot therapy, the intentional introduction by a health care practitioner of live, disinfected maggots into nonhealing wounds. Maggots dissolve only necrotic, infected tissue; disinfect the wound by killing bacteria; and stimulate wound healing. Maggot therapy has been shown to accelerate debridement of necrotic wounds and reduce the bacterial load of the wound, leading to earlier healing, reduced wound odor and less pain. The combination and interactions of these actions make maggots an extremely potent tool in chronic wound care.
[0061] Negative pressure wound therapy (NPWT) is a treatment that improves ischemic tissues and removes wound fluid used by bacteria. This therapy, also known as vacuum- assisted closure, reduces swelling in tissues, which brings more blood and nutrients to the area, as does the negative pressure itself. The treatment also decompresses tissues and alters the shape of cells, causes them to express different mRNAs and to proliferate and produce ECM molecules.
2.3.1.2 TREATING TRAUMA AND PATNFUU WOUNDS
[0062] Persistent chronic pain associated with non-healing wounds is caused by tissue (nociceptive) or nerve (neuropathic) damage and is influenced by dressing changes and chronic inflammation. Chronic wounds take a long time to heal and patients can suffer from chronic wounds for many years. Chronic wound healing may be compromised by coexisting underlying conditions, such as venous valve backflow, peripheral vascular disease, uncontrolled edema and diabetes mellitus.
2.3.1.3 TREATING TSCHEMTA AND HYPOXTA
[0063] Blood vessels constrict in tissue that becomes cold and dilate in warm tissue, altering blood flow to the area. Thus keeping the tissues warm is probably necessary to fight both infection and ischemia. Some healthcare professionals use "radiant bandages" to keep the area warm, and care must be taken during surgery to prevent hypothermia, which increases rates of post-surgical infection.
[0064] Underlying ischemia may also be treated surgically by arterial revascularization, for example in diabetic ulcers, and patients with venous ulcers may undergo surgery to correct vein dysfunction.
[0065] Diabetics that are not candidates for surgery (and others) may also have their tissue oxygenation increased by Hyperbaric Oxygen Therapy, or HBOT, which can compensate for limitations of blood supply and correct hypoxia. In addition to killing bacteria, higher oxygen content in tissues speeds growth factor production, fibroblast growth, and angiogenesis. However, increased oxygen levels also means increased production of ROS. Antioxidants, molecules that can lose an electron to free radicals without themselves becoming radicals, can lower levels of oxidants in the body and have been used with some success in wound healing.
[0066] Low level laser therapy has been repeatedly shown to significantly reduce the size and severity of diabetic ulcers as well as other pressure ulcers.
2.3.1.4 TREATMENT WTTH GROWTH FACTORS- PROTEASE TNHTBTTORS AND HORMONES
[0067] Since some wounds, particularly chronic wounds, underexpress growth factors necessary for healing tissue, chronic wound healing may be speeded by replacing or stimulating those factors and by preventing the excessive formation of proteases like elastase that break them down.
[0068] One way to increase growth factor concentrations in wounds is to apply the growth factors directly, though this takes many repetitions and requires large amounts of the factors. Another way is to spread onto the wound a gel of the patient's own blood platelets, which then secrete growth factors such as vascular endothelial growth factor (VEGF), insulin-like growth factor 1-2 (IGF), PDGF, transforming growth factor-β (TGF-β), and epidermal growth factor (EGF). Other treatments include implanting cultured keratinocytes into the wound to re-epithelialize it and culturing and implanting fibroblasts into wounds.
[0069] Since levels of protease inhibitors are lowered in chronic wounds, some researchers are seeking ways to heal tissues by replacing these inhibitors in them. Secretory leukocyte protease inhibitor (SLPI), which inhibits not only proteases but also inflammation and microorganisms like viruses, bacteria, and fungi, may prove to be an effective treatment. [0070] Research into hormones and wound healing has shown estrogen to speed wound healing in elderly humans and in animals that have had their ovaries removed, possibly by preventing excess neutrophils from entering the wound and releasing elastase. Thus the use of estrogen is a possibility for treating acute and chronic wounds. For information on the hormonal regulation of wound healing see, e.g., Gilliver et al, 2007, Clin. Dermatol. 25:56- 62, which is incorporated herein by reference in its entirety.
2.3.1.5 GRAFTING AND DRESSINGS
[0071] Some patients are treated with artificial skin substitutes that have fibroblasts and keratinocytes in a matrix of collagen to replicate skin and release growth factors. In other cases, skin from cadavers is grafted onto wounds, providing a cover to keep out bacteria and preventing the buildup of too much granulation tissue, which can lead to excessive scarring. Though the allograft (skin transplanted from a member of the same species) is replaced by granulation tissue and is not actually incorporated into the healing wound, it encourages cellular proliferation and provides a structure for epithelial cells to crawl across. On the most difficult chronic wounds, allografts may not work, requiring skin grafts from elsewhere on the patient, which can cause pain and further stress on the patient's system.
[0072] Collagen dressings are another way to provide the matrix for cellular proliferation and migration, while also keeping the wound moist and absorbing exudate.
2.3.2 SCAR REVISION
[0073] Although healing in embryos and some animals even after extended injuries can occur without scarring, no adult human skin scars can be completely removed. Current treatments involve methods that remove the scar by surgical intervention (scar revision) and non-surgical methods that reduce the appearance of the scar. However, no scar treatment to date has been completely effective. As stated by Lalwani AK (2008, Chapter 71, "Scar Revision," IN Current Diagnosis & Treatment in Otolaryngology— Head & Neck Surgery, 2d ed., McGraw-Hill Companies, Inc.), "no technique has been devised to allow total and permanent removal or effacement of scars. Patients should be counseled to understand that the goal of scar revision is to replace one scar for another to improve the appearance and the acceptability of the scar." Clearly, there is a large unmet need for techniques to revise scars, particularly those that form from wounds and burns.
[0074] The only current commonly used surgical scar revision methods are skin grafts or serial expansion of surrounding skin. Other surgical approaches are primary excision/straight line closure, W plasty, Z plasty, and geometric broken line closure. See, Lalwani AK, 2008, Chapter 71, "Scar Revision," IN Current Diagnosis & Treatment in Otolaryngology— Head & Neck Surgery, 2d ed., McGraw-Hill Companies, Inc.
[0075] Skin grafts (either full thickness or split thickness) do not fully address the need for effective scar revision because (1) there is a limited supply of donor tissue (typically buttocks, abdomen, legs, in-front or behind the ear, etc.); (2) scarring occurs at donor sites and contractures {i.e., around the eyes and mouth); and (3) skin grafts typically (but not always) (with the possible exception of scalp) lack qualities of the donor site (porosity, vascularity, color, pigmentation, thickness, texture and overall cosmetic appearance, etc.). In serial expansion, the scar is serially excised, and a balloon is implanted at the wound site, which pushes the tissue around the scar to expand. Surgical excision of hypertrophic or keloid scars is often used with other methods such as pressotherapy or silicone gel sheeting (see below). Lone excision of keloid scars shows a high recurrence rate, close to 45%.
Surgical excision in combination with the immunomodulator imiquimod 5% cream (Aldara) may also have a benefit on scar reduction.
[0076] A number of non-surgical treatments have been postulated to improve the appearance of scars. See, e.g., Zurada et al, 2006, J. Am. Acad. Dermatol. 55: 1024-1031; and Lalwani AK, 2008, Chapter 71, "Scar Revision," ΓΝ Current Diagnosis & Treatment in Otolaryngology— Head & Neck Surgery, 2d ed., McGraw-Hill Companies, Inc.
[0077] Injection of corticosteroids into scars was introduced in the 1960s. A long term course of steroid injections into the scar under medical supervision may help flatten and soften the appearance of keloid or hypertrophic scars. The steroid is injected into the scar itself; since very little is absorbed into the blood stream, side effects of this treatment are minor. However, it does cause thinning of the scar tissue. This treatment is repeated at 4-6 week intervals. Topical steroids are ineffective as scar treatments.
[0078] Other intralesional injections (such as anti-mitotics and collagen) can also be used. For example, collagen injections or other soft tissue fillers can be used to raise sunken scars to the level of surrounding skin. Its effects are temporary, however, and it needs to be regularly repeated. There is also a risk in some people of an allergic reaction.
[0079] Silicone scar treatments improve scar appearance and are often used to prevent and treat hypertrophic scarring. The exact mechanism of action is unknown, though some studies suggest a manipulation of local ionic charges or a decrease in production of "proinflammatory" substances like TGF 2. See, e.g., Kuhn et al, 2001, "Silicone sheeting decreases fibroblast activity and downregulates TGFP2 in hypertrophic scar model," Int J Surg Invest 2:467. Dimethicone silicone gel appears to be is as effective as silicone sheeting in improving scar appearance. See Mustoe TA, 2008, "Evolution of silicone therapy and mechanism of action in scar management," Aesth Plast Surg 32:82-92. Polyurethane bandages are also used.
[0080] Pressure garments are used under supervision by a medical professional. They are most often used for burn scars that cover a large area, and is only effective on recent scars. Pressure garments are usually custom-made from elastic materials, and fit tightly around the scarring. They work best when they are worn 24 hours a day for six to twelve months. It is believed that they work by applying constant pressure to surface blood vessels and eventually causing scars to flatten and become softer.
[0081] Needling is an inexpensive process where the scarred area is continuously needled to promote collagen formation. Once needled the area is allowed to fully heal, and needled again if required depending on the intensity of the scar. Scarring needles and needling rollers are available for home use; however, needling should not be done on parts of the face or areas where major nerves are located without professional medical supervision. Needling at home must also be done in line with hygienic and sterilization requirements.
[0082] Dermabrasion involves the removal of the surface of the skin with specialist equipment and usually involves a general anesthetic. It is useful with raised scars, but is less effective when the scar is sunken below the surrounding skin.
[0083] The use of lasers on scars is a new form of treatment. Several cosmetic lasers have been approved for the treatment of acne scars by using laser resurfacing techniques. Vascular lasers have been proven to greatly reduce the redness of most scars 6-10 weeks after the initial treatment. Fractional lasers and 1064 Nd:YAG lasers have also been shown in clinical studies to benefit scars. Pulsed dye laser has been reported to reduce scar erythema.
[0084] Low-dose, superficial radiotherapy is used to prevent re-occurrence of severe keloid and hypertrophic scarring. It is usually effective, but only used in extreme cases due to the risk of long-term side effects.
[0085] Although several natural remedies to treat scars have been proposed, such as vitamin E and A, research shows the use of vitamin E and onion extract as a treatment for scars is ineffective. Vitamin E causes contact dermatitis in up to 33% of users and in some cases it may worsen scar appearance. See, Baumann & Spencer, 1999, "The effects of topical vitamin E on the cosmetic appearance of scars," Dermatol Surg. 25:31 1-315; and Jenkins et al, 1986, "Failure of topical steroids and vitamin E to reduce postoperative scar formation following reconstructive surgery," J Burn Care Rehabil 7: 309-312. However, there is evidence that vitamin C normalizes collagen production and encourages the production of an organized, healthy collagen framework, which improves scar appearance. Vitamin C and some of its esters also fade the dark pigment associated with some scars. See, Fitzpatrick & Rostan, 2002, "Double-blind, half-face study comparing topical vitamin C and vehicle for rejuvenation of photodamage," Dermatol Surg 28:231-236; and Farris PK, 2005, "Topical vitamin C: a useful agent for treating photoaging and other dermatologic conditions," Dermatol Surg 31 :814-818.
[0086] Thus, scar revision and wound management are limited by the limited
regenerative capacity of adult human skin. Extensive skin damage (e.g., from blast trauma, penetrating munitions, flying debris, or burns) results in severe scars that can limit mobility or function. Current therapies (skin grafting, pressure application) have modest functional and cosmetic results and are limited by the availability of donor skin, and by morbidity of donor and graft sites. There is an urgent need for improved therapies to treat wounds and scars.
3. SUMMARY OF THE INVENTION
[0087] Novel intermittent and pulse lithium treatments that promote wound healing, scar prevention, and scar revision are described. The intermittent and pulse lithium treatments can be administered in situ to acute wounds, chronic wounds, to scars, and/or surrounding skin. The intermittent or pulse lithium treatments can be administered to the wound site or surrounding skin before, at the time of, and/or subsequent to, either acute wounding or, more typically, the wounding that is induced in scar revision. The intermittent and pulse lithium treatments may also be administered to skin-derived cells or skin tissue ex vivo.
[0088] Methods for using intermittent or pulse lithium treatments to enhance deposition of skin adnexal structures into wound sites {e.g. , by inducing hair follicle neogenesis in the site of a scar revision), which in turn enhances wound healing, are described. For example, an intermittent or pulse lithium treatment may be used to enhance hair follicle neogenesis or enhance the re-association of dissociated hair follicle cells into follicles and facilitate their growth and expansion either in situ, or, alternatively, in culture for their implantation into fresh wounds and scar revisions.
[0089] With these methods, traditional approaches to scar revision, such as human skin transplantation, can be efficiently replaced with transplantation of follicular units or other smaller appendage structures from skin. Thus, hair follicles can be introduced to the wound by migration or de novo hair follicle neogenesis, or by transplanting one or more of the following skin elements: full skin (xeno-; autologous human), follicular units, dissociated cells (donor dominance; recipient effects), ex vivo-expanded skin and/or follicular units, or human skin equivalents in vivo (universal donors). Engineered human skin, or human skin equivalents, can also be used for hair follicle neogenesis and scar revision platforms.
[0090] Intermittent lithium treatments or a single pulse lithium treatment are used to revise scars and heal wounds in human subjects. Any pharmaceutically acceptable compound that releases the lithium ion (also referred to herein as lithium cation, Li+, or ionized lithium) can be used for the lithium treatment; such compounds include, but are not limited to lithium gluconate, lithium succinate, and other organic salts/acids; and lithium chloride and other inorganic salts/acids, as described in Section 5.1, infra.
[0091] The intermittent lithium treatment protocol involves multiple courses of lithium treatment interrupted by lithium treatment "holidays" (periods during which no lithium treatment is administered). A lithium treatment holiday is a period of time during which the patient stops the lithium treatment with the intent of resuming treatment. For the single pulse protocol, a dose of lithium is administered over a short period of time.
[0092] The lithium treatment can be administered topically, transdermally, intradermally, cutaneously, subcutaneously, intramuscularly, intravenously, orally, sublingually, or can be bucchal. Topical lithium treatment is a preferred embodiment because high local concentrations can be achieved while minimizing systemic exposure. In one such embodiment, lithium gluconate 8% weight/weight (w/w) gel (e.g., Lithioderm 8% gel) commercially available in France for the treatment of seborrheic dermatitis (Dreno B, 2007, Ann Dermatol Venereol. 134:347-351, incorporated herein by reference) can be used in the treatment methods described herein. In certain preferred embodiments, lithium is formulated into a modified release form that allows controlled release, over time, into the skin. In another preferred embodiment, the lithium is formulated as part of a mesh scaffold that delivers lithium into the skin. More details on these and other lithium formulations and delivery methods for use in the treatment methods described herein are described in Sections 5.1-5.3 infra.
[0093] The intermittent and pulse lithium treatments can be administered alone to wounded skin (e.g., prior to, during, or subsequent to scar revision, or acute skin wounding, or chronic skin wounding) or in combination with other treatments to enhance wound healing or scar revision. The intermittent and pulse lithium treatments can also be administered in combination with other treatments that facilitate hair follicle development and deposition into the wounded skin. Embodiments of the invention include combination therapies, involving the addition of other treatment(s) concurrently with, or during the breaks between, the cycles of intermittent lithium treatments; or the addition of other treatment(s) concurrently with, or before and/or after the pulse lithium treatment. Such combination therapies can include, but are not limited to, the concurrent or sequential use of other chemical agents, or mechanical or physical treatments including but not limited to, laser {e.g., Fraxel), dermatome planing, laser abrasion, electrology, intense pulsed light, or surgical treatments {e.g., skin graft or follicular unit extraction (FUE), etc.) that promote scar revision or wound healing.
[0094] Provided herein are intermittent lithium treatments or pulse lithium treatments in combination with perturbation (e.g., debriding, peeling, or wounding) of the skin and/or other tissues of the integumentary system by methods such as laser treatment, dermabrasion, needling (using, e.g., microneedles), electromagnetic disruption, electroporation, or sonoporation; chemically {e.g., to induce inflammation); or by any other method described herein or known in the art, prior to or concurrent with administration of a lithium formulation described herein. For example, the integumental perturbation procedure can be any
"wounding" procedure used for scar revision. The procedure can be controlled to limit perturbation to the epidermis, or extend deeper into the dermis and/or hypodermis. The occurrence of pinpoint bleeding would indicate removal of the epidermis and portions of the upper layer of the dermis. The occurrence of increased bleeding would indicate deeper penetration (and thus perturbation) into the dermis layer.
[0095] Described herein are intermittent or pulse lithium treatments administered in combination with laser treatment or another approach to scar revision. As discussed in Section 5.4.1 infra, lasers, particularly fractional lasers, and skin graft, follicular unit, and skin component transplant technologies have the capacity to induce regenerative changes in skin that mimic wounding and have applications in revision of scars. In particular, and without being bound by any theory of how the invention works, laser techniques may "mimic" the plastic, embryonic-like, state of the epidermis created by other wound signals, but with laser's precision, versatility, and demonstrated efficacy in small scars.
Consequently, when laser treatments are combined with an intermittent or pulse lithium treatment, the outcome of revising extensive scars, particularly those that limit function {e.g., eye or mouth closure; joint contractions), may be vastly improved.
[0096] Also described herein are intermittent lithium treatments and pulse lithium treatments administered concurrently or in sequential/alternating combination with other agents or treatments that modulate the wound healing process. The intermittent and pulse lithium treatments may be administered with treatments that either promote or delay the wound healing process, such as described in Section 5.4.3 infra.
[0097] Because the invention is based, in part, on the recognition of lithium's ability to mobilize stem cells that promote development of skin adnexal structures (for example, enhancing hair follicle neogenesis and regeneration), the intermittent lithium treatments or pulse lithium treatments described herein can be administered concurrently or alternating sequentially with one or more of the following treatments that prevent follicle senescence, for example, anti-oxidants such as glutathione, ascorbic acid, tocopherol, uric acid, or polyphenol antioxidants); inhibitors of reactive oxygen species (ROS) generation, such as superoxide dismutase inhibitors; stimulators of ROS breakdown, such as selenium; mTOR inhibitors, such as rapamycin; or sirtuins or activators thereof, such as resveratrol, or other SIRT1, SIRT3 activators, or nicotinamide inhibitors.
[0098] The intermittent lithium treatments or a pulse lithium treatment provided herein can also be administered concurrently or alternating sequentially with one or more of the following treatments that promote hair growth, in order to enhance formation of new hair follicles: minoxidil, finasteride, bimatoprost (Latisse), CaCl2, or adenosine, or techniques of integumental perturbation such as, e.g. , by mechanical means, chemical means,
electromagnetic means (e.g. , using a laser such as one that delivers ablative, non-ablative, non-fractional, superficial, or deep treatment, and/or are CCh-based, or Erbium- YAG-based, etc.), irradiation, radio frequency (RF) ablation, or surgical procedures (e.g., hair
transplantation, strip harvesting, follicular unit extraction (FUE), scalp reduction, etc.).
[0099] Treatments that promote hair growth, or, alternatively, treatments that prevent hair growth, may also be used in combination with the intermittent lithium treatments or a pulse lithium treatment described herein in order to promote the establishment of desired hair patterning in the healed wound or revised scar, thereby improving the appearance of the treated skin. For example, treatments that regulate gender-specific specialized human hair follicles, including those under the influence of sex-steroid regulation, or that regulate the differentiation of stem cells into gender-specific specialized human hair follicles, possibly resulting in follicles having features that are different from natural follicles in the target location of skin (e.g., normal sized follicles with terminal hair where previously miniaturized follicles with vellus hair were present) may be administered. For example, treatment of grafted skin with a combination of lithium and a modulator of specific hair patterning may reduce donor dominance and enhance the ability of the graft to acquire properties of the recipient site. Thus, intermittent lithium treatments or a pulse lithium treatment may be used concurrently or in sequential combination with either a treatment that enhances hair growth (described above) or a cytotoxic drug, a hair growth retardant, such as eflornithine HC1 (Vaniqa), 5-fluorouracil (5-FU) (e.g., Efudex 5% cream), or other epilation or depilation methods to prevent or reduce hair growth.
[00100] Success of a pulse or intermittent lithium treatment described herein can be measured by one or more of the following outcomes:
• improvement of pigmentation of the scarred or wounded area
• improved surface contour of the scarred or wounded area
• improved texture of the scarred or wounded area
• improved thickness of the scarred (if the scar started out as depressed) or wounded area
• improved overall cosmetic outcome
• subjective patient measures of improved outcome
• presence of elastin
• proper collagen orientation
• improvement in viscoelasticity
• increased number of hair germs
• hair follicle neogenesis or regeneration
• increased proportion of hair follicles in anagen or decreased proportion of follicles in telogen
• increased numbers of follicular units with 3 or more hair follicles
• reduction in the size of the wound or appearance of the scar compared to a wound or scar not treated with lithium
• conversion of the dermal epidermal junction from a flat junction between the dermis and epidermis (typical of a scar) to rete pegs (epithelial extensions that project into the underlying connective tissue) with interdigitating dermis, as assessed by in vivo scanning laser microscopy
• normalization of blood vessels as assessed using laser Doppler analysis.
[00101] Human subjects who are candidates for the pulse or intermittent lithium treatments described herein include any subject in need of improved wound healing, particularly wound healing without scarring, or scar revision. Human subjects who are candidates for such treatments include any subject for whom improved wound healing or scar revision is desired. Such human subjects include, but are not limited to, subjects with photodamaged skin, acne scars, chicken pox scars, scarring (cicatricial) alopecia, chronic non-healing wounds or scars due to, e.g., diabetes, venous or arterial disease, old age or senescence, infection, medication, chemotherapy, trauma, burns, stress, autoimmune disease, malnutrition, or endocrine dysfunction. Surgical subjects who are candidates for such treatments include, but are not limited to, patients with skin graft, hair transplantation, skin cancer surgery, or Mohs surgery. Subjects who are candidates for such treatments also include subjects with any other form of wounding or scarring or disease or disorder associated with wounding or scarring as discussed infra and/or known in the art. In some embodiments, the subject has a wound or scar on a cosmetically sensitive location, such as the face or neck.
[00102] The invention is based in part on the recognition that the timing of the administration of lithium is important for it to function as an effective modulator of wound healing (and thus, scar revision) in human subjects. For example, lithium treatment results, indirectly, in increasing Wnt signaling, but agents that increase Wnt signaling have had conflicting effects on hair follicle development and wound healing. When continuously present, they stimulate follicle morphogenesis but also induce hair follicle tumors (Gat et al. , 1998, Cell 95: 605-614), and lead to decreased hair growth (Millar et al., 1999, Dev. Biol. 207: 133-149). In the case of lithium, it has been shown to arrest mitosis (Wolniak, 1987, Eur. J. Cell Biol. 44: 286-293; and Wang, 2008, World J. Gastroenterol. 14:3982-3989), cause pathological hair loss when systematically administered (see, e.g., Mercke et al., 2000, Ann. Clin. Psych. 12:35-42), induce a psoriatic (i.e., chronically wounded) state (see, Wolf e? al., 2000, J. Eur. Acad. Dermatol. Venereol. 14:97-99; Stojadinovic et al., 2005, Am. J. Pathol. 167:59-69) or, at best, stimulate the generation of only rudiments of hair follicles (which also leads to the formation of epithelial cysts) (Fathke et al., 2006, BMC Cell Biol. 7:4). These apparently discrepant roles of lithium as a stimulator of Wnt signaling and a negative regulator of the cell cycle are resolved in the present invention. By using lithium in formulations for intermittent or pulse treatments described herein, for example, before, concurrently with, or after integumental perturbation or another treatment that modulates wound healing, it functions as an effective treatment for wounds and scar revisions in humans. It is thus also possible that the timing of exposure to other compounds with Wnt agonist activity may be important for wound healing and scar revision.
[00103] The invention is also based, in part, on the principle that human skin is replenished by bone-marrow derived and tissue-derived stem cells throughout life. In some embodiments, the lithium treatment(s) is used in combination with methods that mobilize tissue stem cells (e.g., using integumental perturbation) and/or methods that mobilize bone marrow-derived stem cells (e.g., growth factors such as G-CSF and/or chemical agents such as plerixafor (Mozobil®)). In some embodiments, the lithium treatments described herein are used together with methods that regulate the differentiation of these stem cells into specialized human hair follicles in order to facilitate the desired hair patterning at the acceptor site, using agents such as finasteride, fluconazole, spironolactone, flutamide, diazoxide, 11 -alpha-hydroxyprogesterone, ketoconazole, RU58841, dutasteride, fluridil, or QLT-7704, an antiandrogen oligonucleotide, cyoctol, topical progesterone, topical estrogen, cyproterone acetate, ru58841, combination 5 alpha reductase inhibitors, oral contraceptive pills, and others in Poulos & Mirmirani, 2005, Expert Opin. Investig. Drugs 14: 177-184, incorporated herein by reference, or any other antiestrogen, an estrogen, or estrogen-like drug (alone or in combination with agents that increase stem cell plasticity; e.g., such as valproate), etc. , known in the art. Such combination treatments can further include the use of agents that modulate hair growth or that modulate wound healing.
[00104] The methods of the invention are illustrated by the examples described in Section 6 to 19.
3.1 GLOSSARY OF TERMS
[00105] The following terms are used herein consistently with their art-accepted meanings summarized below.
[00106] Anagen: Growth stage of the hair Follicle Cycle.
[00107] Bulb: Lowermost portion of the hair follicle, containing rapidly proliferating matrix cells that produce the hair.
[00108] Bulge: Portion of the outer-root sheath of the hair follicle, located at the region of the insertion of the arrector pili muscle; thought to contain epithelial stem cells responsible for regenerating follicles in the anagen stage.
[00109] Catagen: Stage of the hair cycle characterized by regression and involution of the follicle.
[00110] Cicatricial (scarring) Alopecia: Abnormal hair loss with scarring. Caused by destruction of hair follicles and replacement with scar tissue as a result of inflammation, trauma, fibrosis, or unknown causes; examples include lichen planopilaris and discoid lupus erythematosus.
[00111] Exogen: Phase of the hair Follicle Cycle where hair shaft is shed from the follicle. [00112] Follicle cycle: Hair growth in each follicle occurs in a cycle that includes the following phases: anagen (growth phase), catagen (involuting/regressing stage), telogen (the quiescent phase), exogen (shedding phase), and re-entry into anagen.
[00113] Integumental: Pertaining to the integumentary system, which comprises the skin (epidermis, dermis, hypodermis (or subcutanea)) and all cells contained therein regardless of origin, and its appendages (including, e.g., hair and nails).
[00114] Kenogen: Latent phase of hair cycle after hair shaft has been shed and growth is suspended in follicle.
[00115] With regard to the concentrations of lithium (including its concentration in formulations, in tissue, in serum, etc., and as a salt form, as ionized lithium in solution, etc.) described herein, since ionized lithium is a monovalent cation, the concentration of lithium expressed in millimolar units (mM) is equal to its concentration expressed in
milliequivalents (mEq) {i.e., to avoid any doubt, 1 mM Li+ = 1 mEq Li+), as is sometimes used in the art.
[00116] Telogen: Resting stage of the hair cycle; club hair is the final product and is eventually shed.
[00117] Telogen effluvium: Excessive shedding of hair caused by an increased proportion of follicles entering the telogen stage; common causes include drugs and fever.
[00118] Terminal hair: Large, usually pigmented hairs on scalp and body.
[00119] Vellus hair: Very short, nonpigmented hairs (e.g., those found diffusely over nonbeard area of face and bald scalp as a result of miniaturization of terminal hairs).
4. DESCRIPTION OF THE FIGURES
[00120] Figure 1. Types of human hair follicles.
[00121] Figure 2. Architecture of the skin.
[00122] Figure 3. Diagram of human hair follicle.
[00123] Figure 4. Cellular structure of the human hair bulb.
[00124] Figure 5. Permeation of lithium ions (also referred to as "Li ions") through the dermis (y-axis) from Formulation 35 A' (lithium chloride emulsion cream; see Table 2) is plotted over time, in hours (x-axis). Cadaver skin was dermabraded with a standard dermabrader to remove the stratum corneum and epidermis prior to administration of the lithium compound. [00125] Figure 6. Permeation of Li ions through intact cadaver skin (y-axis) from
Formulation 35A' (lithium chloride emulsion cream; see Table 2) is plotted over time, in hours (x-axis).
[00126] Figure 7. Release of Li ions through dermis (y-axis) from Formulation BX (lithium chloride gel; see Table 2) is plotted over time, in hours (x-axis). Cadaver skin was dermabraded, with a standard dermabrader to remove the stratum corneum and epidermis prior to administration of the lithium compound.
[00127] Figure 8. Release of Li ions through dermis (y-axis) from Formulation BV-001- 003A (lithium chloride hydrogel; see Table 2) is plotted over time, in hours (x-axis).
Cadaver skin was dermabraded with a standard dermabrader to remove the stratum corneum and epidermis prior to administration of the lithium compound.
[00128] Figure 9. Release of Li ions through dermis (y-axis) from Formulation 28A (lithium chloride topical dispersion cream; see Table 2) is plotted over time, in hours (x-axis). Cadaver skin was dermabraded, with a standard dermabrader to remove the stratum corneum and epidermis prior to administration of the lithium compound.
[00129] Figure 10. Pharmacokinetic analysis of lithium concentrations in skin and plasma with once daily topical dosing with lithium gluconate hydrogel ("lithium gluconate") 8% and lithium chloride hydrogel ("lithium chloride") 8% following dermabrasion (DA). Lithium ion concentrations were measured by ICP/MS/MS, using a validated method (see Section 13.2 infra). Shown are lithium concentrations in skin (top) and blood (bottom), with once daily topical dosing with 80 mg/ml lithium on dermabraded mouse skin. Doses were given at Time (T)=0, T=24h, T=48h, and T=72 h. Skin and Blood samples were taken at T=0, 4-6h, 24h, 25h, 28h, 48h, 49h, 52h, 72h, 73h, 76h, and 96h. Arrows indicate peak levels of Lithium ion in skin one hour post dosing. N=2 per time point— error bars denote range. To obtain peak levels, mice were sacrificed and skin and blood obtained ~ 1 hour after dosing on the 5th day. To obtain trough levels, mice were sacrificed and skin and blood obtained ~1 hour without dosing, on the 5th day.
[00130] Figure 11. Linearity of calibration curves, validation of bioanalytical ICP method described in Section 13.2 infra.
[00131] Figure 12. Skin lithium concentrations calculated in mM, as a function of increasing doses of a formulation of lithium chloride dissolved in isotonic saline in mg/kg administered subcutaneously to mice dermabraded prior to dosing. "Peak samples" were taken 1 h post last dosing. [00132] Figure 13. Comparison of Peak lithium concentrations in plasma and skin upon subcutaneous administration of a formulation of lithium chloride dissolved in isotonic saline following DA.
[00133] Figure 14. A: Lithium concentrations calculated in mM, in total blood (red blood cells (RBC) + plasma), as a function of increasing doses of a formulation of lithium chloride dissolved in isotonic saline in mg/kg, administered subcutaneously to mice dermabraded prior to dosing. B: Skin lithium concentrations calculated in μg/kg, as a function of increasing doses in mg/kg. In the wounded groups, skin was dermabraded prior to administration of the formulation of lithium chloride dissolved in isotonic saline. Non-wounded comparisons are shown (square, diamond) with dermabrasion wounded groups (cross, triangle). * It is noted that in this experiment, dermabrasion was accomplished using a microdermabrasion device.
[00134] Figure 15. Skin lithium concentrations calculated in mM, as a function of increasing doses of a formulation of lithium chloride dissolved in isotonic saline in mg/kg. The lithium formulation was administered subcutaneously following full thickness excision (FTE) of skin. Dosing was started on the day of scab detachment (-11-14 days post-FTE). Lithium ion concentrations were measured by a validated bioanalytical ICP method (see Section 13.2 infra).
[00135] Figure 16. Plasma lithium Concentrations calculated in mM, as a function of increasing doses of a formulation of lithium chloride dissolved in isotonic saline in mg/kg. The lithium formulation was administered subcutaneously following FTE. Dosing was started on the day of scab detachment (~11-14 days post-FTE). Lithium concentrations were measured by a validated bioanalytical ICP method (see Section 13.2 infra).
[00136] Figure 17. Comparison of Peak lithium concentrations in plasma and skin upon subcutaneous administration of a formulation of lithium chloride dissolved in isotonic saline following FTE.
[00137] Figure 18. Pharmacokinetic analysis of lithium concentrations in skin and plasma with once daily topical dosing of 8% lithium chloride or 8% lithium gluconate hydrogel ("lithium gluconate") following FTE. Lithium ion concentrations were measured by
ICP/MS/MS, using a validated method (see Section 13.2 infra). Single dose administered at Oh, 24 h, 48 h, 72 h. Tissue samples (skin and blood taken at T=0, 4-6h, 24h, 25h, 28h, 48h, 49h, 52h, 72h, 73h, 76h, 96h). Arrows indicate peak levels of Lithium ion in skin one hour post dosing. N=2 per time point— error bars denote range [00138] Figure 19. Pharmacokinetic analysis of lithium concentrations in skin and plasma with twice daily topical dosing of lithium gluconate, 1%; lithium gluconate, 8%; and lithium gluconate, 16% following DA.
[00139] Figure 20. Topical lithium 8% increases the proportion of mature neogenic hair follicles in healed FTE wounds, based on histologic examination. A: Diagrams of selected stages of hair follicle development. B: Percentage of stageable neogenic hair follicles at stage 5 or greater following administration of topical lithium gluconate hydrogel ("lithium gluconate"), 1%, 8%, or 16% or lithium chloride hydrogel ("LiCl"), 8%. Numbers in the bars indicate the number of mice per group that were used for quantitation. Ratios above the bars indicate the number of NHF (neogenic hair follicles) > stage 5 divided by the total number of stageable NHF.
[00140] Figure 21. Topical lithium 8% increases maturation of neogenic hair follicles as shown by histology (13x = mouse ID; lOx magnification). Tissues analyzed following administration of topical lithium gluconate hydrogel ("lithium gluconate"), 1%, 8%, or 16% or lithium chloride hydrogel ("LiCl"), 8%.
[00141] Figure 22. Topical lithium 8% increases both the number and maturation of neogenic hair follicles in FTE wounds, as measured following administration of topical lithium gluconate hydrogel ("lithium gluconate"), 1%, 8%, or 16% or lithium chloride hydrogel ("LiCl"), 8%.
[00142] Figure 23. No adverse systemic effects of topical lithium as indicated by equal weight gain. Weight gain profile of mice administered topical lithium gluconate hydrogel ("lithium gluconate"), 1%, 8%, or 16%, or lithium chloride hydrogel ("LiCl"), 8%, following FTE.
[00143] Figure 24. Topical lithium 8% increases shaft thickness of regenerated hair follicles following DA. Tissues analyzed following administration of topical lithium gluconate hydrogel ("lithium gluconate"), 1%, 8%, or 16% or lithium chloride hydrogel ("LiCl"), 8%.
[00144] Figure 25. Topical lithium gluconate 8% results in a 16% increase thickness of regenerated hair shafts following DA. Tissues analyzed following administration of topical lithium gluconate hydrogel ("lithium gluconate"), 1%, 8%, or 16% or lithium chloride hydrogel ("LiCl"), 8%. Median + first and third quartile shown. P-values for comparisons of lithium treatments to placebo are for 1-sided tests for superiority, and should < 0.0125 for statistical significance with a family-wise error rate of alpha = 5% adjusted by the Bonferroni method for 4 comparisons to placebo. Graph on right side shows simultaneous 90% confidence intervals, corrected for 4 comparisons by the Treatment Groups Bonferroni method.
[00145] Figure 26. Healed FTE wounds treated with topical LiCl 8% have increased numbers of neogenic hair follicles, as assessed by in vivo scanning laser microscopy, imaging the wounded area approximately 60-80 μιη beneath the skin surface.
[00146] Figure 27. Topical LiCl 8% results in a 1.82 fold increase in number of neogenic hair follicles per FTE wound. Median + first and third quartiles shown, p-value is for onesided Wilcoxon test for superiority to placebo, p < 0.0125 for statistical significance with a family -wise error rate of a = 5 % corrected by the Bonferroni method for 4 comparisons to placebo. Right graph: Hodges-Lehman estimate of median difference. Simultaneous 90% confidence intervals, corrected for 4 comparisons by the Bonferroni method.
[00147] Figure 28. Topical LiCl 8% increases the total number of neogenic hair follicles (also referred to as "HF") per FTE wound by 3-fold, based on histology of tissue sections. Left graph: Median + first and third quartiles shown. Numbers above columns =total number of neogenic hair follicles ("NHF") combined from individual mice (parentheses indicate number of NHF that could not be staged). Numbers below columns =total number of slides analyzed where one slide =one mouse (parentheses indicate slides not analyzed due to technical issues), p-value is for one-sided Wilcoxon test for superiority to placebo, p < 0.0125 for statistical significance with a family-wise error rate of a =5% corrected by the Bonferroni method for 4 comparisons to placebo. Right graph: Hodges-Lehman estimate of median difference. Simultaneous 90% confidence intervals, corrected for 4 comparisons by the Bonferroni method.
[00148] Figure 29. Topical LiCl 8% increase the percentage of FTE wound covered with neogenic hair follicles, based on in vivo scanning confocal microscopy.
[00149] Figure 30. Topical LiCl 8% results in a 1.57 fold increase over placebo in coverage of the FTE wound with neogenic hair follicles. Median + first and third quartiles shown, p-value is for one-sided Wilcoxon test for superiority to placebo, p < 0.0125 for statistical significance with a family-wise error rate of a = 5% corrected by the Bonferroni method for 4 comparisons to placebo. Right graph: Hodges-Lehman estimate of median difference. Simultaneous 90% confidence intervals, corrected for 4 comparisons by the Bonferroni method.
[00150] Figure 31. Following FTE, topical LiCl 8%, as compared to placebo, does not affect the density of neogenic hair follicles in the region where follicles are forming. Median + first and third quartiles shown, p-value is for one-sided Wilcoxon test for superiority to placebo, p < 0.0125 for statistical significance with a family-wise error rate of a = 5% corrected by the Bonferroni method for 4 comparisons to placebo. Right graph: Hodges- Lehman estimate of median difference. Simultaneous 90% confidence intervals, corrected for 4 comparisons by the Bonferroni method.
[00151] Figure 32. Topical LiCl 8% does not affect density of regenerated hair follicles following DA. Median + first and third quartiles shown. Right graph: Hodges-Lehman estimate of median difference. Simultaneous 90% confidence intervals, corrected for 4 comparisons by the Bonferroni method.
[00152] Figure 33. Topical LiCl 8% and 16% decreases the area of healed FTE wounds. 1.5 cm2 wounds were induced by FTE, and then allowed to heal. Shown are median + first and third quartiles. P-values for comparisons of lithium treatments to placebo are for onesided tests for superiority, and should be p < 0.0125 for statistical significance with a family- wise error rate of a = 5% adjusted by the Bonferroni method for 4 comparisons to placebo. In the right graph, a Hodges-Lehman estimate of median difference is shown, with simultaneous 90% confidence intervals, corrected for 4 comparisons by the Bonferroni method.
[00153] Figure 34. Increased total number of neogenic HF is a factor that accounts for the increased coverage of wounds with LiCl 8% as compared to placebo. Shown is mean + SEM. p-values for comparison of lithium treatments to placebo for are for one-sided tests for superiority, and should be p < 0.0125 for statistical significance with a family-wise error rate of a = 5% adjusted by the Bonferroni method for 4 comparisons to placebo. Shown in the right graph: Mean + SEM. Treatment with LiCl 8% leads to an increased number of NHF. Treatment with LiCl 8% or Lithium gluconate 16% result in the same, reduced, total wound area. LiCl 8% has increased % of wound coverage by new hair follicles compared to Lithium gluconate 16%.
[00154] Figure 35. Complexed Lithium Gluconate encapsulated within biodegradable poly (D,L-lactide-co-glycolide) ("PLG") microspheres.
[00155] Figure 36. Non-Complexed Lithium Gluconate encapsulated within
biodegradable PLG microspheres.
[00156] Figure 37. Synthetic Biodegradable Matrices from PLA/PLG Blends. A:
Scanning electron micrograph (SEM) of a 100% PLA matrix. B: SEM of a 100% PLG matrix. C: In vitro profiles of lithium ion release from biodegradable matrices comprised of different ratios of PLA and PLG. 5. DESCRIPTION OF THE INVENTION
5.1 LITHIUM COMPOSITIONS
[00157] Any compound or composition that can release a lithium ion (also referred to herein as lithium cation, Li+, or ionized lithium) is suitable for use in the compositions and methods. Such compounds include but are not limited to a pharmaceutically acceptable prodrug, salt or solvate {e.g., a hydrate) of lithium (sometimes referred to herein as "lithium compounds"). Optionally, the lithium compounds can be formulated with a pharmaceutically acceptable vehicle, carrier, diluent, or excipient, or a mixture thereof. Additionally, lithium- polymer complexes can be utilized to developed various sustained release lithium matrices.
[00158] Any form of lithium approved for pharmacological use may be used in the intermittent lithium treatments or a pulse lithium treatment. For example, lithium is best known as a mood stabilizing drug, primarily in the treatment of bipolar disorder, for which lithium carbonate (L12CO3), sold under several trade names, is the most commonly used. Other commonly used lithium salts include lithium citrate (L13C6H5O7), lithium sulfate (L12SO4), lithium aspartate, and lithium orotate. A lithium formulation well-suited for use in the methods disclosed herein is lithium gluconate, for example, a topical ointment of 8% lithium gluconate (Lithioderm™), is approved for the treatment of seborrheic dermatitis. See, e.g., Dreno and Moyse, 2002, Eur J Dermatol 12:549-552; Dreno et al, 2007, Ann Dermatol Venereol 134:347-351 (abstract); and Ballanger et al, 2008, Arch Dermatol Res 300:215- 223, each of which is incorporated by reference herein in its entirety. Another lithium formulation for use in the methods disclosed herein is lithium succinate, for example, an ointment comprising 8% lithium succinate, which is also used to treat seborrheic dermatitis. See, e.g., Langtry et al, 1996, Clinical and Experimental Dermatology 22:216-219; and Cuelenaere et al, 1992, Dermatology 184: 194-197, each of which is incorporated by reference herein in its entirety. In one embodiment, the lithium formulation is an ointment comprising 8% lithium succinate and 0.05% zinc sulfate (marketed in the U.K. as Efalith). See, e.g., Efalith Multicenter Trial Group, 1992, J Am Acad Dermatol 26:452-457, which is incorporated by reference herein in its entirety. Examples of lithium succinate formulations and other lithium formulations for use in the intermittent lithium treatments or pulse lithium treatment described herein are also described in U.S. Patent No. 5,594,031, issued January 14, 1997, which is incorporated herein by reference in its entirety. 5.1.1 LITHIUM SALTS
[00159] Any pharmaceutically acceptable lithium salt may be used as a source of lithium ions in the intermittent lithium treatments or a pulse lithium treatment. It will be understood by one of ordinary skill in the art that pharmaceutically acceptable lithium salts are preferred. See, e.g., Berge et al, J. Pharm. Sci. 1977, 66: 1-19; Stahl & Wermuth, eds., 2002, Handbook of Pharmaceutical Salts, Properties, and Use, Zurich, Switzerland: Wiley -VCH and VHCA; Remington 's Pharmaceutical Sciences, 1990, 18th eds., Easton, PA: Mack Publishing;
Remington: The Science and Practice of Pharmacy, 1995, 19th eds., Easton, PA: Mack Publishing.
[00160] In some embodiments, the compositions used for intermittent lithium treatment or a pulse lithium treatment comprise mixtures of one or more lithium salts. For example, a mixture of a fast-dissolving lithium salt can be mixed with a slow dissolving lithium salt proportionately to achieve the release profile. In certain embodiments, the lithium salts do not comprise lithium chloride.
[00161] In some embodiments, the lithium salt can be the salt form of anionic amino acids or poly(amino) acids. Examples of these are glutamic acid, aspartic acid, polyglutamic acid, polyaspartic acid.
[00162] By reciting lithium salts of the acids set forth above, applicants do not mean only the lithium salts prepared directly from the specifically recited acids. In contrast, applicants mean to encompass the lithium salts of the acids made by any method known to one of ordinary skill in the art, including but not limited to acid-base chemistry and cation-exchange chemistry.
[00163] In another embodiment, lithium salts of anionic drugs that positively affect hair growth, such as prostaglandins can be administered. In another embodiment, a large anion or multianionic polymer such as polyacrylic acid can be complexed with lithium, then complexed with a cationic compound, such as finasteride, to achieve a slow release formulation of both lithium ion and finasteride. Similarly, a lithium complex with a polyanion can be complexed further with the amines of minoxidil, at pHs greater than 5.
[00164] Lithium compounds for use in the methods provided herein may contain an acidic or basic moiety, which may also be provided as a pharmaceutically acceptable salt. See, Berge et al, J. Pharm. Sci. 1977, 66: 1-19; Stahl & Wermuth, eds., 2002, Handbook of Pharmaceutical Salts, Properties, and Use Zurich, Switzerland: Wiley-VCH and VHCA.
5.1.2 ORGANIC LITHIUM SALTS [00165] In some embodiments, the lithium salts are organic lithium salts. Organic lithium salts for use in these embodiments include lithium 2,2-dichloroacetate, lithium salts of acylated amino acids {e.g., lithium -acetylcysteinate or lithium N-stearoylcysteinate), a lithium salt of poly(lactic acid), a lithium salt of a polysaccharides or derivative thereof, lithium acetylsalicylate, lithium adipate, lithium hyaluronate and derivatives thereof, lithium polyacrylate and derivatives thereof, lithium chondroitin sulfate and derivatives thereof, lithium stearate, lithium linoleate, lithium oleate, lithium taurocholate, lithium cholate, lithium glycocholate, lithium deoxycholate, lithium alginate and derivatives thereof, lithium ascorbate, lithium L-aspartate, lithium benzenesulfonate, lithium benzoate, lithium 4- acetamidobenzoate, lithium (+)-camphorate, lithium camphorsulfonate, lithium (+)-(15)- camphor-10-sulfonate, lithium caprate, lithium caproate, lithium caprylate, lithium cinnamate, lithium citrate, lithium cyclamate, lithium cyclohexanesulfamate, lithium dodecyl sulfate, lithium ethane- 1,2-disulfonate, lithium ethanesulfonate, lithium 2-hydroxy- ethanesulfonate, lithium formate, lithium fumarate, lithium galactarate, lithium gentisate, lithium glucoheptonate, lithium D-gluconate, lithium D-glucuronate, lithium L-glutamate, lithium a-oxoglutarate, lithium glycolate, lithium hippurate, lithium (+)-L-lactate, lithium (±)-DL-lactate, lithium lactobionate, lithium laurate, lithium (-)-L-malate, lithium maleate, lithium malonate, lithium (±)-DL-mandelate, lithium methanesulfonate, lithium naphthalene- 2-sulfonate, lithium naphthalene- 1,5-disulfonate, lithium 1 -hydroxy-2-naphthoate, lithium nicotinate, lithium oleate, lithium orotate, lithium oxalate, lithium palmitate, lithium pamoate, lithium L-pyroglutamate, lithium saccharate, lithium salicylate, lithium 4-amino-salicylate, sebacic acid, lithium stearate, lithium succinate, lithium tannate, lithium (+)-L-tartarate, lithium thiocyanate, lithium p-toluenesulfonate, lithium undecylenate, or lithium valerate. In some embodiments, the organic lithium salt for use in these embodiments is lithium (S)-2- alkylthio-2-phenylacetate or lithium (R)-2-alkylthio-2-phenylacetate {e.g., wherein the alkyl is C2-C22 straight chain alkyl, preferably C8-16). See, e.g., International Patent Application Publication No. WO 2009/019385, published February 12, 2009, which is incorporated herein by reference in its entirety.
[00166] In some embodiments, the organic lithium salts used for intermittent lithium treatment or a pulse lithium treatment comprise the lithium salts of acetic acid, 2,2- dichloroacetic acid, acetylsalicylic acid, acylated amino acids, adipic acid, hyaluronic acid and derivatives thereof, polyacrylic acid and derivatives thereof, chondroitin sulfate and derivatives thereof, poly(lactic acid-co-glycolic acid), poly(lactic acid), poly(glycolic acid), pegylated lactic acid, stearic acid, linoleic acid, oleic acid, taurocholic acid, cholic acid, glycocholic acid, deoxycholic acid, alginic acid and derivatives thereof, anionic derivatives of polysaccharides, poly(sebacic anhydride)s and derivatives thereof, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(15)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, cyclohexanesulfamic acid, dodecylsulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D- glucuronic acid, L-glutamic acid, a-oxoglutaric acid, glycolic acid, hippuric acid, (+)-L-lactic acid, (±)-DL-lactic acid, lactobionic acid, lauric acid, maleic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene- 1,5-disulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, L-pyroglutamic acid, saccharic acid, salicylic acid, 4-amino- salicylic acid, sebacic acid, stearic acid, succinic acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, or valeric acid. Other organic lithium salts for use in these embodiments is the lithium salt of (S)-2-alkylthio-2-phenylacetic acid or the lithium salt of (R)-2-alkylthio-2-phenylacetic acid {e.g., wherein the alkyl is C2- C22 straight chain alkyl, preferably C8-16). See, e.g., International Patent Application Publication No. WO 2009/019385, published February 12, 2009, which is incorporated herein by reference in its entirety.
5.1.2.1 SUSTAINED RELEASE ORGANTC ETTHTTJM SALTS
[00167] In some embodiments, the organic lithium salt can be modified to create sustained release lithium salts. Due to the size of the lithium ion, it is possible that the residence time of ion at the treatment site will be short. In efforts to generate sustained release lithium salts, the hydrophobicity of the salt can be enhanced and made "lipid-like," to, for example, lower the rate of ionization of the salt into lithium ions. For example, lithium chloride has a much faster rate of ionizing into lithium ions, than lithium stearate or lithium orotate. In that regard, the lithium salt can be that of a cholesterol derivative, or a long chain fatty acids or alcohols. Lipid complexed lithium salts of size less than 10 microns can also be effectively targeted to the hair follicles and "tethered" to the sebaceous glands, by hydrophobic-hydrophobic interactions.
[00168] In some embodiments, the organic lithium salt can be in the form of complexes with anionic compounds or anionic poly(amino acids) and other polymers. The complexes can be neutral, wherein all of the negative charges of the complexation agent are balanced by equimolar concentrations of Li ions. The complexes can be negatively charged, with Lithium ions bound to an anionic polymer. The complexes can be in the form of nano-complexes, or micro-complexes, small enough to be targeted to the hair follicles. If the complexes are targeted to the dermis, the charged nature of the complexes will "tether" the complexes to the positively charged collagen. This mode of tethering holds the Li ions at the site of delivery, thereby hindering fast in-vivo clearance. Examples of negatively charged polymers that can be used in this application are poly(acrylates) and its copolymers and derivatives thereof, hyaluronic acid and its derivatives, alginate and its derivatives, etc. In one variation, the anionic lithium complexes formed as described above can be further complexed with a cationic polymer such as chitosan, or polyethylimine form cell-permeable delivery systems.
[00169] In some embodiments, particularly for administration of the lithium formulation to the skin, the salt can be that of a fatty acid, e.g. , lithium stearate, thereby promoting absorption through skin tissues and extraction into the lipid compartments of the skin. In another example, the lithium salt of sebacic acid can be administered to the skin for higher absorption and targeting into structures of the skin, such as hair follicles.
5.1.3 INORGANIC LITHIUM SALTS
[00170] In some embodiments, the lithium salts are inorganic lithium salts. Inorganic lithium salts for use in these embodiments include halide salts, such as lithium bromide, lithium chloride, lithium fluoride, or lithium iodide. In one embodiment, the inorganic lithium salt is lithium fluoride. In another embodiment, the inorganic lithium salt is lithium iodide. In certain embodiments, the lithium salts do not comprise lithium chloride. Other inorganic lithium salts for use in these embodiments include lithium borate, lithium nitrate, lithium perchlorate, lithium phosphate, or lithium sulfate.
[00171] In some embodiments, the inorganic lithium salts used for intermittent lithium treatment or a pulse lithium treatment comprise the lithium salts of boric acid, hydrobromic acid, hydrochloric acid, hydrofluoric acid, hydroiodic acid, nitric acid, perchloric acid, phosphoric acid, or sulfuric acid.
5.2 LITHIUM FORMULATIONS AND MODES OF DELIVERY
5.2.1 LITHIUM FORMULATIONS
[00172] The lithium compounds used for intermittent lithium treatment or a pulse lithium treatment may be formulated with a pharmaceutically acceptable carrier (also referred to as a pharmaceutically acceptable excipients), i.e., a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, an encapsulating material, or a complexation agent. In one embodiment, each component is "pharmaceutically acceptable" in the sense of being chemically compatible with the other ingredients of a pharmaceutical formulation, and biocompatible, when in contact with the biological tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio. See, Remington: The Science and Practice of Pharmacy, 2005, 21st ed., Philadelphia, PA: Lippincott Williams & Wilkins; Rowe et al, eds., 2005, Handbook of Pharmaceutical Excipients, 5th ed., The Pharmaceutical Press and the American
Pharmaceutical Association; Ash & Ash eds., 2007, Handbook of Pharmaceutical Additives, 3rd ed., Gower Publishing Company; Gibson ed., 2009, Pharmaceutical Preformulation and Formulation, 2nd ed., Boca Raton, FL: CRC Press LLC, each of which is incorporated herein by reference.
[00173] Suitable excipients are well known to those skilled in the art, and non-limiting examples of suitable excipients are provided herein. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art, including, but not limited to, the method of administration. For example, forms for topical administration such as a cream may contain excipients not suited for use in transdermal or intravenous administration. The suitability of a particular excipient depends on the specific active ingredients in the dosage form.
Exemplary, non-limiting, pharmaceutically acceptable carriers for use in the lithium formulations described herein are the cosmetically acceptable vehicles provided in
International Patent Application Publication No. WO 2005/120451, which is incorporated herein by reference in its entirety.
[00174] The lithium compounds suitable for use in intermittent lithium treatments or a pulse lithium treatment may be formulated to include an appropriate aqueous vehicle, including, but not limited to, water, saline, physiological saline or buffered saline (e.g., phosphate buffered saline (PBS)), sodium chloride for injection, Ringers for injection, isotonic dextrose for injection, sterile water for injection, dextrose lactated Ringers for injection, sodium bicarbonate, or albumin for injection. Suitable non-aqueous vehicles include, but are not limited to, fixed oils of vegetable origin, castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenated soybean oil, and medium-chain triglycerides of coconut oil, lanolin oil, lanolin alcohol, linoleic acid, linolenic acid and palm seed oil. Suitable water- miscible vehicles include, but are not limited to, ethanol, wool alcohol, 1,3-butanediol, liquid polyethylene glycol (e.g., polyethylene glycol 300 and polyethylene glycol 400), propylene glycol, glycerin, N-methyl-2-pyrrolidone (ΝΜΡ), N,N-dimethylacetamide (DMA), and dimethyl sulfoxide (DMSO). In one embodiment, the water-miscible vehicle is not DMSO.
[00175] The lithium compounds for use in the methods disclosed herein may also be formulated with one or more of the following additional agents. Suitable antimicrobial agents or preservatives include, but are not limited to, alkyl esters of p-hydroxybenzoic acid, hydantoins derivatives, propionate salts, phenols, cresols, mercurials, phenyoxyethanol, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride (e.g. , benzethonium chloride), butyl, methyl- and propyl-parabens, sorbic acid, and any of a variety of quarternary ammonium compounds. Suitable isotonic agents include, but are not limited to, sodium chloride, glycerin, and dextrose. Suitable buffering agents include, but are not limited to, phosphate, glutamate and citrate. Suitable antioxidants are those as described herein, including ascorbate, bisulfite and sodium metabisulfite. Suitable local anesthetics include, but are not limited to, procaine
hydrochloride, lidocaine and salts thereof, benzocaine and salts thereof and novacaine and salts thereof, and may be used with epinephrine. Suitable suspending and dispersing agents include but are not limited to sodium carboxymethylcelluose (CMC), hydroxypropyl methylcellulose (HPMC), polyvinyl alcohol (PVA), and polyvinylpyrrolidone (PVP).
Suitable emulsifying agents include but are not limited to, including polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate 80, and triethanolamine oleate. Suitable sequestering or chelating agents include, but are not limited to, EDTA. Suitable pH adjusting agents include, but are not limited to, sodium hydroxide, hydrochloric acid, citric acid, and lactic acid. Suitable complexing agents include, but are not limited to, cyclodextrins, including a-cyclodextrin, β-cyclodextrin, hydroxypropyl-P-cyclodextrin, sulfobutylether-β- cyclodextrin, and sulfobutylether 7-P-cyclodextrin (CAPTISOL®, CyDex, Lenexa, KS).
[00176] Soothing preparations, e.g., for topical administration, may contain sodium bicarbonate (baking soda), and coal tar based products. Formulations may also optionally contain a sunscreen or other skin protectant, or a waterproofing agent.
[00177] A product for application to skin may additionally be formulated so that it has easy rinsing, minimal skin/eye irritation, no damage to existing skin or hair, has a thick and/or creamy feel, pleasant fragrance, low toxicity, and good biodegradability. [00178] In particular embodiments, commercially available preparations of lithium can be used, such as, e.g., lithium gluconate - for example, 8% lithium gluconate (Lithioderm™), which is approved for the treatment of seborrheic dermatitis (see, e.g., Dreno and Moyse, 2002, Eur J Dermatol 12:549-552; Dreno et al., 2007, Ann Dermatol Venereol 134:347-351 (abstract); and Ballanger et al, 2008, Arch Dermatol Res 300:215-223, each of which is incorporated by reference herein in its entirety); 8% lithium succinate (see, e.g., Langtry et al, 1996, Clinical and Experimental Dermatology 22:216-219; and Cuelenaere et al, 1992, Dermatology 184: 194-197, each of which is incorporated by reference herein in its entirety); or 8% lithium succinate with 0.05% zinc sulfate (marketed in the U.K. as Efalith; see, e.g., Efalith Multicenter Trial Group, 1992, J Am Acad Dermatol 26:452-457, which is incorporated by reference herein in its entirety). In some embodiments, a preparation of lithium or lithium salt comprises an anionic polymer (such as, e.g., crosslinked polyacrylic acid), which may form a gel. For example, a preparation provided in the examples of Sections 16-19 below may be used.
5.2.2 MODIFIED RELEASE FORMS
[00179] The lithium compounds for use in intermittent lithium treatments or a pulse lithium treatment can be formulated as a modified release dosage form. As used herein, the term "modified release" refers to a dosage form in which the rate or place of release of the lithium or other active ingredient(s) is different from that of an immediate dosage form when administered by the same route. Modified release dosage forms include, but are not limited to, delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated- and fast-, targeted-, programmed-release, and gastric retention dosage forms. The compositions in modified release dosage forms can be prepared using a variety of modified release devices and methods known to those skilled in the art, including, but not limited to, matrix controlled release devices, osmotic controlled release devices, multiparticulate controlled release devices, ion-exchange resins, enteric coatings, multilayered coatings, microspheres, liposomes, and combinations thereof. The release rate of the active ingredient(s) can also be modified by varying the particle sizes and polymorphism of the active ingredient(s). In some embodiments, the controlled release is achieved by using an adjuvant that causes a depot effect, i.e., that causes an active agent or antigen to be released slowly, leading to prolonged exposure to a target cell or tissue (e.g., cells of the follicle, or, in the case of
immunostimulatory adjuvants, prolonged exposure to the immune system). [00180] Examples of formulations for modified release to skin or hair include those described in International Patent Application Publication No. WO 2008/1 15961, published September 25, 2008, which is incorporated herein by reference in its entirety. Other examples of modified release include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598, 123; 4,008,719; 5,674,533; 5,059,595;
5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,639,480; 5,733,566; 5,739, 108; 5,891,474; 5,922,356; 5,958,458; 5,972,891; 5,980,945; 5,993,855; 6,045,830; 6,087,324; 6,1 13,943; 6,197,350; 6,248,363; 6,264,970; 6,267,981; 6,270,798; 6,375,987; 6,376,461; 6,419,961; 6,589,548; 6,613,358; 6,623,756; 6,699,500; 6,793,936; 6,827,947; 6,902,742; 6,958, 161; 7,255,876; 7,416,738; 7,427,414; 7,485,322; Bussemer et al. , Crit. Rev. Ther. Drug Carrier Syst. 2001, 18, 433-458; Modified-Release Drug Delivery Technology, 2nd ed.; Rathbone et al, Eds.; Marcel Dekker AG: 2005; Maroni et al, Expert. Opin. Drug Deliv. 2005, 2, 855-871 ; Shi et al, Expert Opin. Drug Deliv. 2005, 2, 1039-1058; Polymers in Drug Delivery; Ijeoma et al, Eds.; CRC Press LLC: Boca Raton, FL, 2006; Badawy et al., J.
Pharm. Sci. 2007, 9, 948-959; Modified-Release Drug Delivery Technology, supra; Conway, Recent Pat. Drug Deliv. Formul. 2008, 2, 1-8; Gazzaniga et al, Eur. J. Pharm. Biopharm. 2008, 68, 1 1-18; Nagarwal et al, Curr. Drug Deliv. 2008, 5, 282-289; Gallardo et al, Pharm. Dev. Technol. 2008, 13, 413-423; Chrzanowski, AAPS PharmSciTech. 2008, 9, 635-638; Chrzanowski, AAPS PharmSciTech. 2008, 9, 639-645; Kalantzi et al, Recent Pat. Drug Deliv. Formul. 2009, 3, 49-63; Saigal et al, Recent Pat. Drug Deliv. Formul. 2009, 3, 64-70; and Roy et al, J. Control Release 2009, 134, 74-80, each of which is incorporated by reference herein in its entirety.
5.2.2.1 MATRTX CONTROT T FD RFT F ASF DFVTCFS
[00181] The modified release dosage form can be fabricated using a matrix controlled release device known to those skilled in the art. See, Takada et al, 1999, in Encyclopedia of Controlled Drug Delivery, Mathiowitz E, ed., Vol. 2, Wiley.
[00182] In certain embodiments, the modified release dosage form is formulated using an erodible matrix device, which is water-swellable, erodible, or soluble polymers, including, but not limited to, synthetic polymers, and naturally occurring polymers and derivatives, such as polysaccharides and proteins. Materials useful in forming an erodible matrix include, but are not limited to, chitin, chitosan, dextran, and pullulan; gum agar, gum arabic, gum karaya, locust bean gum, gum tragacanth, carrageenans, gum ghatti, guar gum, xanthan gum, and scleroglucan; starches, such as dextrin and maltodextrin; hydrophilic colloids, such as pectin; phosphatides, such as lecithin; alginates; propylene glycol alginate; gelatin; collagen;
cellulosics, such as ethyl cellulose (EC), methylethyl cellulose (MEC), carboxymethyl cellulose (CMC), CMEC, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), cellulose acetate (CA), cellulose propionate (CP), cellulose butyrate (CB), cellulose acetate butyrate (CAB), CAP, CAT, hydroxypropyl methyl cellulose (HPMC), HPMCP, HPMCAS, hydroxypropyl methyl cellulose acetate trimellitate (HPMCAT), and ethyl hydroxyethyl cellulose (EHEC); polyvinyl pyrrolidone; polyvinyl alcohol; polyvinyl acetate; glycerol fatty acid esters; polyacrylamide; polyacrylic acid; copolymers of ethacrylic acid or methacrylic acid (EUDRAGIT®, Rohm America, Inc., Piscataway, NJ); poly(2-hydroxyethyl- methacrylate); polylactides; copolymers of L-glutamic acid and ethyl-L-glutamate;
degradable lactic acid-glycolic acid copolymers; poly-D-(-)-3-hydroxybutyric acid; and other acrylic acid derivatives, such as homopolymers and copolymers of butylmethacrylate, methyl methacrylate, ethyl methacrylate, ethylacrylate, (2-dimethylaminoethyl)methacrylate, and (trimethylaminoethyl)methacrylate chloride.
[00183] In certain embodiments, the compositions are formulated with a non-erodible matrix device. The active ingredient(s) is dissolved or dispersed in an inert matrix and is released primarily by diffusion through the inert matrix once administered. Materials suitable for use as a non-erodible matrix device include, but are not limited to, insoluble plastics, such as polyethylene, polypropylene, polyisoprene, polyisobutylene, polybutadiene,
polymethylmethacrylate, polybutylmethacrylate, chlorinated polyethylene, polyvinylchloride, methyl acrylate-methyl methacrylate copolymers, ethylene-vinyl acetate copolymers, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, vinyl chloride copolymers with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubbers, epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, ethylene/vinyloxyethanol copolymer, polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, silicone rubbers, polydimethylsiloxanes, and silicone carbonate copolymers; hydrophilic polymers, such as ethyl cellulose, cellulose acetate, crospovidone, and cross- linked partially hydrolyzed polyvinyl acetate; and fatty compounds, such as carnauba wax, microcrystalline wax, and triglycerides.
[00184] In a matrix controlled release system, the desired release kinetics can be controlled, for example, via the polymer type employed, the polymer viscosity, the particle sizes of the polymer and/or the active ingredient(s), the ratio of the active ingredient(s) versus the polymer, and other excipients or carriers in the compositions. [00185] The modified release dosage forms can be prepared by methods known to those skilled in the art, including direct compression, dry or wet granulation followed by compression, and melt-granulation followed by compression.
5.2.2.2 OSMOTTC CONTROLLED RELEASE DEVICES
[00186] The modified release dosage form can be fabricated using an osmotic controlled release device, including, but not limited to, one-chamber system, two-chamber system, asymmetric membrane technology (AMT), and extruding core system (ECS). In general, such devices have at least two components: (a) a core which contains an active ingredient; and (b) a semipermeable membrane with at least one delivery port, which encapsulates the core. The semipermeable membrane controls the influx of water to the core from an aqueous environment of use so as to cause drug release by extrusion through the delivery port(s).
[00187] In addition to the active ingredient(s), the core of the osmotic device optionally includes an osmotic agent, which creates a driving force for transport of water from the environment of use into the core of the device. One class of osmotic agents is water- swellable hydrophilic polymers, which are also referred to as "osmopolymers" and
"hydrogels." Suitable water-swellable hydrophilic polymers as osmotic agents include, but are not limited to, hydrophilic vinyl and acrylic polymers, polysaccharides such as calcium alginate, polyethylene oxide (PEO), polyethylene glycol (PEG), polypropylene glycol (PPG), poly(2-hydroxyethyl methacrylate), poly(acrylic) acid, poly(methacrylic) acid,
polyvinylpyrrolidone (PVP), crosslinked PVP, polyvinyl alcohol (PVA), PVA/PVP copolymers, PVA/PVP copolymers with hydrophobic monomers such as methyl methacrylate and vinyl acetate, hydrophilic polyurethanes containing large PEO blocks, sodium croscarmellose, carrageenan, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), carboxymethyl cellulose (CMC) and carboxyethyl, cellulose (CEC), sodium alginate, polycarbophil, gelatin, xanthan gum, and sodium starch glycolate.
[00188] The other class of osmotic agents is osmogens, which are capable of imbibing water to affect an osmotic pressure gradient across the barrier of the surrounding coating. Suitable osmogens include, but are not limited to, inorganic salts, such as magnesium sulfate, magnesium chloride, calcium chloride, sodium chloride, lithium chloride, potassium sulfate, potassium phosphates, sodium carbonate, sodium sulfite, lithium sulfate, potassium chloride, and sodium sulfate; sugars, such as dextrose, fructose, glucose, inositol, lactose, maltose, mannitol, raffinose, sorbitol, sucrose, trehalose, and xylitol; organic acids, such as ascorbic acid, benzoic acid, fumaric acid, citric acid, maleic acid, sebacic acid, sorbic acid, adipic acid, edetic acid, glutamic acid, p-toluenesulfonic acid, succinic acid, and tartaric acid; urea; and mixtures thereof.
[00189] Osmotic agents of different dissolution rates can be employed to influence how rapidly the active ingredient(s) is initially delivered from the dosage form. For example, amorphous sugars, such as MA OGEM™ EZ (SPI Pharma, Lewes, DE) can be used to provide faster delivery during the first couple of hours to promptly produce the desired therapeutic effect, and gradually and continually release of the remaining amount to maintain the desired level of therapeutic or prophylactic effect over an extended period of time. In this case, the active ingredient(s) is released at such a rate to replace the amount of the active ingredient metabolized and excreted.
[00190] The core can also include a wide variety of other excipients and carriers as described herein to enhance the performance of the dosage form or to promote stability or processing.
[00191] Materials useful in forming the semipermeable membrane include various grades of acrylics, vinyls, ethers, polyamides, polyesters, and cellulosic derivatives that are water- permeable and water-insoluble at physiologically relevant pHs, or are susceptible to being rendered water-insoluble by chemical alteration, such as crosslinking. Examples of suitable polymers useful in forming the coating, include plasticized, unplasticized, and reinforced cellulose acetate (CA), cellulose diacetate, cellulose triacetate, CA propionate, cellulose nitrate, cellulose acetate butyrate (CAB), CA ethyl carbamate, CAP, CA methyl carbamate, CA succinate, cellulose acetate trimellitate (CAT), CA dimethylaminoacetate, CA ethyl carbonate, CA chloroacetate, CA ethyl oxalate, CA methyl sulfonate, CA butyl sulfonate, CA p-toluene sulfonate, agar acetate, amylose triacetate, beta glucan acetate, beta glucan triacetate, acetaldehyde dimethyl acetate, triacetate of locust bean gum, hydroxylated ethylene-vinylacetate, EC, PEG, PPG, PEG/PPG copolymers, PVP, HEC, HPC, CMC, CMEC, HPMC, HPMCP, HPMCAS, HPMCAT, poly(acrylic) acids and esters and poly- (methacrylic) acids and esters and copolymers thereof, starch, dextran, dextrin, chitosan, collagen, gelatin, polyalkenes, polyethers, polysulfones, polyethersulfones, polystyrenes, polyvinyl halides, polyvinyl esters and ethers, natural waxes, and synthetic waxes.
[00192] A semipermeable membrane can also be a hydrophobic microporous membrane, wherein the pores are substantially filled with a gas and are not wetted by the aqueous medium but are permeable to water vapor, as disclosed in U.S. Pat. No. 5,798, 1 19. Such hydrophobic but water- vapor permeable membrane are typically composed of hydrophobic polymers such as polyalkenes, polyethylene, polypropylene, polytetrafluoroethylene, polyacrylic acid derivatives, polyethers, polysulfones, polyethersulfones, polystyrenes, polyvinyl halides, polyvinylidene fluoride, polyvinyl esters and ethers, natural waxes, and synthetic waxes.
[00193] The delivery port(s) on the semipermeable membrane can be formed post-coating by mechanical or laser drilling. Delivery port(s) can also be formed in situ by erosion of a plug of water-soluble material or by rupture of a thinner portion of the membrane over an indentation in the core. In addition, delivery ports can be formed during coating process, as in the case of asymmetric membrane coatings of the type disclosed in U.S. Pat. Nos.
5,612,059 and 5,698,220.
[00194] The total amount of the active ingredient(s) released and the release rate can substantially by modulated via the thickness and porosity of the semipermeable membrane, the composition of the core, and the number, size, and position of the delivery ports.
[00195] An osmotic controlled-release dosage form can further comprise additional conventional excipients or carriers as described herein to promote performance or processing of the formulation. The osmotic controlled-release dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art. See Remington: The Science and Practice of Pharmacy, supra; Santus and Baker, J. Controlled Release 1995, 35, 1-21 ; Verma et al., Drug Development and Industrial Pharmacy 2000, 26, 695-708; and Verma et al., J. Controlled Release 2002, 79, 7-27.
[00196] In certain embodiments, the compositions are formulated as AMT controlled- release dosage form, which comprises an asymmetric osmotic membrane that coats a core comprising the active ingredient(s) and other pharmaceutically acceptable excipients or carriers. See, U.S. Patent No. 5,612,059 and International Publication No. WO 2002/17918. The AMT controlled-release dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art, including direct compression, dry granulation, wet granulation, and a dip-coating method. In certain embodiments, the compositions are formulated as ESC controlled-release dosage form, which comprises an osmotic membrane that coats a core comprising the active ingredient(s), a hydroxylethyl cellulose, and other pharmaceutically acceptable excipients or carriers.
5.2.2.3 IN ,S7777GFJ J JNG DRUG DFT TVFRY SYSTEMS
[00197] In one embodiment, the lithium-containing compound can be loaded into a polymeric solution that consists of a water-soluble polymer that is a solution at room temperature (20-25°C) and below, but gels at physiological temperatures of 32-37°C. In one application the lithium-containing solution can be cooled to 2-8°C to impart a soothing effect, while being sprayed as a liquid spray on the tissue surface. Once sprayed on, the lithium- loaded solution will thicken into a gel, releasing the lithium-containing compound slowly over time. Examples of these thermo-gelling polymers are poly(isopropyl acrylamide), poly(EO)x-(PO)y-(EO)x and poly(PO)x-(EO)y-(PO)x, wherein EO=ethylene oxide and PO=propylene oxide. Other examples include, but are not limited to, PLA-PEO-PLA polymers, wherein PLA=polylactic acid, PEO=polyethylene oxide, poly(sebacic anhydride)- poly(ethylene oxide)-poly(sebacic anhydride) and poly(stearate)-poly(ethylene oxide)- poly(stearate). In a variation of the idea, the lithium-loaded solution can be injected as a liquid, to form an in situ depot within the tissue. In another variation of the concept, the lithium-loaded solution can be delivered as a solution, which can flow into orifices of the tissue, such as hair follicles and then, form a gel to release lithium for follicle-associated conditions, such as MPHL, folliculitis, or another condition described herein. The temperature and time of gelation can be correlated to the concentration of the polymers and the length of the polymer blocks that constitute the polymers.
5.2.2.4 MUETTPARTTCUEATE CONTROLLED RELEASE DEVTCES
[00198] The a modified release dosage form can be fabricated as a multiparticulate controlled release device, which comprises a multiplicity of particles, granules, or pellets, ranging from about 10 μιη to about 3 mm, about 50 μιη to about 2.5 mm, or from about 100 μιη to about 1 mm in diameter. Such multiparticulates can be made by the processes known to those skilled in the art, including microfluidization, membrane-controlled emulsification, oil-in-water, water-oil-water and oil-in oil emulsification and homogenization processes, complex coacervation, wet-and dry-granulation, extrusion/spheronization, roller-compaction, melt-congealing, and by spray-coating seed cores. See, for example, Ghebre-Sellassie, ed., 1994, Multiparticulate Oral Drug Delivery, Marcel Dekke; and Ghebre-Sellassie ed., 1989, Pharmaceutical Pelletization Technology, Marcel Dekker.
[00199] Other excipients or carriers as described herein can be blended with the compositions to aid in processing and forming the multiparticulates. The resulting particles can themselves constitute the multiparticulate device or can be coated by various film- forming materials, such as enteric polymers, water-swellable, and water-soluble polymers. The multiparticulates can be further processed as a capsule or a tablet. 5.2.3 TARGETED DELIVERY
[00200] The lithium compounds for use herein may be formulated with a carrier that delivers the lithium to the site of action, for example, a follicle in a particular tissue. Such targeted delivery may be preferable in formulations for systemic administration, in order to reduce side effects associated with lithium therapy and/or ensure that the lithium reaches only follicles of particular tissues. The carrier may be an aptamer targeted to a particular protein or cell type in the follicle, an antibody or antigen-binding fragment thereof, a virus, virus-like particle, virosome, liposome, micelle, microsphere, nanoparticle, or any other suitable compound.
[00201] Compositions for use in the methods provided herein can also be formulated to be targeted to a particular tissue, follicle, or other area of the body of the subject to be treated, including liposome-, resealed erythrocyte-, and antibody-based delivery systems. Examples include, but are not limited to, those disclosed in U.S. Pat. Nos. 5,709,874; 5,759,542;
5,840,674; 5,900,252; 5,972,366; 5,985,307; 6,004,534; 6,039,975; 6,048,736; 6,060,082; 6,071,495; 6,120,751; 6,131,570; 6,139,865; 6,253,872; 6,271,359; 6,274,552; 6,316,652; and 7, 169,410.
[00202] In some embodiments, targeting is accomplished by the attachment of specific targeting moieties to the delivery systems containing the drug. Targeting moieties can be in the form of antibodies, aptamers or small molecules that bind to specific proteins expressed in specific tissues. Specific or guided targeting can "channel" the drug only to the specific tissue type, thus minimizing distribution to all tissues. This concept is especially useful if the drug causes side effects. For hair follicle drug delivery, microspheres and nanospheres have been utilized, to deliver drugs into the hair follicle. Entry into the hair follicle is governed by the size of the drug-containing spheres, with microspheres of size 0.5-0.7 microns of ideal size for entry. However, out- flux of sebaceous fluid from the hair follicle can result in a short residence time of the delivery systems in the follicle. To minimize this, the surface of the microspheres can be functionalized with moieties that bind to specific surfaces in the follicular orifice to "retain" them at the site. These moieties can be non-specific, such as hydrophobic coatings, or cationic coatings, in order to be bioadhesive to cells within the follicle. The moieties can be specific and targeted to certain proteins that are expressed specifically on specific cell membranes. For example, proteins over-expressed on the follicular lymphoma cell surfaces can be targeted by delivery systems that have antibodies or aptamers designed to bind to these proteins. The surface of the delivery systems can also be functionalized with cell-penetrating moieties such as cell-permeable peptides, positively charged polymers that bind to anionic cell surfaces.
5.2.4 LOCAL DELIVERY
[00203] Common side effects of systemic lithium treatment include muscle tremors, twitching, ataxia, and hypothyroidism. Long term use has been linked to
hyperparathyroidism, hypercalcemia (bone loss), hypertension, kidney damage, nephrogenic diabetes insipidus (polyuria and polydipsia), seizures, and weight gain. There also appears to be an increased risk of Ebstein (cardiac) Anomaly in infants born to women taking lithium during the first trimester of pregnancy. In order to circumvent these side effects, the dosage of systemically administered lithium is tightly controlled. The intermittent lithium treatments or a pulse lithium treatment described herein may have a decreased risk of such side effects because of the intermittent or temporary nature of the treatment. Another way in which such side effects may be circumvented is to deliver the intermittent lithium treatment or a pulse lithium treatment locally to the site where hair growth modulation is desired.
[00204] The intermittent lithium treatments or a pulse lithium treatment described herein may be delivered locally to any part of the subject in which wound healing or scar revision is desired, including, e.g., the head (e.g., the scalp, cheek, chin, lips, ears, nose, eyelid or eyebrow), neck, abdomen, chest, breast, back, arms, armpits, stomach, genital area, buttocks, legs, hands, or feet of a subject. In one embodiment, the intermittent lithium treatment or a pulse lithium treatment is applied to wounded or scarred skin. In one embodiment, the intermittent lithium treatment or a pulse lithium treatment is applied before the skin is wounded or scarred.
[00205] Such local delivery of the intermittent lithium treatment or a pulse lithium treatment can be achieved by topical administration, transdermal, intradermal, subcutaneous (depot effect), or by intramuscular, intravenous and oral routes of delivery in formulations for targeting systemically delivered lithium to desired follicles. Such modes of delivery are discussed supra.
5.2.5 DELIVERY VIA SCAFFOLDS FOR MODULATING WOUND HEALING
[00206] In some embodiments, enhancement of wound healing or scar revision in wounded or otherwise integumentally perturbed skin (such as, e.g., as occurs during scar revision; see Section 5.4.1 infra) is accomplished by a lithium treatment described herein in combination with a pre-designed biomaterial dressing that may serve as a substrate to encourage a step-wise attachment of keratinocytes and epithelial cells to it, such that formation of an organized extra-cellular matrix (ECM) is enhanced in order to promote wound healing. Without being bound by any theory, formation of an organized extracellular matrix leads to less granular epithelialization of the wound and, therefore, less scarring. Furthermore, and also without being bound by any theory, it is thought that the presence of a "scaffold" at the wounded or perturbed site prevents rapid wound contraction, whereupon the edges of the wound contract in a rapid, haphazard manner to produce granular collagen-rich skin devoid of any adnexal structures such as follicles or sweat glands, and rapid wound contraction by secondary intention almost always results in fibrous tissue that is sub-optimal in temperature regulation, tensile and compressive strength and barrier function.
[00207] The scaffold for use in combination with lithium treatment may be comprised of a mesh of a biocompatible, bioabsorbable material that cells recognize and attach to, preferably with ease. For example, these materials can be collagen type I/III, hyaluronic acid, chitosan, alginates, or combinations and derivatives thereof or any other such material described herein or known in the art. The mesh scaffold may be neutral, or charged. If the mesh is positively charged, it may permit cells (which are negatively charged) to adhere to it more effectively. If the mesh scaffold is negatively charged, it may contain signaling moieties that the cells will recognize and attach to. For example, polymers such as hyaluronic acid are present already in skin, and thus a mesh comprised of this material is thought to be compatible with cells.
[00208] In some embodiments, the scaffold is pre-fabricated with a fine microstructure that is of the dimension of cells, for example, red blood cells that will initially diffuse throughout the scaffold, or epithelial cells and keratinocytes from surrounding tissue.
Moreover, it is envisioned that the "epithelial tongue" can move with greater ease and organization by crawling on the scaffold mesh.
[00209] In some embodiments, the mesh scaffold has an "open-cell" structure, with the pores inter-connected, much like an open-celled foam. The open, interconnecting nature of the scaffold may allow free diffusion of oxygen and cells, so that optimal organized wound healing can occur.
[00210] In some embodiments, the mesh scaffold has the capacity to hydrate and remain hydrated throughout the wound healing period. This is useful because, without being bound by any theory, drying out of the wound results in a impermeable granular structure that the keratinocytes cannot "crawl upon." [00211] In some embodiments, the mesh scaffold has moieties that act as molecular signals to the cells, for example, to aid their proliferation. These moieties include, but are not limited to, peptidoglycans and RGD integrin recognition sequences that encourage cell attachment and subsequent proliferation.
[00212] In some embodiments, the mesh scaffold has incorporated within it one or more active agents, for example, a small molecule, or a nucleic acid, or a protein. In some embodiments, the additional active agent is a protein, such as noggin or WNT, or is a nucleic acid that encodes noggin or WNT. In some embodiments, a small molecule is incorporated into the scaffold, such as, e.g. , a GSK inhibitor, BMP inhibitor, or PPAR antagonist.
[00213] In some embodiments, the compound incorporated in the mesh scaffold is a compound considered for use in the combination therapies described herein, for example, in Section 5.4, especially Sections 5.4.2 to 5.4.4. For example, the scaffold may incorporate superoxide dismutase, a free radical quenching molecule that functions in the reduction of inflammation. In other embodiments, compounds are included in the mesh scaffold that alter the kinetics of wound healing, for example, that slow wound healing. Such compounds are known in the art and described elsewhere herein. Other compounds that may be incorporated in the mesh scaffold include growth factors that aid in cell proliferation and tissue regeneration. In some embodiments, the compounds aid in hair follicle migration or hair follicle neogenesis in the wound site.
[00214] In some embodiments, the lithium compound itself is incorporated within the mesh scaffold. In some embodiments, the lithium compound is incorporated within one or more layers of a multilayered mesh scaffold. For example, in one embodiment the mesh scaffold contains the lithium compound in alternating layers, which may achieve a pulsatile delivery of lithium. In some embodiments, the lithium compound in incorporated in microspheres in the scaffold, enabling a controlled release of lithium from the scaffold.
[00215] In another embodiment, the mesh scaffold can be fibrin gels that additionally contain lithium. A fibrin network is the first scaffold that a cell encounters as it performs its role in healing wounds due to trauma or other insults to tissue. Unlike the extracellular matrices and basement membranes that are formed by collagen, laminin and proteoglycans, which assemble slowly in an ordered manner, the fibrin network (the "scab") assemble rapidly by a modified polycondensation reaction from fibrinogen, an abundant constituent of blood plasma, as soon as the protease thrombin is activated in the clotting cascade— the result is a three-dimensional network of branching fibers, What is envisioned is a fibrin delivery matrix containing lithium, fibrinogen and thrombin, that "gels" in-situ. One issue that is encountered is the ability of lithium to diffuse through the fibrin "scab" - making the drug part of the scab solves this issue.
[00216] In another embodiment, the mesh scaffold is a synthetic biodegradable dressing and lithium delivery system that also acts as a "sponge" and absorbs the exudates/bloods from a wound. These exudates intercalating with the synthetic scaffold contain an abundance of fibrinogen, thrombin, fibronectin, cell adhesion proteins, growth factors and hyaluronic acid, all of which create an integrated structure that is an attractive matrix for cell attachment /differentiation and delivery of lithium. The release rate of lithium can be modulated by varying the composition of polymers that comprise the synthetic scaffold, or sponge. For example, a synthetic scaffold fabricated out of poly(lactide)-co-(glycolide) (PLG) and poly(lactide) (PLA) can be developed to have varied release profiles of lithium. Changing the ratio of PLA to PLG will change the release profile of the lithium from the scaffold. Other polymers that can utilized to generate synthetic scaffolds are chitosan, carregenan, alginate, poly(vinyl alcohol), poly(ethylene oxide) (PEO), poly(ethylene oxide)-co-poly(propylene oxide)-co-poly(ethylene oxide) (PEO-PPO-PEO), poly(acrylates) and poly(vinyl pyrrolidone) (PVP). By varying the composition of polymers, the rate of lithium release from the formulation {e.g. , scaffold or sponge) can be controlled, so that it takes anywhere from 2 hours to 30 days for most (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 100%) of the lithium ion to be released. In some embodiments, most of the lithium is released from the formulation within 2 hours, within 4 hours, within 8 hours, within 16 hours, within 24 hours, within 36 hours, within 48 hours, within 3 days, within 5 days, within 7 days, within 10 days, within 14 days, within 30 days, or within 2 months or more. For a specific example of such formulations that may be used in accordance with such embodiments, see the example of Section 19 infra.
[00217] In some embodiments, the mesh scaffold releases the aforementioned compounds in a timed release manner, acting as a controlled release formulation such as described in Section 5.2.2 above. For example, the compounds may be bound to the mesh scaffold, and are then released at a sustained release manner as a result of de-binding kinetics from the mesh. In some embodiments, the compound may be bound to a polymer, which is then incorporated to the mesh scaffold, and which may allow the compound to diffuse from the mesh at a slow rate, resulting in sustained release.
[00218] In some embodiments, the mesh scaffold is extruded as a gel, with certain components of the gel precipitating out to form a mesh in situ. Alternatively, in some embodiments, the in situ mesh can be sprayed on the wounded or otherwise perturbed surface, such as tissue that has been extensively burned. A large area can be covered in this manner.
[00219] In some embodiments, the mesh scaffold is pre-fabricated as a dressing or a wrap, to cover large areas of wounded tissue. In some such embodiments, the mesh scaffold can be cut to size to fit the size of the wound to present a compatible surface for favorable movement of the epithelial tongue.
[00220] In some embodiments, the scaffold is prepared by melt spinning, electrospinning, micromachining, weaving, or other methods known in the art in which open cell foams are fabricated. Using starting materials that are United States Pharmacopeia (USP)-approved, the mesh scaffold can be fabricated by these methods, with the optional incorporation of additional compound(s) (which are optionally sterilized), then sterilized by gentle ethylene oxide sterilization. In some embodiments, the additional compounds are sterilized, and then added to the sterile mesh scaffold.
[00221] In a particular embodiment, a combinatorial strategy that uses a biodegradable scaffold combined with administration of a lithium formulation described herein (alone or in combination with another treatment, such as described in Section 5.4, especially Sections 5.4.2 to 5.4.4) is applied, which may result in the in situ generation of embryonic stem cells or recruitment of cells required for wound healing following wounding. This approach may be used together with a form of integumental perturbation described in Section 5.4.1 {e.g. , dermabrasion accomplished by a standard dermabrader or a laser, deep full-thickness excision (as for deep burns) accomplished by a bulk ablative laser) or integumental perturbation by acute wounds, chronic wounds, or wounds generated for the purpose of scar revision. While not being bound by any theory of how the invention works, such
integumental perturbation in combination with a scaffold that administers drug results in the in situ generation of stem cells or recruitment of other cells required for the wound healing process and may facilitate more effective wound healing with little or no scarring.
5.2.5.1 BIODEGRADABLE PROPERTIES OF THE
SCAFFOT )
[00222] In one embodiment, the scaffold is biodegradable. Placement of a 3-dimensional biodegradable scaffold in the wound assists the attachment, growth and differentiation of cells. Historically, tissue repair has been by autologous cell/tissue transplantation— however, autografts are associated with donor site morbidity and limited availability. An alternative is allografts, but these are susceptible to immune responses and also carry the risk of disease transfer. Thus, tissue engineering has emerged as an interdisciplinary field that makes use of biomaterials, cells and factors either alone, or in combination to restore tissues. The tissue engineering strategy generally involves isolation of healthy cells from a patient, followed by their expansion in vitro. These expanded cells are then seeded onto three-dimensional biodegradable frameworks that provide structural support for the cells and allow cellular infiltration, attachment, proliferation and growth ultimately leading to new tissue. In a sense, natural wound healing utilizes a "scaffold" as well— the fibrin clot. A fibrin network is the natural network that forms rapidly due to a polycondensation reaction from fibrinogen, an abundant constituent of blood plasma, as soon as the protease thrombin is activated in the clotting cascade. The fibrin clot then forms a three-dimensional network for cells to attach, for re-epithelialization.
[00223] In some embodiments, the biodegradability of the scaffold is modulated. Ideally, the biodegradability of the scaffold should be matched to the formation of the new epithelium due to wound healing. One skilled in the art would know how to measure whether a synthetic matrix is biodegradable. For example, biodegradability can be measured ex vivo in implants or using rats or another animal model, by histological and HPLC analysis. In one embodiment, biodegradability by hydrolysis can be assessed. In such an embodiment, the scaffold structure of choice is incubated in phosphate buffered saline, pH 7.4 and 37 °C. For degradation by enzymolysis, the incubation buffer includes enzymes. The scaffolds are weighed prior to incubation. The scaffolds are retrieved two-at-a-time at predetermined time points and dried in a vacuum oven. The scaffolds are weighed at each time point and a plot of weight versus time is generated to develop the rate of biodegradability. In one embodiment, the biodegradability of the scaffold matrix is modulated to coincide with the healing process, and can be modulated by changing the composition of polymers utilized to fabricate the mesh. For example, a percentage of polyethylene glycol (PEG) can be included in a composition with PLG (e.g., described in the example in Section 19) to increase
biodegradation (for example, see ASTM El 279 - 89, 2008, Standard Test Method for Biodegradation By a Shake-Flask Die-Away Method).
5.2.5.2 BTOMTMETTC PROPERTIES OF THE SCAFFOLD
[00224] Biodegradable synthetic matrices can be created to mimic the extra-cellular micro- environment for the enhanced cellular attachment necessary for tissue regeneration. In some embodiments, cell-recognition motifs such as RGD peptides may be incorporated to encourage cells to attach themselves to the scaffold. [00225] One skilled in the art would know how to measure whether the biodegradable synthetic matrix has biomimetic properties. For example, in one embodiment, the biomimetic nature of the scaffold is judged on the basis of the content of the mesh and resultant intercalating fibrin.
5.2.5.3 PHYSTCAE PROPERTIES OF THE SCAFFOLD
[00226] The properties of the synthetic scaffold are dependent upon the three-dimensional geometry, matching of the modulus of the matrix with the tissue type and the porosity. It has been shown that the differentiation process can be modulated if the modulus of the tissue type is matched with the modulus of the scaffold.
[00227] One skilled in the art would know how to measure whether the biodegradable synthetic matrix has optimal physical properties. For example, in one embodiment, the modulus of the scaffold is matched with the modulus of the tissue type. In general, the compressive modulus of a scaffold or hydrogel can be measured by a standard Instron instrument {e.g., using the TA Instruments DMA Q800).
5.2.5.4 BTOCOMPATTBTETTY OF THE SCAFFOT )
[00228] Further, the micro-environment created by the cells is optimally highly biocompatible to the cells present at the site, namely keratinocytes and stem cells derived from the dermal papilla. In one embodiment, this can be accomplished through the use of hydrophilic components that can absorb water. Use of hydrophobic components such as petrolatum is likely to be occlusive and prevent rapid cell proliferation.
[00229] One skilled in the art would know how to measure whether the biodegradable synthetic matrix is biocompatible. For example, in one embodiment, the scaffold is incubated with human foreskin fibroblasts (HFF) in vitro and the scaffold is considered to be biocompatible if the cells maintain their shape and attach appropriately. See, e.g. , the following reference for studies on the biocompatibility of materials: Altankov et ah, 1996, Journal of Biomedical Materials Research Part A; 30:385-391, which is incorporated by reference herein in its entirety.
5.2.5.5 OXYGEN PERMEABTETTY OF THE SCAFFOTT)
[00230] In some embodiments, the biodegradable scaffold is permeable to water, nutrients, oxygen and growth factors, enabling easy exchange of nutrients between tissues and cells (see, e.g., ASTM D39857). In some embodiments, a non-occlusive, non-permeable barrier is avoided.
5.2.5.6 TJTTETTY OF THE SCAFFOLD ΪΝ DEEP WOUNDS
[00231] In one embodiment, the scaffold is used to "fill" a deep wound, as is common in a deep burn, to provide a matrix for the cells to attach, grow and differentiate - existence of the scaffold will likely minimize the scar formation normally observed in deep, large-area wounds.
5.2.5.7 COMBTNED BTOEOGTCAE/SYNTHETTC MESH
[00232] In another embodiment, a loose, dry, highly porous network or scaffold or mesh is placed in the bleeding site of the wound to gently absorb the blood and the cell adhesion proteins released at the site, as a result of wounding. This will result in creation of a highly rich environment that consists of a combination of a 3 -dimensional scaffold combined with fibrinogen and thrombin, which will ultimately result in a highly biocompatible hydrogel suitable for cell attachment and growth. In some embodiment, inclusion of blood components and cell adhesion proteins into the network is critical for establishment of the ECM
(extracellular matrix) necessary to form continuous tissue in-growth, particularly in the case of large-area and deep wounds.
[00233] A dry scaffold has the added advantage of absorbing the blood at the wound site. Thus, a person's own blood components can be used to create a combined synthetic/natural ECM. In practical terms, the scaffold has an added advantage of serving as a blood absorbing gauze.
[00234] In another embodiment, the scaffold has cell-recognition motifs, such as RGD peptides, to recruit cells to the site and attachment, thereof. Once attached, cells will proliferate. Without being bound by any theory, it is hypothesized that the primary attachment of cells to the scaffold is a critical step to prevent premature cell death.
[00235] In one embodiment, a dry, sterile biodegradable scaffold is placed onto the freshly formed wound. The properties of the scaffold will be such that it will transform into an adherent hydrogel upon water absorption.
5.2.5.8 FABRTCATTNG AND APPEYTNG THE SCAFFOTT)
[00236] Methods that may be employed to fabricate the scaffold are known in the art, and include electrospinning, micromachining, and others. Nano-fiber meshes fabricated by electrospinning, hydrogel imprint technologies have been utilized to create three-dimensional microstructures that match the supramolecular architecture of the tissue type. In situ forming scaffolds are also contemplated.
[00237] In some embodiments, the active agents (e.g., lithium alone or in a combination described herein) are administered using an active agent-containing spray-on hydrogel. In one such embodiment, after placement of the biodegradable scaffold, the active agent is sprayed on the tissue. The active agent (or combination of active agents, e.g., lithium and another stem cell signaling agent) may be incorporated into a spray-on hydrogel that will be sprayed on as a liquid, but which transforms into a hydrogel after it is sprayed on the tissue. This will be especially useful if the area of the wound is large and uniform coverage is needed.
[00238] In some embodiments, the active agent-containing spray-on hydrogel is applied on the wound site, forming a cross-linked hydrogel that releases active agent over the time period of healing or a shorter or longer time period, as necessary. Depending upon the release characteristics that are required, the active agent will either be incorporated in microencapsulates or nano-encapsulates and suspended into the pre-hydrogel solution. The active agent can also be dissolved into the pre-hydrogel solution. The "pre-hydrogel" solution is defined as the solution that will be sprayed on the tissue and which also contains the active agent.
[00239] In some embodiments, the active agent is contained within microspheres that can be positively charged to rapidly bind themselves to the negatively charged collagen present in the dermis. Binding the microspheres to the dermis renders the active agent-releasing moiety immobile at the site.
[00240] In a variation of the foregoing embodiments, the wound may be covered with a breathable, non-occlusive spray-on hydrogel to cover the wound from infection during healing.
5.2.6 MODES OF ADMINISTRATION
[00241] The intermittent lithium treatments or a pulse lithium treatment can be provided by administration of the lithium compound (or combination treatments, discussed in Section 5.4 infra) in forms suitable for topical {e.g., applied directly to the skin, transdermal, or intradermal), subcutaneous, intramuscular, intravenous or by other parenteral means, oral administration, sublingual administration, or bucchal administration. In some embodiments, the topical {e.g. , applied directly to the skin, transdermal, or intradermal) administration is accomplished with the use of a mechanical device, such as, e.g., an iontophoretic device. The lithium compounds (or combination treatment) can also be formulated as modified release dosage forms, including delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated-, fast-, targeted-, programmed-release, and gastric retention dosage forms. These dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art (see, Rathbone et al, eds., 2008, Remington: The Science and Practice of Pharmacy, supra; Modified-Release Drug Delivery Technology, 2nd ed., New York, NY: Marcel Dekker, Inc.). The intermittent lithium treatments or a pulse lithium treatment can be administered by a health care practitioner or by the subject. In some embodiments, the subject administers the intermittent lithium treatments or a pulse lithium treatment to him or herself.
5.2.6.1 TOPTCAT, ADMTNTSTRATTON
[00242] In a preferred embodiment, topical administration is to the skin, either to the skin surface, transdermally, or intradermally. Topical administration can be with or without occlusion with a bandage or other type of dressing. In some embodiments, topical administration is to orifices or mucosa, or conjunctival, intracorneal, intraocular, ophthalmic, auricular, nasal, vaginal, urethral, respiratory, and rectal administration. The formulation used for topical administration can be designed to retain the lithium in the skin or to deliver a dose of lithium systematically. In some embodiments, topical administration of a lithium compound is combined with another treatment described herein, such as, but not limited to, a technique of integumental perturbation described in Section 5.4.1 infra.
[00243] Dosage forms that are suitable for topical administration for preferably local but also possible systemic effect, include emulsions, solutions, suspensions, creams, gels, hydrogels, ointments, dusting powders, dressings, elixirs, lotions, suspensions, tinctures, pastes, powders, crystals, foams, films, aerosols, irrigations, sprays, suppositories, sticks, bars, ointments, sutures, bandages, wound dressings, microdermabrasion or dermabrasion particles, drops, and transdermal or dermal patches. The topical formulations can also comprise micro- and nano-sized capsules, liposomes, micelles, microspheres, microparticles, nanosystems, e.g., nanoparticles, nano-coacervates and mixtures thereof. See, e.g.,
International Patent Application Publication Nos. WO 2005/107710, published November 17, 2005, and WO 2005/020940, published March 10, 2005, each of which is incorporated herein by reference in its entirety. In one embodiment, the nano-sized delivery matrix is fabricated through a well-defined process, such as a process to produce lithium encapsulated in a polymer. In another embodiment, the lithium-releasing compound is spontaneously assembled in aqueous solutions, such as in liposomes and micelles. In some embodiments, the formulation for topical administration is a shampoo or other hair product, tanning product or sun protectant, skin lotion, or cosmetic.
[00244] The selected formulation will penetrate into the skin and reach the hair follicle. Thus, in some embodiments, the stratum corneum and/or epidermis have been or are removed by a method of integumental perturbation described herein (including by wounding or scar revision procedure, by laser, or by dermabrasion or microdermabrasion, which is a less vigorous form of dermabrasion), permitting application of the dosage form for topical administration directly into the exposed dermis. In some embodiments, the formulation for topical administration will be lipid-based, so that it will penetrate the stratum corneum. In some embodiments, the formulation for topical administration will contain a skin penetrant substance, such as, e.g., propylene glycol or transcutol. See, e.g., International Patent Application Publication No. WO 2004/103353, published December 2, 2004, which is incorporated herein by reference in its entirety. The ability to penetrate into the skin can be tested using any method known in the art, such as, e.g., the method described in International Patent Application Publication No. WO 2005/107710, which is incorporated herein by reference in its entirety. In one embodiment, a formulation in ointment form comprises one or more of the following ingredients: wool alcohol (acetylated lanolin alcohol), hard paraffin, white soft paraffin, liquid paraffin, and water. See, e.g., Langtry et al, supra. In some embodiments, the selected formulation is inconspicuous when applied to the skin, for example, is colorless, odorless, quickly-absorbing, etc. In some embodiments, the selected formulation is applied on the skin surface as a solution, which can crosslink into a hydrogel within a few minutes, thus creating a biocompatible dressing. In one application, the hydrogel may be biodegradable. In another embodiment, the solution will absorb into the skin and crosslink into depots releasing drug. In another embodiment, the lithium ion will be used to crosslink the polymer, with release of the lithium ion controlled by the rate of degradation of the hydrogel.
[00245] Pharmaceutically acceptable carriers and excipients suitable for use in topical formulations include, but are not limited to, aqueous vehicles, water-miscible vehicles, nonaqueous vehicles, antimicrobial agents or preservatives against the growth of
microorganisms, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, penetration enhancers, cryoprotectants, lyoprotectants, thickening agents, and inert gases.
[00246] Forms for topical administration can also be in the form of ointments, creams, and gels. Suitable ointment vehicles include oleaginous or hydrocarbon vehicles, including lard, benzoinated lard, olive oil, cottonseed oil, mineral oil and other oils, white petrolatum, paraffins; emulsifiable or absorption vehicles, such as hydrophilic petrolatum, hydroxystearin sulfate, and anhydrous lanolin; water-removable vehicles, such as hydrophilic ointment; water-soluble ointment vehicles, including polyethylene glycols of varying molecular weight; emulsion vehicles, either water- in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, including cetyl alcohol, glyceryl monostearate, lanolin, wool alcohol (acetylated lanolin alcohol), and stearic acid {see, Remington: The Science and Practice of Pharmacy, supra). These vehicles are emollient but generally require addition of antioxidants and preservatives.
[00247] Suitable cream base can be oil-in-water or water-in-oil. Suitable cream vehicles may be water-washable, and contain an oil phase, an emulsifier, and an aqueous phase. The oil phase is also called the "internal" phase, which is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation may be a nonionic, anionic, cationic, or amphoteric surfactant.
[00248] Gels are semisolid, suspension-type systems. Single-phase gels contain organic macromolecules distributed substantially uniformly throughout the liquid carrier. Suitable gelling agents include, but are not limited to, crosslinked acrylic acid polymers, such as carbomers, carboxypolyalkylenes, and CARBOPOL®; hydrophilic polymers, such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers, and polyvinylalcohol; cellulosic polymers, such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methylcellulose; gums, such as tragacanth and xanthan gum; sodium alginate; and gelatin. In order to prepare a uniform gel, dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing, and/or stirring.
[00249] In particular embodiments, commercially available preparations of lithium can be used for topical administration in the methods described herein. These include, e.g., lithium gluconate, e.g., 8% lithium gluconate (Lithioderm™), approved for the treatment of seborrheic dermatitis {see, e.g., Dreno and Moyse, 2002, Eur J Dermatol 12:549-552; Dreno et al, 2007, Ann Dermatol Venereol 134:347-351 (abstract); and Ballanger et al, 2008, Arch Dermatol Res 300:215-223, each of which is incorporated by reference herein in its entirety); 8% lithium succinate (see, e.g., Langtry et al, 1996, Clinical and Experimental Dermatology 22:216-219; and Cuelenaere et al, 1992, Dermatology 184: 194-197, each of which is incorporated by reference herein in its entirety); or 8% lithium succinate with 0.05% zinc sulfate (marketed in the U.K. as Efalith; see, e.g., Efalith Multicenter Trial Group, 1992, J Am Acad Dermatol 26:452-457, which is incorporated by reference herein in its entirety). Other means of topical administration, including mechanical means
[00250] Other means of topical administration of lithium compounds are also
contemplated. Each of these methods of topical administration may be used alone to administer lithium compounds or in combination with one or more other treatments as described in Section 5.4 infra.
[00251] In some embodiments, topical administration is by electrical current, ultrasound, laser light, or mechanical disruption or integumental perturbation. These include electroporation, RF ablation, laserporation, laser ablation (fractional or non- fractional), non- ablative use of a laser, iontophoresis, phonophoresis, sonophoresis, ultrasound poration, or using a device that accomplishes skin abrasion, or microneedle or needle-free injection, such as topical spray or POWDERJECT™ (Chiron Corp., Emeryville, CA), BIOJECT™ (Bioject Medical Technologies Inc., Tualatin, OR), or JetPeel™ (from TavTech, Tel Aviv, Israel), which uses supersonically accelerated saline to remove epidermis. Means of topical administration that can be used in accordance with the methods described herein are known in the art and are described in, e.g., U.S. Patent Nos. 5,957,895, 5,250,023, 6,306,1 19, 6,726,693, and 6,764,493, and International Patent Application Publication Nos. WO 2009/061349, WO 1999/003521, WO 1996/017648, and WO 1998/011937, each of which is incorporated herein by reference in its entirety.
[00252] In some embodiments, the device for topical administration of lithium compounds is an automatic injection device worn continuously but delivers lithium intermittently. In some embodiments, the device for topical administration of lithium compounds is an automatic injection device that is inconspicuous, for example, can be worn without undue discomfort under clothes, in the hair, under a hairpiece, etc. In some embodiments, a device for administration of the intermittent lithium treatment or a pulse lithium treatment delivers the lithium at a controlled depth in the skin so that it reaches hair follicles, but entry into the circulation is minimized.
[00253] Other methods for administration of the lithium compounds described herein, used alone or in combination with other treatments described in Section 5.4.1 (e.g., in combination with integumental perturbation methods such as dermabrasion, laser treatment, or partial thickness or full thickness excision) include use of a transdermal particle injection system, such as, e.g., a "gene gun." Such systems typically accelerate drug or drug particles to supersonic velocities and "shoot" a narrow stream of drug through the stratum corneum. In some embodiments, the stratum corneum and epidermis is previously removed using a method of integumental perturbation (or by integumental perturbation as a result of wounding) described herein, and thus the required delivery pressures and velocities can be reduced. This reduction reduces the required complexity of the firing mechanisms. In some embodiments, a narrow firing stream is used, particularly to accomplish systemic delivery. In other embodiments, the particle injection system administers the lithium compound over a broad area of skin. An exemplary particle delivery device compatible with broad-based skin delivery (in some embodiments, for use in conjunction with integumental perturbation, wherein the surface of skin to which drug is administered corresponds to the perturbed area) includes a low pressure / low velocity firing mechanism with a spray nozzle designed to deliver to a broad area. For example, a single-shot device that delivers to a 25 -cm2 area could be fired or used multiple times on the scalp or other skin surface until the entire area is treated.
[00254] In another embodiment, a dry particle spraying mechanism similar to an airbrush or miniature grit-blaster can be used to "paint" drug or drug particles onto the perturbed, wounded, or scarred area. In some embodiments, the stratum corneum and epidermis are already removed, e.g., by a method of integumental perturbation (e.g., wounding) described herein, and thus permits effective use of the mechanism using lowered pressure and velocity requirements to achieve dermal delivery.
[00255] In another embodiment, the lithium compound (and/or additional drug) is present in an aqueous suspension, permitting use of standard aerosol spray can technology to deliver the lithium compound to the desired skin area.
[00256] Specific embodiments of modes of administration using a device that combines integumental perturbation and lithium compound delivery follow. An advantage of using such a device is that it offers a convenient one step process for administration of the lithium compound.
[00257] In one embodiment, dermabrasion (e.g., using a mechanical device, including microdermabrasion devices that can be used to dermabrade, or alumina-, silica- or ice-based dermabrasion (as described by Webber, U.S. 6,764,493; U.S. 6,726,693; and U.S. 6,306, 119) is customized to include a drug particle delivery feature using methods readily known in the art. As the device fires ablation particles at the skin, it could also fire smaller drug particles that would simultaneously embed in the exposed dermis. Alternatively, via an internal valve control, the device could switch over to firing drug particles once it is determined that adequate skin disruption has occurred. See, International Patent Application Publication No. WO 2009/061349, which is incorporated herein by reference in its entirety.
[00258] In another embodiment, a standard dermabrasion device can be modified to incorporate any of the devices described above, e.g., a spraying/painting device. In one embodiment, a spray nozzle is located behind the dermabrasion wheel such that drug is sprayed into the dermis as it is exposed by the wheel. Alternatively, the dermabrasion device, via internal controls, could turn off the abrasion wheel once it is determined that adequate skin disruption has occurred, and switch on the drug spray to convert to drug painting mode.
[00259] In one embodiment, a pulsed dye laser (585-595 nm) is combined with drug spraying either before or without skin perturbation, in conjunction with skin perturbation, or following skin perturbation.
[00260] In another embodiment, a non- fractional C(¾ or Erbium- YAG laser is combined with drug spraying either without or before skin disruption, in conjunction with skin disruption, or following skin disruption.
[00261] In another embodiment, a fractional non-ablative laser (e.g., an Erbium- YAG laser used at 1540-1550 nm) is combined with drug spraying either before or without skin perturbation, in conjunction with skin perturbation, or following skin perturbation.
[00262] In another embodiment, a fractional ablative laser (e.g., an Erbium-YAG laser used at 2940 nm or a CO2 laser used at 10,600 nm) is combined with drug spraying either before or without skin perturbation, in conjunction with skin perturbation, or following skin perturbation.
[00263] In another embodiment, fractional ablative laser treatment of the skin (e.g., an Erbium-YAG laser used at 2940 nm or a C(¾ laser used at 10,600 nm) is combined with lithium compound delivery. For example, by invoking inkjet technology, a fractional laser could be combined with a precise delivery means such that as the laser forms a hole in the skin, the inkjet-like delivery component could fill that same hole with drug. One of skill in the art would appreciate that adequate integrated hardware and software controls are required such that the laser ablation and drug delivery are properly timed resulting in each newly formed hole being properly filled with drug. In another embodiment, fractional ablative laser treatment of the skin (e.g., an Erbium-YAG laser used at 2940 nm or a CO2 laser used at 10,600 nm) is combined with lithium compound delivery. For example, by invoking inkjet technology, use of a non-ablative, fractional laser could be combined with a precise delivery means such that as the laser forms a hole in the skin, the inkjet-like delivery component could fill that same hole with drug. One of skill in the art would appreciate that adequate integrated hardware and software controls are required such that the laser treatment and drug delivery are properly timed resulting in each newly formed hole being properly filled with drug.
[00264] In some embodiments, topical administration comprises administration of lithium- containing particles. The particles can be delivered to the skin in combination with any of the means above and described elsewhere infra. Additionally, the particles can be designed for intermittent or pulse delivery of lithium. In one embodiment, particles with different release properties are be delivered simultaneously to achieve pulse delivery.
[00265] In another embodiment, topical administration comprises administration of a lithium-containing formulation that is delivered through channels that are created by the use of needling or micro-needle technology. The formulation can be, e.g., a liquid, a gel or a dry spray. In another variation, topical administration may be through delivery of a lithium- containing formulation through hollow needles.
[00266] In another embodiment, topical administration comprises administration of a lithium-containing formulation that is delivered into the skin by an iontophoretic patch. In one example of this embodiment, a patch can be developed in which the lithium-containing formulation is incorporated.
[00267] In another embodiment, topical administration comprises administration of a lithium-containing formulation that is incorporated into micro-needle shaped biodegradable polymers. In one such embodiment, the biodegradable microneedles penetrate the targeted skin tissue, and are optionally left in place to deliver the lithium ions in a sustained fashion over time.
5.2.6.2 PARENTERAL ADMTNTSTRATTON
[00268] Administration can be parenterally by injection, infusion, or implantation, for local or systemic administration. Parenteral administration, as used herein, includes intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial, intravesical, and subcutaneous administration. Compositions for parenteral administration can be formulated in any dosage forms that are suitable for parenteral administration, including solutions, suspensions, emulsions, micelles, liposomes, microspheres, nanosystems, and solid forms suitable for solutions or suspensions in liquid prior to injection. Such dosage forms can be prepared according to conventional methods known to those skilled in the art of pharmaceutical science (see, Remington: The Science and Practice of Pharmacy, supra). Compositions intended for parenteral administration can include one or more pharmaceutically acceptable carriers and excipients, including, but not limited to, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, antimicrobial agents or preservatives against the growth of microorganisms, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, cryoprotectants, lyoprotectants, thickening agents, pH adjusting agents, and inert gases. All such compositions must be sterile, as known in the art. The compositions for parenteral administration can be formulated as a suspension, solid, semi-solid, or thixotropic liquid, for administration as an implanted depot. In one embodiment, the compositions are dispersed in a solid inner matrix, which is surrounded by an outer polymeric membrane that is insoluble in body fluids but allows the active ingredient in the pharmaceutical compositions diffuse through. Suitable inner matrixes include, but are not limited to, polymethylmethacrylate, polybutyl-methacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers, such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinyl alcohol, and cross-linked partially hydrolyzed polyvinyl acetate.
Suitable outer polymeric membranes include but are not limited to, polyethylene,
polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride, vinyl chloride copolymers with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, and ethylene/vinyloxyethanol copolymer.
5.2.6.3 ORAL ADMTNTSTRATTON
[00269] Pharmaceutical compositions comprising lithium compounds for oral
administration can be provided in solid, semisolid, or liquid dosage forms for oral administration. As used herein, oral administration also includes buccal, lingual, and sublingual administration. Suitable oral dosage forms include, but are not limited to, tablets, fastmelts, chewable tablets, capsules, pills, strips, troches, lozenges, pastilles, cachets, pellets, medicated chewing gum, bulk powders, effervescent or non-effervescent powders or granules, oral mists, solutions, emulsions, suspensions, wafers, sprinkles, elixirs, and syrups. In addition to the active ingredient(s), the pharmaceutical compositions can contain one or more pharmaceutically acceptable carriers or excipients, including, but not limited to, binders, fillers, diluents, disintegrants, wetting agents, lubricants, glidants, coloring agents, dye-migration inhibitors, sweetening agents, flavoring agents, emulsifying agents, suspending and dispersing agents, preservatives, solvents, non-aqueous liquids, organic acids, and sources of carbon dioxide. Compositions for oral administration can be also provided in the forms of liposomes, micelles, microspheres, or nanosystems. Micellar dosage forms can be prepared as described in U.S. Pat. No. 6,350,458.
[00270] In particular embodiments, oral formulations approved for treating mood disorders, e.g., lithium carbonate (L12CO3), sold under several trade names, lithium citrate (L13C6H5O7), lithium sulfate (Li2S04), lithium aspartate, or lithium orotate, may be administered in accordance with the methods described herein.
5.2.7 EX VIVO DELIVERY
[00271] The intermittent and pulse lithium treatments may also be administered to skin- derived cells or skin tissue ex vivo. For example, an intermittent or pulse lithium treatment may be used to enhance the re-association of dissociated hair follicle cells into follicles and their growth and expansion in culture for their implantation into fresh wounds and scar revisions. Thus, in some embodiments, hair follicles promoted by intermittent or pulse lithium treatments are added to the wound before, at the time of, and/or subsequent to, either acute wounding or, more typically, during the wounding that is induced in scar revision. With these methods, traditional approaches to scar revision, such as human skin
transplantation, can be efficiently replaced with transplantation of follicular units or other smaller appendage structures from skin. Thus, hair follicles can be introduced to the wound by migration or de novo hair follicle neogenesis, or by transplanting one or more of the following skin elements: full skin (xeno-; autologous human), follicular units, dissociated cells (donor dominance; recipient effects), ex vivo-expanded skin and/or follicular units, or human skin equivalents in vivo (universal donors). Engineered human skin, or human skin equivalents, can also be used for hair follicle neogenesis and scar revision platforms.
[00272] Human skin equivalents can be grown and assembled in vitro, with the advantage that they can be grown to theoretically to any size/shape; can be comprised of different types of cells, including keratinocytes (hair follicle derived and non-hair follicle derived), dermal cells (hair follicle derived and non-hair follicle derived), other cell types (e.g. , mesenchymal stem cells); can contain cells that are genetically modified to include, e.g., markers or "inducible" signaling molecules; provide an unlimited and uniform source of human cells; from normal skin based on histology and marker studies; are generally devoid of skin appendages; and can be wounded and show similar wound healing events as in vivo.
5.3 LITHIUM TREATMENT REGIMENS
[00273] In the embodiments described herein, the lithium compound or formulation thereof can be administered topically, subcutaneously, orally, etc. Regardless of the route of administration used for lithium ion delivery, the dosing regimen should be adjusted to achieve peak concentrations of lithium in the target skin area of at least about 0.1 mM to 10 mM, and/or peak concentrations of lithium in the blood (serum or plasma samples) of at least about 1 mM (these values are sometimes referred to herein as the "target concentration"). It is noted that, with regard to the concentrations of lithium (including its concentration in formulations, in tissue, in serum, etc., and as a salt form, as an ionized atom in solution, etc.) described herein, since ionized lithium is a monovalent cation, the concentration of lithium expressed in millimolar units (mM) is equal to its concentration expressed in milliequivalents (mEq), as is sometimes used in the art (i.e., 1 mM Li+ = 1 mEq Li+). The peak concentration of lithium can be established by taking samples when peak concentrations are achieved and assaying them for lithium content using techniques well known to those skilled in the art (see, e.g., the examples of Sections 1 1 to 15 and the techniques described therein; see also Wood et al, 1986, Neuropharmacology 25: 1285-1288; and Smith, 1978, Acta Pharmacol et toxicol 43:51-54, each of which is incorporated herein by reference in its entirety). For example, when using oral formulations, samples can be taken when peak blood concentrations are typically achieved - for example, within 1 to 2 hours for standard release formulations, and 4-5 hours for sustained release formulations. The peak concentration times for other formulations, including topical preparations, can be determined for the particular formulation used, and sampling can be adjusted accordingly.
[00274] In some embodiments, the target concentration of lithium should be maintained in the skin and/or blood for at least 1 day; at least 2 days; at least 3 days; at least 5 days; at least 14 days; or at least 21 days; and, in certain embodiments, not more than 21 days. This can be accomplished using, e.g., repeated applications of the lithium compound or a single application of a sustained release or extended release lithium formulation. Either the single pulse protocol or the intermittent treatments can be used to achieve the target concentration of lithium for the shorter maintenance periods (i.e., for at least 1, 2 or 3 days). Maintenance periods longer than 3 days may require repeated application of the intermittent lithium treatments or the single pulse protocol. In some embodiments, it is preferable to allow the concentration of lithium to decline between dosages, in order to achieve a pulsatile effect.
[00275] In some embodiments, topical administration of a lithium compound is preferred over oral or subcutaneous administration. Depending on the formulation used, a topically administered lithium compound may achieve a higher concentration of lithium in skin than in the blood, thereby reducing the risk of toxicity associated with elevated blood levels of lithium. Conversely, and depending on the formulation used, a subcutaneously or orally administered lithium compound may be preferred in order to achieve a controlled release of lithium from the blood to the skin.
[00276] Regardless of the route of administration, care should be taken to avoid toxicity. In this regard, lithium doses should be adjusted on the basis of the blood concentration (serum or plasma) drawn (by convention) 12 or 24 hours after the last dose of the lithium compound; this trough blood concentration should be maintained below 2 mM Li+ and preferably, below about 1.5 mM Li+. In some embodiments, the steady state blood concentration of lithium should not exceed a maximum of 1.5 mM to 2 mM. The relatively stable and characteristic pharmacokinetics of the lithium ion in individual patients makes it possible to predict dosage requirements for that individual based on the results of administration of a single test dose, followed by a skin and/or blood sample assay (plasma or serum) at the peak concentration time; followed by blood sample assays to monitor toxicity at the 12 hour or 24 hour trough concentration; and 24 hours later (when lithium is generally eliminated) which serves as the control value. Once the dose is established for a patient, routine monitoring for toxicity is recommended. For a review of the pharmacokinetics and monitoring of lithium concentrations, see Amdisen, 1980, Ther. Drug. Monit. 2:73-83;
Goodman & Gilman, 1980, "The Pharmacological Basis of Therapeutics" at pp. 430-434; Grandjean & Aubry, 2009, CNS Drugs 23:331-349; and the APA Practice Guideline for the Treatment of Patients with Bipolar Disorder, Second Edition, 2002, each of which is incorporated by reference herein in its entirety.
[00277] In some embodiments, a trough concentration of lithium in the skin of no less than 0.01 mM to 0.05 mM is preferred. In some embodiments, a trough concentration of lithium in the skin of 0.05 mM to 0.1 mM is preferred. In some embodiments, a trough concentration of lithium in the skin of less than 1 mM is preferred. In some embodiments, a trough concentration of lithium in the skin of less than 3 mM is preferred. In some embodiments, lithium concentrations at trough can be increased by twice daily dosing, or more frequent dosing. In such embodiments, topical administration of a lithium compound is preferred. In this regard, a pulsatile effect is achieved by the multiple dosing, but the trough concentrations do not decline as much as when once daily dosing is used. In some embodiments, a trough skin concentration of lithium is maintained at 0.25 mM or higher, for example from 0.25 mM to 0.5 mM or 0.5 mM to 0.75 mM. In some embodiments, the trough concentration is maintained at approximately 0.6 mM to 1.4 mM lithium. In some such embodiments, a trough skin concentration is maintained at 1 mM to 3 mM lithium. In some such
embodiments, the trough skin concentration is maintained at less than 0.5 mM, or less than 0.75 mM, or less than 1 mM, or less than 2 mM, or less than 3 mM of lithium.
[00278] In specific embodiments, an effective amount of a lithium compound is administered such that the target concentration of lithium ions in plasma or serum, as measured 30 minutes to 1 hour after the lithium treatment, is 0.10-0.20 μΜ, 0.20-0.50 μΜ, 0.50-1.0 μΜ, 1.0-5.0 μΜ, 5.0-10 μΜ, 10-20 μΜ, 20-50 μΜ, 50-100 μΜ, 100-500 μΜ, 0.1- 0.5 mM, 0.5-1.0 mM, 1.0 mM-2.0 mM, 2.0-2.5 mM, 2.5-3.0 mM, 3.0-4.0 mM, 4.0 mM-5.0 mM, 5.0-7.0 mM, or 7.0 mM or greater. In some embodiments, an effective amount of lithium is administered such that the plasma or serum lithium ion concentration measured either 8 hours, 16 hours, 1 day, 1 week, 2 weeks, or 1 month after the lithium treatment, is 0.1 to 0.5 μΜ, 0.1 to 1.0 μΜ, 0.5 to 1.0 μΜ, 0.5 to 1.5 μΜ, 1 to 10 μΜ, 10 to 50 μΜ, 50 to 100 μΜ, 100 to 150 μΜ, 150 to 200 μΜ, 250 to 300 μΜ, 100 to 250 μΜ, 100 to 500 μΜ, 200 to 400 μΜ, 500 to 1000 μΜ; or 1000 to less than 1500 μΜ. In one embodiment, the plasma or serum lithium concentration reaches at least 1 μΜ. In one embodiment, the plasma or serum lithium concentration reaches at least 100 μΜ. In one embodiment, the plasma or serum lithium concentration reaches at least 1 mM. In one embodiment, the plasma or serum lithium concentration does not exceed 1 mM. In another embodiments, the plasma or serum concentration of lithium does not exceed 1.5 mM. Serum lithium concentration may be measured using any technique known in the art, such as described in Sampson et ah, 1992, Trace Elements in Medicine 9:7-8.
[00279] In specific embodiments, an amount of a lithium compound is administered such that the target concentration of lithium in the skin is 0.01 to 0.05 μΜ, 0.05 to 0.1 μΜ, 0.1 to 0.5 μΜ, 0.1 to 1 μΜ, 0.5 to 1.0 μΜ, 1.0 to 1.5 μΜ, 1 to 2.5 μΜ, 1 to 5 μΜ, 5 to 10 μΜ, 10 to 50 μΜ, 50 to 100 μΜ, 100 to 150 μΜ, 150 to 200 μΜ, 250 to 300 μΜ, 100 to 250 μΜ, 100 to 500 μΜ, 200 to 400 μΜ, 500 to 1000 μΜ, 1 to 10 mM, 1 to 5 mM, 5 to 10 mM, 10 to 100 mM, 100 to 200 mM, or 500 to 1000 mM. In some embodiments, the concentration of lithium achieved in the skin is greater than 0.1 mM. In some embodiments, the concentration of lithium achieved in the skin is greater than 1.0 mM. In some embodiments, the concentration of lithium achieved in the skin is greater than 1.5 mM. In one embodiment, the amount of lithium achieved in the skin is approximately 1 mM to 5 mM. In one embodiment, the amount of lithium achieved in the skin is approximately 5 mM to 10 mM. In one embodiment, the amount of lithium achieved in the skin is approximately 100 to 200 mM. In one embodiment, the amount of lithium achieved in the skin does not exceed 5 mM. In one embodiment, the amount of lithium achieved in the skin does not exceed 10 mM. In one embodiment, the amount of lithium achieved in the skin does not exceed 50 mM. In some embodiments, an amount of lithium is administered such that the concentration of lithium delivered to the stratum corneum is 0.1 to 0.5 mM, 0.5 to 1 mM, 1 to 10 mM, 10 to 100 mM, 100 to 200 mM, or 500 to 1000 mM. In some embodiments, the concentration of lithium delivered to the stratum corneum is greater than 1.5 mM. In one embodiment, the amount of lithium achieved in the stratum corneum is approximately 100 to 200 mM. In one embodiment, the amount of lithium achieved in the stratum corneum does not exceed 5 mM. In one embodiment, the amount of lithium achieved in the stratum corneum does not exceed 10 mM. One of skill in the art would be able to measure lithium concentrations in skin using techniques known in the art, for example, mass spectroscopy, e.g., inductively coupled plasma mass spectroscopy (ICP-MS). For example, the concentration of lithium in skin can be measured using the method provided in the example of Section 13.2 below or equivalent methods.
[00280] In other embodiments, the lithium concentration is measured in the hair shaft using techniques known in the art, e.g., Tsanaclis & Wicks, 2007, Forensic Science Intl. 176: 19-22, which is incorporated by reference herein in its entirety.
[00281] Specific, non-limiting, formulations of lithium for topical, subcutaneous, and oral administration are provided in Sections 5.3.1-5.3.3 below.
5.3.1 TOPICAL FORMS FOR ADMINISTRATION
[00282] In the embodiments described in the subsections that follow, lithium can be applied topically, e.g., as a cream, gel, ointment, or other form for topical administration as described in Section 5.2 supra. Topical lithium may be administered to wounded or unwounded skin.
[00283] In some embodiments, the lithium formulation for topical administration (e.g., gel, cream, ointment, salve, etc.) comprises lithium (or monovalent lithium salt) at a concentration of 50 niM, 75 niM, 100 niM, 125 niM, 150 niM, 175 niM, 200 niM, 250 niM, 300 niM, 350 niM, 400 niM, 450 niM, 500 niM, 550 niM, 600 niM, 650 niM, 700 niM, 750 niM, 800 niM, 900 niM, 1 M, 1.1 M, or 1.2 M, or more. As used herein, a monovalent lithium salt {e.g., lithium gluconate, lithium chloride, lithium stearate, lithium orotate, etc.) refers to a salt form of lithium in which there is one lithium cation for each anion of the salt. A divalent lithium salt {e.g., in some embodiments, lithium succinate, lithium carbonate) refers to a salt form of lithium in which there are two lithium cations for each anion of the salt. A trivalent lithium salt {e.g., in some embodiments, lithium citrate), refers to a salt form of lithium in which there are three lithium cations for each anion of the salt. In some embodiments, a lithium formulation comprising lithium (or monovalent lithium salt) at a concentration in the range of 50 mM to 200 mM is chosen for use in the embodiments described herein. In some embodiments, a lithium formulation comprising lithium (or monovalent lithium salt) at a concentration in the range of 200 mM to 400 mM is used. In some embodiments, a lithium formulation comprising lithium (or monovalent lithium salt) at a concentration in the range of 400 mM to 600 mM is used. In some embodiments, a lithium formulation comprising lithium (or monovalent lithium salt) at a concentration in the range of 600 mM to 800 mM is used. The concentration of lithium in a particular topical lithium formulation to deliver the intended dose of lithium will depend on the release properties of the lithium ion, the hydrophobicity of the lithium salt form, the partition coefficient of the lithium salt form, etc.
[00284] Lithium formulations comprising the foregoing lithium (or monovalent lithium salt) concentrations may be achieved using, for example, a formulation comprising, w/w, lithium ions at a concentration of 0.10% lithium, 0.15% lithium, 0.20% lithium, 0.25% lithium, 0.30% lithium, 0.35% lithium, 0.40% lithium, 0.45% lithium, 0.50% lithium, 0.55% lithium, 0.60% lithium, 0.65% lithium, 0.70% lithium, 0.75% lithium, 0.80% lithium, 0.85% lithium, 0.90% lithium, 0.95% lithium. In some embodiments, the form of lithium for topical administration comprises, w/w, 0.1% to 0.5% lithium ions, 0.2% to 0.5% lithium ions, 0.5% to 1% lithium ions, or more.
[00285] The amount of a salt form of lithium to generate a topical lithium formulation with one of the aforementioned concentrations of lithium ion is readily deducible by one of ordinary skill in the art, and depends upon several factors including, e.g. , the valency of the salt form, the stability of the salt form, the ability of the salt form to release the lithium ion, the hydrophobicity or hydrophilicity, etc. For example, Lithioderm (Labcatal) comprises 8% lithium gluconate, which corresponds to 0.275% lithium ion {i.e., 274.8 mg Li+/100 g gel). It is noted that a formulation of topical 8% lithium gluconate, w/w, contains approximately 80 mg/ml lithium gluconate, which is approximately 400 mM lithium gluconate (and, thus, 400 mM lithium ion). Thus, in some exemplary embodiments, a formulation for topical administration comprises a salt form of lithium {e.g., lithium gluconate or other form described in Section 5.1 above) at a concentration, w/w, of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 16%, 18%, 20%, or more. In some embodiments, a salt form of lithium for topical administration comprises, w/w, 1% to 2% lithium salt {e.g., lithium gluconate or other form described in Section 5.1 above), 2% to 5% lithium salt, 5% to 10% lithium salt, 10% to 15% lithium salt, 15% to 20% lithium salt, 20% to 25% lithium salt, or 25% to 50% lithium salt. In one embodiment, the form of lithium for topical administration is 1% to 20% w/w lithium salt.
[00286] In some embodiments, a topical formulation of lithium comprises l%-4% lithium gluconate (w/w). In some embodiments, a topical formulation of lithium comprises 4%-8% lithium gluconate (w/w). In some embodiments, a topical formulation of lithium comprises 8%-16% or more lithium gluconate (w/w). In some embodiments, a topical formulation of lithium comprises 0.2%-l%, or l%-5%, or more lithium chloride (w/w). In some embodiments, a topical formulation of lithium comprises 0.5%-2%, or 2%-4%, or 4%-8%, or 8%-16, or more lithium succinate (w/w). In some embodiments, a topical formulation of lithium comprises 0.5%-6%, 6%-12%, or 12%-25%, or more lithium stearate (w/w). In some embodiments, a topical formulation of lithium comprises l%-4%, 4%-8%, or 8%-16%, or more lithium orotate (w/w). In some embodiments, a topical formulation of lithium comprises 0.25%-0.75%, 0.75%-1.5%, or 1.5%-3%, or more lithium carbonate (w/w). In some embodiments, a topical formulation of lithium comprises 0.25%- 1.5%, 1.5%-3.0%, or 3%-6%, or more 8% lithium citrate (w/w).
[00287] In an exemplary embodiment, a 50 kg patient is administered a single droplet - approximately 0.1 ml - of 8% (w/w) lithium gluconate at 3 sites, twice daily. This corresponds to approximately 8 mg lithium gluconate (0.274 mg Li+) per site, i.e., 0.16 mg/kg lithium gluconate (0.005 mg/kg Li+) per site. Over three sites twice daily, this corresponds to approximately 0.96 mg/kg lithium gluconate (0.033 mg/kg Li+) per day. Thus, in some embodiments, a patient {e.g., a 50 kg patient) is administered about 30-50 mg, about 50-75 mg, or about 75-100 mg topical lithium gluconate/day, which is equivalent to about 1-1.7 mg, 1.7-2.2 mg, or 2.2-3.5 mg, respectively, Li+/day. In some embodiments, a topical lithium formulation is administered once daily. In some embodiments, a topical lithium formulation is administered twice daily. In some embodiments of a twice daily treatment regimen, doses are administered 6 hours apart, or 7 hours apart, or 8 hours apart, or 9 hours apart, or 10 hours apart, or 11 hours apart, or 12 hours apart. In a particular embodiment, the doses are administered 7 to 8 hours apart.
[00288] In some embodiments when lithium is administered topically, an amount of lithium is administered such that the peak lithium concentration in skin is between 0.01 mM and 0.05 mM, 0.05 mM and 0.1 mM, 0.1 mM and 0.5 mM or between 0.5 mM and 10 mM, for example, between 0.1 and 0.5 mM, 0.5 mM and 1 mM, 1 mM and 2 mM, between 2 mM and 5 mM, 5 mM to 10 mM, or 10 mM to 50 mM. In some such embodiments, the peak lithium concentration in blood may be one or more orders of magnitude lower than the peak concentration in skin (for example, 0.001 mM to 0.01 mM, 0.01 mM to 0.1 mM, or 0.1 mM to 0.5 mM, 0.5 mM to 1.0 mM, or 1.0 mM to 10 mM). In some such embodiments, the steady state blood concentration of lithium should not exceed a maximum of 1.5 mM to 2 mM.
5.3.2 SUBCUTANEOUS FORMS FOR ADMINISTRATION
[00289] In some embodiments, a formulation of lithium described herein (by non-limiting e.g., lithium gluconate, lithium chloride, lithium succinate, lithium carbonate, lithium citrate, lithium stearate, lithium orotate, etc.) is administered subcutaneous ly, to either wounded or unwounded skin.
[00290] In some embodiments, the form of lithium for subcutaneous administration is administered at a dose comprising 0.001 mg lithium ion per kg of patient weight. In some embodiments, the dose is 0.001 mg/kg, 0.002 mg/kg, 0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg, 0.010 mg/ kg, 0.020 mg/kg, 0.025 mg/kg, 0.050 mg/kg, 0.075 mg/kg, 0.10 mg/kg, 0.15 mg/kg, 0.20 mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.40 mg/kg, 0.50 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5 mg/kg, 9 mg/kg, 9.5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, or 50 mg/kg or more of lithium ions. In some embodiments, the dose does not exceed 50 mg/kg. The lower ranges of dosages may be preferably used for bolus dosing. For a controlled release (e.g., a delayed release or a sustained release) dosage form, the maximum dosage that may be administered at any one time may vary depending on the release kinetics of the lithium and the concentration of efficacy of the formulation.
[00291] The concentration of a salt form of lithium required to generate a subcutaneously administered formulation that delivers lithium ions at one of the aforementioned dosages is readily deducible by one of ordinary skill in the art, and depends upon several factors including, e.g., the valency of the salt form, the stability of the salt form, the ability of the salt form to release the lithium ion, the hydrophobicity or hydrophilicity, etc. For example, to achieve an equivalent dosage of lithium ions, a formulation comprising lithium gluconate may be subcutaneously administered at a dosage of approximately 10 mg lithium gluconate per kg of patient weight (mg/kg), 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 650 mg/kg, 700 mg/kg, 750 mg/kg, 800 mg/kg, 850 mg/kg, 900 mg/kg, 950 mg/kg, or 1000 mg/kg. In some embodiments, the formulation for subcutaneous administration contains a dose of 10 mg/kg to 50 mg/kg, 50 mg/kg to 100 mg/kg, 100 mg/kg to 200 mg/kg, 200 mg/kg to 400 mg/kg, 400 mg/kg to 600 mg/kg, or 100 mg/kg to 600 mg/kg of lithium gluconate. In one embodiment, the formulation for subcutaneous administration contains a dose in the range of 30 mg/kg to 150 mg/kg lithium gluconate. In one embodiment, the formulation for subcutaneous administration contains a dose in the range of about 30 mg/kg to 300 mg/kg lithium gluconate. In one embodiment, the dose for subcutaneous administration does not exceed 300 mg/kg lithium gluconate. In another embodiment, the dose for subcutaneous administration does not exceed 600 mg/kg lithium gluconate. The lower ranges of dosages may be preferably used for bolus dosing. For a controlled release (e.g., a delayed release or a sustained release) dosage form, the maximum dosage that may be administered at any one time may vary depending on the release kinetics of the lithium and the concentration of efficacy of the formulation.
[00292] In some embodiments, the lithium formulation is administered subcutaneously once daily. In some embodiments, the lithium formulation is administered subcutaneously twice daily. In some embodiments of a twice daily treatment regimen, doses are administered 6 hours apart, or 7 hours apart, or 8 hours apart, or 9 hours apart, or 10 hours apart, or 11 hours apart, or 12 hours apart. In a particular embodiment, the doses are administered 7 to 8 hours apart.
[00293] In some embodiments when lithium is administered subcutaneously (for example, once daily, although smaller doses may be administered more than once daily), an amount of lithium is administered such that the peak lithium concentration in skin is between 0.1 μΜ and 0.2 μΜ, 0.2 μΜ and 0.5 μΜ, 0.5 and 1 μΜ, 1 μΜ and 2 μΜ, 2 μΜ to 10 μΜ, 10 μΜ to 100 μΜ, 100 μΜ to 500 μΜ, 500 μΜ to 1000 μΜ. These peak values will depend on the lithium release properties of the formulation, the hydrophobicity of the lithium salt form, the partition coefficient of the lithium salt form, etc. In some embodiments, the peak concentration in skin is 0.2 μΜ to 1.5 μΜ lithium. In some embodiments, the peak concentration in skin should not exceed 1 μΜ or 1.5 μΜ lithium. In some embodiments, the peak concentration in skin is 10 μΜ to 100 μΜ lithium. In some embodiments, the peak concentration in skin is 100 μΜ to 1000 μΜ lithium. In some such embodiments, the peak lithium concentration in blood may be several orders of magnitude higher, for example, 0.1 mM to 0.5 mM, or 0.5 mM to 1.1 mM, 1.1 to 1.5 mM, 1.5 mM to 5 mM, 5 mM to 10 mM, 10 mM to 50 mM, or 50 mM to 100 mM. These peak values will depend on the lithium release properties of the formulation, the hydrophobicity of the lithium salt form, the partition coefficient of the lithium salt form, etc. In some such embodiments, the steady state blood concentration of lithium should not exceed a maximum of 1.5 mM to 2 mM.
5.3.3 ORAL FORMS FOR ADMINISTRATION
[00294] In some embodiments, a formulation of lithium described herein (by non-limiting e.g., lithium gluconate, lithium chloride, lithium succinate, lithium carbonate, lithium citrate, lithium stearate, lithium orotate, etc.) is administered orally, for example, once daily, or twice daily as determined by the medical practitioner and in accordance with Section 5.3 above.
[00295] In some embodiments, an oral formulation comprising of 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or more, but preferably less than 10 mM, of lithium ions (or monovalent lithium salt) is administered. In some embodiments, an oral formulation comprising lithium ions or a monovalent lithium salt in the range of 0.1 to 0.5 mM, 0.4 to 0.6 mM, 0.5 to 1 mM, 0.6 to 1.2 mM, or 1 to 1.5 mM, is administered.
[00296] Administration of the foregoing amounts of lithium may be achieved by oral administration of a lithium formulation at a dosage comprising 0.001 mg lithium ion per kg of patient weight. In some embodiments, the dose is 0.001 mg/kg, 0.002 mg/kg, 0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg, 0.010 mg/ kg, 0.020 mg/kg, 0.025 mg/kg, 0.050 mg/kg, 0.075 mg/kg, 0.10 mg/kg, 0.15 mg/kg, 0.20 mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.40 mg/kg, 0.50 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5 mg/kg, 9 mg/kg, 9.5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, or 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, or 50 mg/kg or more of lithium ions. In some embodiments, the dose does not exceed 50 mg/kg Li+. For a controlled release {e.g., a delayed release or a sustained release) dosage form, the maximum dosage that may be administered at any one time may vary depending on the release kinetics of the lithium and the concentration of efficacy of the formulation.
[00297] The concentration of a salt form of lithium required to generate an orally administered formulation that delivers lithium ions at one of the aforementioned dosages is readily deducible by one of ordinary skill in the art, and depends upon several factors including, e.g., the valency of the salt form, the stability of the salt form, the ability of the salt form to release the lithium ion, the hydrophobicity or hydrophilicity, etc. For example, to achieve an equivalent dosage of Li+, a formulation comprising lithium carbonate, which is a divalent lithium salt {e.g., trade names Eskalith CR, Eskalith, Lithobid), may be orally administered at a dosage of approximately 2 mg lithium carbonate per kg of patient weight (mg/kg), 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, or 250 mg/kg or more is administered. In some embodiments, the oral formulation contains a dose of 2 mg/kg to 10 mg/kg, 10 mg/kg to 25 mg/kg, 25 mg/kg to 50 mg/kg, 50 mg/kg to 100 mg/kg, 100 mg/kg to 200 mg/kg, or 200 mg/kg to 500 mg/kg of lithium carbonate. In one embodiment, the oral formulation contains a dose in the range of 5 mg/kg to 100 mg/kg lithium carbonate. In one embodiment, the oral formulation contains a dose in the range of about 5 mg/kg to 50 mg/kg lithium carbonate. In one embodiment, the oral formulation contains a dose in the range of about 10 mg/kg to 100 mg/kg lithium carbonate. In one embodiment, the oral formulation contains a dose that does not exceed 300 mg/kg lithium carbonate. For a controlled release {e.g., a delayed release or a sustained release) dosage form, the maximum dosage that may be administered at any one time may vary depending on the release kinetics of the lithium and the concentration of efficacy of the formulation.
[00298] In some embodiments when the lithium formulation is for oral administration (for example, for once daily administration, although smaller doses may be administered more than once daily), an amount of lithium compound is administered such that the peak lithium concentration in skin is between 0.1 μΜ and 0.2 μΜ, 0.2 μΜ and 0.5 μΜ, 0.5 and 1 μΜ, 1 μΜ and 2 μΜ, 2 μΜ to 10 μΜ, 10 μΜ to 100 μΜ, 100 μΜ to 500 μΜ, 500 μΜ to 1000 μΜ. These peak values will depend on the lithium release properties of the formulation, the hydrophobicity of the lithium salt form, the partition coefficient of the lithium salt form, etc. In some embodiments, the peak concentration in skin is 0.2 μΜ to 1.5 μΜ lithium. In some embodiments, the peak concentration in skin should not exceed 1 μΜ or 1.5 μΜ lithium. In some embodiments, the peak concentration in skin is 10 μΜ to 100 μΜ lithium. In some embodiments, the peak concentration in skin is 100 μΜ to 1000 μΜ lithium. In some such embodiments, the peak lithium concentration in blood may be several orders of magnitude higher, for example, 0.1 mM to 0.5 mM, or 0.5 mM to 1.1 mM, 1.1 to 1.5 mM, 1.5 mM to 5 mM, 5 mM to 10 mM, 10 mM to 50 mM, or 50 mM to 100 mM. These peak values will depend on the lithium release properties of the formulation, the hydrophobicity of the lithium salt form, the partition coefficient of the lithium salt form, etc. In some such embodiments, the steady state blood concentration of lithium should not exceed a maximum of 1.5 mM to 2 mM.
5.3.4 PULSE TREATMENT
[00299] The pulse lithium treatment can be administered one time, or multiple times at intervals of time. It is understood that the precise dosage and duration of treatment may vary with the age, weight, and condition of the patient being treated, and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test or diagnostic data. It is further understood that for any particular individual, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations. For example, in the treatment of bipolar disorder, therapeutically useful amounts of lithium (~ 0.4 to 1.2 mM) are only slightly lower than toxic amounts (>1.5 mM), so the skilled practitioner knows that the blood levels of lithium must be carefully monitored during treatment to avoid toxicity.
[00300] In some embodiments, a pulse lithium treatment is administered at the time of integumental perturbation. In some embodiments, a pulse lithium treatment is administered following integumental perturbation. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered before scab formation. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered during scab formation. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered periscab detachment. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered immediately after scab detachment. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered 1 hour after scab detachment. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered up to 6 hours after scab detachment. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered 6- 12 hours after scab detachment. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered 12-18 hours after scab detachment. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered 18- 24 hours after scab detachment. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered 1 day after scab detachment. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered 2 days after scab detachment. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered 3 days after scab detachment. In some embodiments, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered within 3 days, 5 days, 7 days, 10 days, 2 weeks, or 3 weeks after integumental perturbation.
[00301] In one embodiment, the pulse lithium treatment is administered at the time of integumental perturbation and then maintained for 3 or 4 or 5 days thereafter (in some embodiments, a scab forms during this time). In some embodiments, a pulse lithium treatment is administered as soon as the scab falls of and maintained for 3 or 4 or 5 days. In some embodiments, the pulse lithium treatment is administered in order to modulate the neoepidermis that forms underneath the scab. In some such embodiments, the pulse lithium treatment is administered at the time of integumental perturbation and is maintained up to some time after scab falls off, for example, between 5 - 14 days following integumental perturbation. In some embodiments, the course of treatment with lithium is short, for example, limited to a few days just following scab detachment, or even continued only for as long as the scab is still attached. The timing of integumental perturbation and lithium administration is preferably monitored and adjusted so that optimal results are achieved. [00302] In some embodiments, a pulse treatment is combined with a form of integumental perturbation that does not lead to formation of a scab. In one such embodiment, the pulse lithium treatment is administered at the time of integumental perturbation. In some embodiments, a pulse lithium treatment is administered following integumental perturbation. In some embodiments, in which a pulse lithium treatment is administered following an integumental perturbation that does not lead to formation of a scab, the pulse lithium treatment is administered within 15 minutes of, or 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 10 days, 2 weeks, or 3 weeks after integumental perturbation.
[00303] The Examples in Sections 6 to 19 provide exemplary protocols for carrying out the aforementioned embodiments, although the invention is not meant to be so limited.
5.3.5 INTERMITTENT TREATMENTS
[00304] The intermittent lithium treatment can be administered one time {e.g., using a controlled release formulation), or multiple times at intervals of time. It is understood that the precise dosage and duration of treatment may vary with the age, weight, and condition of the patient being treated, and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test or diagnostic data. It is further understood that for any particular individual, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations.
[00305] In one embodiment, lithium can be administered daily {e.g., once, twice or three times daily) for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 7 days; and in some embodiments not more than 14 days. Holidays can be interspersed for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 7 days; and in some embodiments not more than 14 days.
[00306] In some embodiments, an intermittent lithium treatment is begun at the time of integumental perturbation. In some embodiments, an intermittent lithium treatment is begun following integumental perturbation. In one embodiment, in which an intermittent lithium treatment is begun following an integumental perturbation that leads to formation of a scab, the intermittent lithium treatment is begun before scab formation. In one embodiment, in which an intermittent lithium treatment is begun following an integumental perturbation that leads to formation of a scab, the intermittent lithium treatment is begun during scab formation. In one embodiment, in which an intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium in the intermittent lithium treatment is periscab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium is immediately after scab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium is up to 6 hours after scab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium is 6-12 hours after scab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium is 12-18 hours after scab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium is 18-24 hours after scab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium is 1 day after scab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium is 2 days after scab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium is 3 days after scab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium is administered immediately after scab detachment, followed by another administration each day for several days to 1 week. In some embodiments, in which an intermittent lithium treatment is begun following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is begun within 3 days, 5 days, 7 days, 10 days, 2 weeks, or 3 weeks after integumental perturbation.
[00307] In one embodiment, the intermittent lithium treatment is begun at the time of integumental perturbation and then administered daily (or twice daily) for 5 days thereafter (in some embodiments, a scab forms during this time). In some embodiments, the intermittent lithium treatment is begun as soon as the scab falls off, and administered daily for 5 days. In some embodiments, the intermittent lithium treatment is to modulate the neoepidermis that forms underneath the scab. In some such embodiments, the intermittent lithium treatment is begun at the time of integumental perturbation and is continued with daily dosing up to some time after scab falls off, for example, between 5 - 14 days following integumental perturbation. In some embodiments, the course of treatment with lithium is short, for example, limited to daily doses for a few days just following scab detachment, or even continued only for as long as the scab is still attached. The timing of integumental perturbation and lithium administration is preferably monitored and adjusted so that optimal results are achieved.
[00308] In some embodiments, an intermittent lithium treatment is combined with a form of integumental perturbation that does not lead to formation of a scab. In one such embodiment, the intermittent lithium treatment is begun at the time of integumental perturbation. In some embodiments, an intermittent lithium treatment is begun following integumental perturbation. In some embodiments, in which an intermittent lithium treatment is begun following an integumental perturbation that does not lead to formation of a scab, the intermittent lithium treatment is begun within 15 minutes of, or 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 10 days, 2 weeks, or 3 weeks after integumental perturbation.
[00309] The Examples in Sections 6 to 19 provide exemplary protocols for carrying out the aforementioned embodiments, although the invention is not meant to be so limited.
5.4 COMBINATION TREATMENTS
[00310] Intermittent lithium treatment or a pulse lithium treatment in combination with other methods, including conventional methods, for enhancing wound healing or revising scars enhances the effectiveness of these methods. The effect that each drug offers could be an additive or synergistic improvement, or a combination of two different pharmacologically defined effects, to achieve the desired end result. The combined modality of treatment could involve alternating treatment of each dosage form or concurrent or simultaneous treatment. Synergism occurs when the combination has an effect that is more than would be expected from merely the additive effect of each element in the combination, for example, if branched hair follicles or more hair follicles per pore were produced by the combination and not by either alone.
[00311] The intermittent lithium treatments or the pulse lithium treatment described herein may be in combination with any additional treatment(s) described or incorporated by reference herein or determined to be appropriate by the medical practitioner. The amount of an additional treatment(s) will depend on the desired effect and the additional compound that is selected. Dosages and regimens for administering such additional treatment(s) are the dosages and regimens commonly in use, which can be easily determined by consulting, for example, product labels or physicians' guides, such as the Physicians' Desk Reference ("PDR") (e.g., 63rd edition, 2009, Montvale, NJ: Physicians' Desk Reference).
[00312] In one embodiment, the combination treatment comprises lithium and an additional compound(s) formulated together. The lithium in such formulations may be released concurrently with or separately from the additional compound(s), or may be released and/or delivered to the tissue site with different pharmacokinetics. For example, in some embodiments, one or more of the compounds in the formulation undergoes controlled release, whereas one or more of the other compounds does not. For example, one or more of the compounds in the formulation undergoes sustained release whereas one or more of the other compounds undergoes delayed release.
[00313] In another embodiment, the combination treatment comprises lithium and an additional compound(s) formulated separately. The separate formulations may be administered concurrently, sequentially, or in alternating sequence. For example, the lithium compound may be administered sequentially, or concurrently with another compound to achieve the desired effect of improved wound healing or scar revision.
[00314] In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more treatments selected from, e.g., cell therapy (such as a stem cell), a formulation for gene therapy (such as, e.g., a virus, virus-like particle, virosome), an antibody or antigen-binding fragment thereof, an herb, a vitamin (e.g., a form of vitamin E, a vitamin A derivative, such as, e.g., all-trans retinoic acid (ATRA), a B vitamin, such as, e.g., inositol, panthenol, or biotin, or a vitamin D3 analog), a mineral, essential oils, an antioxidant or free radical scavenger, amino acids or amino acid derivatives, a shampoo ingredient (e.g. , ammonium chloride, ammonium lauryl sulfate, glycol, sodium laureth sulfate, sodium lauryl sulfate, ketoconazole, zinc pyrithione, selenium sulfide, coal tar, a salicylate derivative, dimethicone, or plant extracts or oils), a conditioning agent, a soap product, a moisturizer, a sunscreen, a waterproofing agent, a powder, talc, or silica, an oil-control agent, alpha-hydroxy acids, beta-hydroxy acids (e.g., salicylic acid), poly-hydroxy acids, benzoyl peroxide, antiperspirant ingredients, such as astringent salts (e.g., zinc salts, such as zinc pyrithione, inorganic or organic salts of aluminum, zirconium, zinc, and mixtures thereof, aluminum chloride, aluminum
chlorohydrate, aluminum chlorohydrex, aluminum chlorohydrex PEG, aluminum chlorohydrex PG, aluminum dichlorohydrate, aluminum dichlorohydrex PEG, aluminum dichlorohydrex PG, aluminum sesquichlorohydrate, aluminum sesquichlorohydrex PEG, aluminum sesquichlorohydrex PG, aluminum sulfate, aluminum zirconium
octachlorohydrate, aluminum zirconium octachlorohydrex GLY (abbreviation for glycine), aluminum zirconium pentachlorohydrate, aluminum zirconium pentachlorohydrex GLY, aluminum zirconium tetrachlorohydrate, aluminum zirconium trichlorohydrate, aluminum zirconium tetrachlorohydrate GLY, and aluminum zirconium trichlorohydrate GLY, potassium aluminum sulphate, (also known as alum (KAl(S04)2l2H20)), aluminum undecylenoyl collagen amino acid, sodium aluminum lactate+ aluminum sulphate
(Na2HAl(OOCCHOHCH3)2-(OH)6) + A12(S04)3), sodium aluminum chlorohydroxylactate, aluminum bromohydrate (A^B^OH^nSLO), aluminum chloride (AICI36H2O), complexes of zinc salt and of sodium salt, complexes of lanthanum and cerium, and the aluminum salt of lipoamino acids (R— CO— H— CHR'— CO— OA1— (OH)2 with R = C6-n and R'=amino acid), retinoids (e.g., retinoic acid, retinol, retinal, or retinyl esters), sunscreens (e.g., derivatives of para-aminobenzoic acid (PABA), cinnamate and salicylate, avobenzophenone (Parsol 1789®), octyl methoxycinnamate (Parsol™ MCX) and 2-hydroxy-4-methoxy benzophenone (also known as oxybenzone and available as Benzophenone™ , and preservatives), an anti-acne medication, an anti-age cream, a sebum production inhibitor and/or pore size reducing agent (e.g., carboxyalkylates of branched alcohols and/or alkoxylates thereof, e.g. , tridecyl carboxy alkylates, cerulenin or a cerulenin analog, including pharmaceutically acceptable salts or solvates thereof, another fatty acid synthase inhibitor, such as triclosan or analogs thereof, a polyphenol extracted from green tea (EGCG), available from Sigma Corporation (St. Louis, Missouri), or a-methylene-Y-butyrolactone), a massage agent, an exfoliant, an anti-itch agent, a pro-inflammatory agent, an immunostimulant (e.g., interferon, cytokines, agonists or antagonists of various ligands, receptors and signal transduction molecules of the immune system, immunostimulatory nucleic acids, an adjuvant that stimulates the immune response and/or which causes a depot effect). In certain embodiments, adjuvants and/or other stimulators of local cytokines are used in conjunction with the intermittent lithium treatment or pulse lithium treatment. Without being bound by any theory, one rationale for administering adjuvants and/or other stimulators of local cytokines in conjunction with the intermittent lithium treatment or pulse lithium treatment is that the production of local cytokines may induce changes in the hair follicle cell cycle and recruit new follicle stem cells to follicles. [00315] In other embodiments, the combination ntreatment comprises lithium in ccombination with a cell cycle regulator, a hormonal agonist, a hormonal antagonist (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), an inhibitor of hormone biosynthesis and processing, a steroid (e.g., dexamethasone, retinoids, deltoids, betamethasone, Cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, hydrocortisone, mineralocorticoids, estrogen, testosterone, progestins), antigestagens (e.g., mifepristone, onapristone), an antiandrogen (e.g., cyproterone acetate), an antiestrogen, an antihistamine (e.g., mepyramine,
diphenhydramine, and antazoline), an anti-inflammatory (e.g., corticosteroids (such as, e.g., Dermatop®), NTHEs, and COX-2 inhibitors, adrenocorticoids, beclomethasone, budesonide, flunisolide, fluticasone, triamcinolone, methylprednisolone, prednisolone, prednisone, hydrocortisone), an anesthetic (e.g., vocal anesthesia, lidocaine, bupivacaine, etidocaine, etc., with or without epinephrine or sodium bicarbonate) a retinoid (e.g., 13-cis-retinoic acid, adapalene, all-trans-retinoic acid, and etretinate), PMMA, Restylane, poly-L-lactic acid, collagen, hyaluronic acid, which may be present in microspheres, or other skin fillers, a cosmetic (e.g., intended to increase soft tissue volume), an immunosuppressant (e.g., cyclosporine, tacrolimus, rapamycin, everolimus, and pimecrolimus), an anti-cancer agent (such as, e.g., fluorouracil (5-FU or f5U) or other pyrimidine analogs, methotrexate, cyclophosphamide, vincristine), a mood stabilizer (e.g., valproic acid or carbamazepine), an antimetabolite, non-steroidal anti-inflammatory drugs ( e.g. , aspirin, ibuprofen, diclofenac, and COX-2 inhibitors), pain relievers, leukotreine antagonists (e.g., montelukast, methyl xanthines, zafirlukast, and zileuton), beta2-agonists (e.g. , albuterol, biterol, fenoterol, isoetharie, metaproterenol, pirbuterol, salbutamol, terbutalin formoterol, salmeterol, and salbutamol terbutaline), anticholinergic agents (e.g., ipratropium bromide and oxitropium bromide), sulphasalazine, penicillamine, dapsone, antihistamines, anti-fungal agents, antimalarial agents (e.g., hydroxychloroquine), anti-viral agents (e.g., nucleoside analogs (e.g., zidovudine, acyclovir, gangcyclovir, vidarabine, idoxuridine, trifluridine, and ribavirin), foscarnet, amantadine, rimantadine, saquinavir, indinavir, ritonavir, and AZT), antibiotics (e.g., a polymyxin, dactinomycin (formerly actinomycin), bleomycin, erythomycin, penicillin, mithramycin, and anthramycin (AMC)), and antimicrobials (e.g., benzyl benzoate, benzalkonium chloride, benzoic acid, benzyl alcohol, butylparaben, ethylparaben, methylparaben, propylparaben, camphorated metacresol, camphorated phenol,
hexylresorcinol, methylbenzethonium chloride, cetrimide, chlorhexidine, chlorobutanol, chlorocresol, cresol, glycerin, imidurea, phenol, phenoxyethanol, phenylethylalcohol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, potassium sorbate, sodium benzoate, sodium proprionate, sorbic acid, and thiomersal (thimerosal)).
[00316] In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more narcotic analgesics, selected from the group of, e.g., alfentanil, benzylmorphine, codeine, codeine methyl bromide; codeine phosphate, codeine sulfate, desomorphine, dihydrocodeine,
dihydrocodeinone enol acetate, dihydromorphine, ethylmorphine, hydrocodone,
hydromorphone, methadone hydrochloride, morphine, morphine hydrochloride, morphine sulfate, nicomorphine, normethadone, normorphine, opium, oxycodone, oxymorphone, phenoperidine, and propiram. In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more non-narcotic analgesics, selected from the group of, e.g., aceclofenac, acetaminophen, acetanilide, acetylsalicylsalicylic acid; aspirin, carbamazepine, dihydroxyaluminum acetylsalicylate, fenoprofen, fluproquazone, ibufenac, indomethacin, ketorolac, magnesium acetylsalicylate, morpholine salicylate, naproxen, phenacetin, phenyl salicylate, salacetamide, salicin, salicylamide, sodium salicylate, and tolfenamic acid. Other pain treatments that may be used in combination with the lithium treatments described herein include nerve blocks or non-traditional pain medications, such as, e.g., Lyrica (pregabalin) or Neurontin (gabapentin).
5.4.1 COMBINATION TREATMENTS COMPRISING
INTEGUMENTAL PERTURBATION
[00317] The present invention is based, in part, on the appreciation that hair follicles play a role in wound healing. Inducing the formation of new hair follicles in wounds, or enhancing the entry of hair follicles into wounds (for example, by transplanting hair follicles into wounds) harnesses their regenerative capacity and provides a transformational approach to scar revision and the management of wounds. The approaches described herein permit scar revision under sterile and controlled conditions that recreates and harnesses the fetal skin's plastic and regenerative capacity.
[00318] The physical disruption of the skin (integumental perturbation) provides a signal for the formation of new hair follicles. Wounding is itself a form of integumental perturbation. Consequently, scar revision (which involves wounding) also provides a signal for hair follicle neogenesis and/or migration into the wound site. For instance, as discussed in Section 2.3.2 above, a current method for scar revision is serial expansion. We believe that serial expansion is one example of the regenerative capacity of skin, since the physical tension induced by balloons induces integumental perturbation and results in either migration of hair follicles into the wound site or division and differentiation of hair follicle stem cells and formation of new hair follicles. Other current methods of scar revision have similarly involved integumental perturbation. One method involves the surgical excision of the wound and surrounding normal tissue. The newly formed wound is then re-closed by primary intent. In some cases, a jagged surgical incision is created so that the lines of tension of the skin are parallel to the incisions (since perpendicular incisions to the lines of tension heal poorly.) Another method involves dermabrasion to remove epidermis and papillary dermis. This treatment has been used for acne (atrophic scars), but is not widely used today. Needling is an antiquated technique that employs repeated "needle" injury to the scar to "loosen" it. In subcision, a technique developed by Norman and David Orentreich that has not been widely accepted, cuts are made "under" scars to loosen the connective tissue that might be anchoring scar.
[00319] Laser treatment, such as by pulsed dye laser and, more recently, nonablative fractional laser, has also been reported to improve the appearance of surgical scars. See Tierney et al. , 2009, Dermatol Surg 35: 1 172- 1180 (incorporated herein by reference in its entirety). We have found that another example of the regenerative capacity of skin underlies the capacity of fractional lasers to effect resurfacing and mediate wound revision. Fractional laser treatment of scarred tissue creates areas of small micro-injuries with intact epidermis in- between, and the integumental perturbation of the laser activates hair follicle deposition into the injury sites, either by migration from the intact epidermis or by inducing hair follicle neogenesis in the wound. In one embodiment, laser- induced wounding of columns (the non- ablative coagulum is a preferred embodiment) triggers the regenerative capacity of the intervening normal skin stem cells. This technique may have utility in, for example, revising small scars (to improve texture, pigmentation and other features).
[00320] Without being bound by any theory, one advantage of using combinations comprising integumental perturbation is that the perturbation provides a signal for hair follicle deposition and/or deposition of other adnexal structures into the wound site, e.g., by their migration and/or by generation of new hair follicles (hair follicle neogenesis) or adnexal structures. Again while not being bound by any theory, whether or not a wound heals by scarring may depend on the efficiency of hair follicle or other adnexal structure deposition into the wound. If these structures, e.g., hair follicles, are not timely deposited into the healing wound, the process will result in a scar. Thus, wound healing without scarring (and scar revision) may be effected by improving the efficiency of adnexal structure {e.g., hair follicles) deposition into the wound or by slowing wound healing in order to allow sufficient time for deposition of these structures into the wound site.
[00321] In certain embodiments, enhancement of wound healing or scar revision is accomplished by lithium treatment alone, for example, in acutely wounded skin or skin affected by a chronic non-healing wound, i.e., skin already subjected to integumental perturbation. In some embodiments, the lithium treatment is administered to skin that has been damaged and which no longer contains follicles. In such embodiments, the lithium treatment may restore follicle production in that area of skin. In one such embodiment, an area of skin containing a wound that has not healed correctly, such as a scar {e.g., a keloid scar), is administered a lithium treatment in order to restore hair follicles and/or hair growth to that area of skin. These effects may be accomplished by modulating the dosage of lithium.
[00322] In certain other embodiments, enhancement of wound healing or scar revision is accomplished by a combination of integumental perturbation and a pulse or intermittent lithium treatment. In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with integumental perturbation or, optionally, also comprises another treatment known in the art or described herein.
Combinations comprising integumental perturbation are preferred for skin that is not already acutely wounded, since wounding itself is a form of integumental perturbation. Integumental perturbation can be achieved by any means known in the art or described herein, such as, for example, using chemical or mechanical means.
[00323] In one embodiment, integumental perturbation comprises disrupting the skin of the subject (for example, resulting in the induction of re-epithelialization of the skin of the subject). In some embodiments, a certain area of the epithelium is partially or wholly disrupted. In some embodiments, a certain area of both the epithelium and stratum corneum are partially or wholly disrupted. For a discussion of skin disruption and re-epithelialization, including methods for disrupting skin and inducing and detecting re-epithelialization, see PCT Publication Nos. WO 2008/042216 and WO 2006/105109, each of which is
incorporated herein by reference. Integumental perturbation can be used to induce, for example, a burn, excision, dermabrasion, full-thickness excision, or other form of abrasion or wound. In specific embodiments, the combination of integumental perturbation and lithium treatment is administered to skin that has been damaged and which no longer contains hair follicles. In such embodiments, the combination of integumental perturbation and lithium treatment restores follicle production in that area of skin. In one such embodiment, an area of skin containing a wound that has not healed correctly, such as a scar {e.g. , a keloid scar), is administered a combination treatment of integumental perturbation and lithium in order to restore hair follicles and/or growth of hair to that area of skin.
[00324] Mechanical means of integumental perturbation include, for example, use of sandpaper, a felt wheel, ultrasound, supersonically accelerated mixture of saline and oxygen, tape-stripping, spiky patch, or peels. Chemical means of integumental perturbation can be achieved, for example, using phenol, trichloroacetic acid, or ascorbic acid. Electromagnetic means of integumental perturbation include, for example, use of a laser (e.g., using lasers, such as those that deliver ablative, non-ablative, fractional, non-fractional, superficial or deep treatment, and/or are CC based, or Erbium-YAG-based, etc.). Integumental perturbation can also be achieved through, for example, the use of visible, infrared, ultraviolet, radio, or X-ray irradiation. In one embodiment, integumental perturbation is by light energy, such as described in Leavitt et al, 2009, Clin. Drug. Invest. 29:283-292. Electrical or magnetic means of disruption of the epidermis can be achieved, for example, through the application of an electrical current, or through electroporation or RF ablation. Electric or magnetic means can also include the induction of an electric or a magnetic field, or an electromagnetic field. For example, an electrical current can be induced in the skin by application of an alternating magnetic field. A radiofrequency power source can be coupled to a conducting element, and the currents that are induced will heat the skin, resulting in an alteration or disruption of the skin. Integumental perturbation can also be achieved through surgery, for example, a biopsy, a skin transplant, skin graft, follicular unit extraction, hair transplant, cosmetic surgery, open- heart surgery, etc.
[00325] In some embodiments, integumental perturbation is by laser treatment, as discussed below. Exemplary laser treatments for integumental perturbation include Fraxel, laser abrasion, Erbium- YAG laser, Ultrapulse CO2 fractional laser, Ultrapulse CO2 ablative laser, Smooth Peel Full-ablation Erbium laser (Candela), as described, for example, in the examples of Section 8 below. In one embodiment, a laser treatment is chosen in which the integumental perturbation achieved most resembles that achieved by dermabrasion (for example, a dermabrasion method described herein). In a preferred embodiment, integumental perturbation by laser treatment is by a fractional laser. See, e.g., the laser treatments described in U.S. Provisional Application Nos. 61/262,820, 61/262,840, 61/262,831, each of which is incorporated herein by reference in its entirety. One example of a fractional laser treatment is treatment with an Erbium- YAG laser at around 1540 nm or around 1550 nm (for example, using a Fraxel® laser (Solta Medical)). Treatment with an Erbium-YAG laser at 1540 or 1550 nm is typically non-ablative, and pinpoint bleeding typical of laser treatment is not observed since the stratum corneum is left in tact. The column of dead (epidermal and/or dermal) cells in the path of the laser treatment is termed a "coagulum." In another embodiment, integumental perturbation by laser treatment is by a fractional laser, using, e.g. , a CO2 laser at 10,600 nm. Treatment with a CO2 laser at 10,600 nm is typically ablative, and typically leads to the appearance of pinpoint bleeding.
[00326] A standard CO2 or Erbium- YAG laser can be used to create superficial and, optionally, broad based, integumental perturbation similar to dermabrasion (discussed below). Although the pinpoint bleeding clinical endpoint may not be achieved due to the coagulation properties of (particularly non-ablative) lasers, use of a laser has an advantage making it possible to select the specific depth of skin disruption to effectively remove the stratum corneum and epidermis, or portions thereof.
[00327] In one embodiment, the laser treatment is ablative. For example, full ablation of tissue is generated by the targeting of tissue water at wavelengths of 10,600 nm by a CO2 laser or 2940 nm by an Erbium- YAG laser. In this mode of laser treatment, the epidermis is removed entirely and the dermis receives thermal tissue damage. The depth of tissue ablation may be a full ablation of the epidermis, or a partial ablation of the epidermis, with both modes causing adequate wounding to the skin to induce the inflammatory cascade requisite for regeneration. In another variation, the depth of ablation may extend partially into the dermis, to generate a deep wound. The denuded skin surface is then treated with a lithium composition described herein; alternatively, the lithium composition can be delivered into the skin after the initial re-epithelialization has occurred already, to prevent clearance and extrusion of the lithium-containing depots from the tissue site by the biological debris- clearance process. In one embodiment, a lithium composition described herein is delivered by a sustained release depot that is comprised of biocompatible, bioabsorbable polymers that are compatible to tissue.
[00328] The standard full thickness excision model is created using scissors or with a scalpel in animal models (see, also, the examples of Section 16 infra). This procedure, while contemplated for use herein, carries with it the risk of scarring. However, various fractional laser modalities could be used to achieve a similarly deep disruption on a grid pattern. A fractional laser can be use to "drill," for example, 1-mm diameter holes with a 1-mm hole spacing (the fractional laser can make holes of smaller dimensions). Although the skin is completely removed within the 1-mm hole, the surrounding intact skin prevents scarring and therefore the full thickness excision model is invoked within each hole. [00329] In some embodiments, the laser treatment is ablative and fractional. For example, fractional tissue ablation can be achieved using a CO2 laser at 10,600 nm or an Erbium- YAG laser at 2940 nm {e.g., the Lux 2940 laser, Pixel laser, or Pro fractional laser). In some such embodiments, the lasing beam creates micro-columns of thermal injury into the skin, at depths up to 4 mm and vaporizes the tissue in the process. Ablative treatment with a fractional laser leads to ablation of a fraction of the skin leaving intervening regions of normal skin intact to rapidly repopulate the epidermis. Approximately 15%— 25% of the skin is treated per session. The density of micro thermal zones (MTZ) can be varied to create a dense "grid" of injury columns surrounded by intact skin and viable cells. The density of the grid on the treatment area plays an important role. The denser the grid, the more the thermal injury and the type of injury begins to approximate full ablation. Therefore, it is appreciated that there may be an "optimum" MTZ density that is appropriate for use in the methods disclosed herein. In one embodiment, a lithium composition described herein is delivered into the dermis immediately after wounding, or after initial re-epithelialization has occurred.
[00330] In another embodiment, the mode of laser treatment is non-ablative, wherein the stratum corneum and the epidermis are intact after treatment, with the dermis selected for the deep thermal treatment required for the requisite injury to tissue. This can be accomplished by cooling the epidermis during the laser treatment. For example, one could use the timed cooling of the epidermis with a cryogen spray while the laser delivers deep thermal damage to the dermis. In this application, the depth of treatment may be 1 mm to 3 mm into the skin. One could also use contact cooling, such as a copper or sapphire tip. Lasers that are non- ablative have emission wavelengths between 1000-1600 nm, with energy fluences that will cause thermal injury, but do not vaporize the tissue. The non-ablative lasers can be bulk, wherein a single spot beam can be used to treat a homogenous section of tissue. In some embodiments, multiple treatments are required to achieve the desired effect. In one embodiment, a lithium composition described herein is delivered deep into the dermis in polymeric micro-depots and released in a sustained fashion. Lasers that are non-ablative include the pulsed dye laser (vascular)(at, e.g., 585-595 nm), the 1064 Nd:YAG laser, or the Erbium- YAG laser at 1540 nm or 1550 nm {e.g., the Fraxel® laser). Use of an Erbium- YAG laser at around 1540 nm or around 1550 nm, as opposed to its use at 2940 nm, "coagulates" zones of dermis and epidermis (forming a "coagulum") and leaves the stratum corneum essentially intact.
[00331] In another embodiment, the mode of laser treatment is fractional and non-ablative. Treatment with a fractional, non-ablative laser leads to perturbation of a fraction of the skin, leaving intervening regions of normal skin intact to rapidly repopulate the epidermis.
Approximately 15%— 25% of the skin is treated per session. As in any non-ablative process, the skin barrier function is maintained, while deep thermal heating of dermis can occur. Thus, zones of dermis and epidermis are coagulated and the stratum corneum is left essentially intact. This process has been coined "fractional photothermolysis" and can be accomplished, e.g., using the Erbium-YAG laser with an emission at or around 1540 nm or 1550 nm. In one embodiment, a lithium composition described herein is delivered immediately after the tissue injury, deep into the dermis. In another embodiment, a combination of bulk and fractional ablation modes of tissue injury are used.
[00332] In another embodiment, a combination treatment comprising use of a laser includes administration to the skin of a compound absorbing light at wavelengths between 1000-1600 nm for the purpose of efficient channeling of light to heat energy. This method of channeling energy may cause micro-zones of thermal injury within the skin. The compound may be delivered to the skin homogenously in the treatment zone, then subsequently irradiated with a non-ablative laser to efficiently capture the vibrational energy of the infrared beam. This method may result in evenly distributed and deep thermal injury, without causing tissue vaporization.
[00333] In another embodiment, a combination treatment comprising use of a laser includes administration of a lithium compound formulation that is encapsulated in matrices that are highly hydrophilic and charged, for example, linked to the dermis by covalent or ionic bonding to prevent the matrices from being cleared by phagocytosis, as part of the wound healing process.
[00334] In another embodiment, a combination treatment comprising use of a laser includes the step of placing a biocompatible, synthetic skin substitute on the newly created wound, especially if the wound is deep, covers large area, and is bulk ablated. This process can help minimize or prevent the rapid wound contraction that occurs after loss of a large area of tissue, frequently culminating in scar tissue formation and loss of skin function. In one embodiment, the biocompatible synthetic skin substitute is be impregnated with depots of a slow releasing lithium formulation described herein. This method of treatment may enable treating a large area in one session at the treatment clinic. In some embodiments, other molecules are also co-eluted at the site through the skin substitute, such as, e.g. , anesthetics and antibiotics, to prevent further pain and minimization of infection, or any other compound described herein. The skin substitute, in the presence or absence of a lithium compound and/or other compounds described herein, may also be pre-cooled and applied to the wound to provide a feeling of comfort to the patient. This mode of lithium or other compound application may prevent the lithium or other compound from being cleared away from the wound site as the wound heals.
[00335] In some embodiments, a fractional like hole pattern (similar to that achieved with a fractional laser or full thickness excision) is achieved with using an array of punch biopsy needles. For example, 1 -mm punch biopsies can be arranged with 1 -mm hole spacing. When inserted into the scalp or other area of skin to be treated, the cored skin samples can be removed and, thus, an effect approximating the full thickness excision model is invoked within each hole. Similarly, and for smaller holes, microneedles (e.g., 19 or 21 gauge needles) and/or micro-coring needles could be used.
[00336] In some embodiments, integumental perturbation is by dermabrasion (also referred to herein as "DA"), a well-established dermatological procedure that has been used for decades as a skin resurfacing technique (Grimes, 2005, Microdermabrasion. Dermatol Surg 31 : 1351-1354). While the popularity of mechanical DA has decreased in recent years with the advent of laser-based procedures, DA is still used for removing facial scars resulting from acne and other trauma. Small, portable mechanical dermabrasion equipment uses interchangeable diamond firaises operated at different rotation speeds to remove the epidermis and dermis to differing skin depths levels. Adult human skin treated with DA completely re- epithelializes in 5-7 days with minor redness lasting up to a few weeks. Dermabrasion may be carried out using any technique known in the art or as described herein, e.g., in the examples of Sections 9, 10 and 16 infra. For example, dermabrasion may be carried out using standard DA with aluminum oxide crystals using the Aseptico Econo-Dermabrader, Advance Microderm DX system, or M2-T system; standard DA with Bell Hand Engine with diamond fraize; etc.
[00337] For example, in some embodiments, DA is carried out using an abrasive wheel. In some embodiments, DA with an abrasive wheel is used in order to achieve pinpoint bleeding. In other embodiments, DA may be carried out using an abrasive wheel to achieve larger globules of bleeding and frayed collagen. In other embodiments, non-powered devices such as abrasive cloths can also be used to achieve the DA, with the optional achievement of the same endpoint(s).
[00338] In some embodiments, DA is accomplished using a device typically used for microdermabrasion. For example, in such DA protocols, a microdermabrasion device is used to remove a greater depth and/or area of skin than is typical for microdermabrasion (also referred to herein as "MDA"). In some embodiments, the microdermabrasion device is used under sterile conditions. In some embodiments, DA is achieved by using a device typically used for microdermabrasion to the point where treatment is stopped upon the observation of pinpoint bleeding, which signals the removal of the stratum corneum and epidermis into the papillary dermis. In other embodiments, DA is achieved by using a device for
microdermabrasion to the point where treatment is stopped upon the observation of larger globules of bleeding and frayed collagen, which signals the removal of the stratum corneum and epidermis into the papillary and reticular dermis. In some embodiments, this extended use is reduced by using a microdermabrasion device with increased output pressure and increased abrasion particle size, which may accelerate the skin removal process.
[00339] In some embodiments, DA is accomplished by removal of surface skin by particle bombardment (also referred to herein as "particle mediated dermabrasion" ("PMDA")), for example, with alumina-, ice- or silica-based particles. In some such embodiments, micron- sized particles are propelled toward the surface of the skin via short strokes of a handpiece, such as a particle gun, as known in the art. The velocity of particles is controlled through positive or negative pressure. The depth of skin removed by particle bombardment DA {e.g., PMDA) is a function of the volume of particles impacting the skin, the suction or positive pressure, the speed of movement of the handpiece, and the number of passes per area of the skin.
[00340] In some embodiments, integumental perturbation by one or more of the aforementioned methods achieves removal of part or all of the epidermis. In some embodiments, integumental perturbation removes the entire epidermis. In some
embodiments, integumental perturbation disrupts the papillary dermis. In some
embodiments, integumental perturbation removes the papillary dermis. In some
embodiments, integumental perturbation removes the reticular dermis. The depth of integumental perturbation depends on the thickness of the skin at a particular treatment area. For example, the skin of the eyelid is significantly thinner than that of the scalp. The occurrence of pinpoint bleeding indicates that the epidermis and portions of the dermis have been removed. Deeper penetration can results in much more bleeding, and the perturbation can go as deeps as the hypodermis.
[00341] In some embodiments, integumental perturbation by one or more of the aforementioned methods is to a skin depth of 60 μιη. In some embodiments, integumental perturbation is to a skin depth of 60-100 μιη. In some embodiments, integumental perturbation is to a skin depth of 100 μιη. In some embodiments, integumental perturbation is to a skin depth of 150 μιη. In some embodiments, integumental perturbation is to a skin depth of 100-500 μηι. In some embodiments, integumental perturbation is to a skin depth of less than 500 μιη. In some embodiments, integumental perturbation is to a skin depth of 500- 1000 μιη. In some embodiments, integumental perturbation is to a skin depth of 1 mm or more. In some embodiments, integumental perturbation is to a skin depth of 1 mm to 3 mm. In some embodiments, integumental perturbation is to a skin depth of 1 mm to 5 mm.
Discussion
[00342] Integumental perturbation, such as occurs during wounding, produces in the affected skin tissue an increase in the number of hair follicle stem cells and in the plasticity of hair follicle cells. The pulse or intermittent lithium treatments cause formation of new hair follicles or enhanced branching, division, or differentiation of existing hair follicles or hair follicle progenitors. Accordingly, and without being bound by any theory for how the invention works, integumental perturbation (or wounding) in combination with a pulse or intermittent lithium treatment provides an environment for the formation of a large number of follicles to enhance wound healing and, preferably, wound healing with reduced scarring.
[00343] Toscani et al. (2009, Dermatol Surg. 35: 1 119-1 125) showed that -70% of bisected hair follicles (either top or bottom half) are capable of forming new hair follicles when bisected surgically ex vivo and transplanted into scalp skin. It is possible that the
integumental perturbation techniques described herein, for example, laser techniques or surgical dissection of follicles, etc., lead to hair follicle transection in vivo. It is thought that at least 40% of follicles, after bisection, form two follicles (which would result if all the "bottoms" from the -30% of failed "tops" successfully yielded a hair fiber and in addition, if all the "tops" from the -30% of failed bottom bisected follicles successfully yielded a hair fiber.) Therefore, the percentage of bisected follicles that produce two new follicles is in the range of -40-70% (with the maximum -70% being the result of all bisections producing two new follicles and no bisections resulting in either a top or a bottom (but not both) producing a follicle). It is expected that this efficiency will be increased when pulsatile lithium is applied because it induces differentiation.
[00344] Moreover, skin rejuvenation following wounding occurs through the action of skin stem cells. See, e.g., Fuchs E, 2009, "Finding one's niche in the skin," Cell Stem Cell 4:499-502. For example, new hair follicles originate from Hair Follicle Stem Cells (FSCs), oligopotent cells whose progeny can differentiate into the highly differentiated specialized cells of the hair follicle (see Amoh Y, et al. Human hair follicle pluripotent stem (hfPS) cells promote regeneration of peripheral-nerve injury: an advantageous alternative to ES and iPS cells. J Cell Biochem, 2009, 107: 1016-1020; and Amoh Y, et al. Nascent blood vessels in the skin arise from nestin-expressing hair-follicle cells. Proc Natl Acad Sci U S A. 2004 Sep 7; 101(36): 13291-5. Epub 2004 Aug 26).
[00345] FSCs originate from one or more of the following: (i) existing follicles ("follicle derived follicle stem cells" or "FDFSC") (see, e.g., Toscani et al, 2009, Dermatol Surg. 35: 1 119-1 125; (ii) the skin ("tissue derived follicle stem cells" or "TDFSC") (see, e.g., Ito M, 2007, Nature 447:316-320); (iii) bone marrow ("bone marrow derived follicle stem cells" or "BMDFSC") (see, e.g., Fathke et al, 2004, Stem Cells 22:812-822; and Rovo et al, 2005, Exp Hematol. 33:909-91 1); and/or (iv) from mesenchymal stem cells such as hair follicle dermal sheath cells and adipocyte stem cells.
[00346] FSCs generate new hair follicles that preserve the type of hair follicle that is typical for each location of skin or scalp. For example, FSCs from the coronal scalp of a male with MPHL typically generate atrophic follicles with vellus or club hairs. In contrast, FSCs from the occipital scalp of the same male typically generate follicles with terminal hair that are not subject to involution in response to DHT
[00347] However, if external signals are provided that interfere with this "default" program, the FSCs responsible for follicle formation may be reprogrammed. FSCs in the process of asymmetric division and subsequent differentiation are susceptible to signals (such as estrogen or testosterone) that alter the determinism of their differentiation program. Thus, a pulse or intermittent lithium treatment in combination with integumental perturbation provides a window during which a third treatment that alters the follicle development program may be administered in order to significantly change the number and quality of follicles in a particular area of skin. For example, any treatment that enhances hair growth or, alternatively, that prevents hair growth or removes excessive hair, that is known in the art or yet to be developed is contemplated for use in such combination treatments. Examples of treatments that promote hair growth include minoxidil, finasteride, bimatoprost (Latisse), CaCl2, or adenosine, or techniques of integumental perturbation such as by mechanical means, chemical means, electromagnetic means (e.g., using a laser such as one that delivers ablative, non-ablative, non-fractional, superficial, or deep treatment, and/or are CCVbased, or Erbium- YAG-based, etc.), irradiation, radio frequency (RF) ablation, or surgical procedures (e.g., hair transplantation, strip harvesting, follicular unit extraction (FUE), scalp reduction, etc.). Examples of treatments that remove unwanted hair or prevent hair growth include, e.g., cytotoxic drugs, hair growth retardants, such as eflornithine HC1 (Vaniqa), 5-fluorouracil (5- FU) (e.g., Efudex 5% cream), or other epilation or depilation methods. Alternatively, a third treatment may comprise treatment with an estrogen or testosterone modulator, such as those described in Poulos & Mirmirani, 2005, Expert Opin. Investig. Drugs 14: 177-184, which is incorporated herein by reference.
[00348] In some embodiments, the third treatment is administered simultaneously with integumental perturbation. In some embodiments, the third treatment is administered after integumental perturbation. In some embodiments, the third treatment is administered 1 day, 2 days, 3 days, 5 days, 7 days, 10 days, or 2 weeks after integumental perturbation. In one embodiment, the third treatment is administered at the time of integumental perturbation and then daily for 5 days thereafter (in some embodiments, a scab forms during this time). In some embodiments, the third treatment is administered daily for 5 days beginning as soon as the scab falls off. In some embodiments, the third treatment is administered in order to modulate the neoepidermis that forms underneath the scab. In some such embodiments, the third treatment is administered at the time of integumental perturbation and up to some time after scab falls off, for example, between 5 - 14 days following integumental perturbation. In some embodiments, the course of treatment with the third treatment is short, for example, limited to a few days just following scab detachment, or even continued only for as long as the scab is still attached. The timing of the integumental perturbation, lithium administration, and the third treatment is preferably monitored and adjusted so that optimal results are achieved.
5.4.2 COMBINATION TREATMENTS TO INCREASE HAIR
FOLLICLES
[00349] This invention is based, in part, on the discovery that there is a correlation between the extent of wound contraction and the deposition of adnexal structures, such as new hair follicles, in wounded areas. In experiments to assess the effect of 8% topical lithium on hair follicle neogenesis following full thickness excision wounding of mouse skin, it was found that there was a direct relationship between increased number of hair follicles and decreased wound size (see the example in Section 16, especially Section 16.4, infra). Thus, without being bound by any theory for how the invention works, intermittent and pulse lithium treatments may promote wound healing and scar revision by, at least in part, promoting the entry of hair follicles into the wound as it heals. This may occur by inducing the generation of new hair follicles and/or promoting migration of hair follicles into the wound site.
[00350] The intermittent and pulse lithium treatments described herein may (i) promote hair follicle neogenesis {e.g. , de novo formation of hair follicles from tissue or bone-marrow derived stem cells or disintegration of preexisting follicles into cells that mix together and reform the hair follicle); and/or (ii) promote branching {e.g., with the assistance of stem cells from dissociated hair follicles) and division of existing hair follicles. Without being bound by any theory, the mechanism by which hair follicles enter the wound site depends on the type of skin that is wounded and the type of wound. For example, in one embodiment, a superficial wound is healed by the assistance of hair follicles remaining in the wound. In another embodiment of a more severe wound, the hair follicles disintegrate and are reorganized and reformed by the presence of stem cells that enter the wound. In another embodiment of severe wounding, such as seen in full thickness excision wounding, hair follicle neogenesis promotes wound healing.
[00351] Thus, in some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more agents that increase the number of hair follicles or that counteract hair follicle cell senescence (also referred to herein as "anti-senescence agents"), for example, anti-oxidants such as glutathione, ascorbic acid, tocopherol, uric acid, or polyphenol antioxidants); inhibitors of reactive oxygen species (ROS) generation, such as superoxide dismutase inhibitors;
stimulators of ROS breakdown, such as selenium; mTOR inhibitors, such as rapamycin; or sirtuins or activators thereof, such as resveratrol, or other SIRT1, SIRT3 activators, or nicotinamide inhibitors.
[00352] In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more agents that induce an immune response or cause inflammation, such as, e.g., tetanus toxoid, topical non-specific irritants (anthralin), or sensitizers (squaric acid dibutyl ester [SADBE] and diphenyl cyclopropenone [DPCP]). While not intending to be bound by any theory, it is thought that by contacting these agents to the skin, lymphocytes and hair follicle stem cells may be recruited to skin.
[00353] In some embodiments, the combination treatment comprises a pulse or intermittent lithium treatment together with a cytokine thought to regulate the activity of Dermal Papillae, which is believed to be the target of androgen regulation of hair growth. Interleukin-1 alpha decreases responses to androgen in cultured dermal papilla cells (Boivin et al, 2006, Exp Dermatol. 15:784-793). TGF-βΙ may mediate androgen-induced hair growth suppression, since in culture, human dermal papilla cells (DPCs) from androgenetic alopecia (AGA) subjects that transiently expressing androgen receptor were co-cultured with keratinocytes (KCs), and secreted TGF-βΙ that inhibited KC growth (Inui et al., 2003, J Investig Dermatol Symp Proc. 8:69-71). Thus, a TGF-B1 inhibitor may be used in a combination treatment.
[00354] Melatonin is a protein hormone secreted by the pineal gland that modulates hair growth, pigmentation and/or molting in many species. Human scalp hair follicles in anagen are important sites of extra-pineal melatonin synthesis. Melatonin may also regulate hair Follicle Cycle control, since it inhibits estrogen receptor-alpha expression (Fischer et al, 2008, Pineal Res. 44: 1-15). Melatonin and the other treatments described herein can be administered, for example, during the lithium treatment "holidays." Alternatively, these treatments can be administered prior to or subsequent to a pulse lithium treatment.
[00355] In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with a chemical or mechanical (such as those discussed infra) treatment that induces an inflammatory process in the skin. While not intending to be bound by any theory, inducing inflammation in the site where hair growth is desired helps to recruit stem cells to the tissues that drive the formation of new follicles.
[00356] In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with an antiapoptotic compound. In one embodiment, the antiapoptotic compound is not a Wnt or a Wnt agonist.
[00357] In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more of stem cell therapy, hair cloning or hair plugs, follicular unit extraction, hair or skin transplantation, massage, a skin graft, or any surgical procedure aimed at skin or hair restoration.
[00358] In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with use of a laser device or other mode of accomplishing "photo-biostimulation" of the hair follicles. For example, the Hairmax Lasercomb or the Leimo laser are non-limiting examples of devices that can be used in combination with the methods described herein.
[00359] In some embodiments, intermittent lithium treatment or a pulse lithium treatment, alone or in combination with other treatments described herein, synchronizes hair follicle cells in the cell cycle. In a specific embodiment, lithium is administered to arrest hair follicle cells in G2/M phase, which synchronizes them; then the lithium treatment is removed; and then their re-entry into the cell cycle and mitotic division is stimulated with other drugs (which leads to anagen follicles and an increased number of follicles). In another embodiment, the lithium treatment arrests hair follicle cells in late prophase or metaphase, which synchronizes them; the lithium treatment is removed; and then their re-entry into the cell cycle and mitotic division is stimulated with other drugs (which leads to anagen follicles and an increased number of follicles). In another embodiment, the lithium treatment arrests hair follicle stem cells in G2/M phase, which synchronizes them; then the lithium treatment is removed; and then their re-entry in to the cell cycle and mitotic division is stimulated with other drugs (which leads to anagen follicles and an increased number of follicles). In another embodiment, the lithium treatment arrests hair follicle stem cells in late prophase or metaphase, which synchronizes them; the lithium treatment is removed; and then their reentry into the cell cycle and mitotic division is stimulated with other drugs (which leads to anagen follicles, and an increased number of follicles).
[00360] In some embodiments, intermittent lithium treatment or a pulse lithium treatment, alone or in combination with the aforementioned combination treatments, synchronizes hair follicle cells in the Follicle Cycle. In one such embodiment, the treatment regimen induces follicles to enter anagen. In another embodiment, the treatment regimen prevents follicles from entering catagen. In one embodiment, the treatment regimen induces follicles in telogen to enter exogen, or induces follicles in exogen to enter anagen.
[00361]
5.4.3 Combination Treatments for Modulation of Wound Healing
[00362] In some embodiments, a combination treatment comprises a pulse or intermittent lithium treatment in combination with another treatment that modulates wound healing, including any treatment described herein or known in the art to modulate wound healing.
[00363] In one embodiment, the pulse or intermittent lithium treatment is administered in combination with a treatment that enhances one or more of the steps of wound healing discussed in Section 2.1.1 above, including any treatment described herein or known in the art to enhance wound healing. By enhancement of a step of wound healing or enhancement of wound healing is meant the hastening of healing, improvement of healing, or reduction of scarring, etc.
[00364] In some embodiments, the pulse or intermittent lithium treatment is administered in combination with a wound dressing or skin replacement, such as, for example, gauze, calcium-alginates, impregnated gauzes, films, foams, hydrogels, hydrocolloids, adsorptive powders and pastes, silicone, mechanical vacuum, dermal matrix replacements, dermal living replacements, or skin living replacements, a collagen dressing, cadaveric skin, or other matrix useful to promote healing of the wound such as described herein or known in the art. See, e.g., Table 10.3 in Lorenz & Longaker, which is incorporated by reference herein in its entirety.
[00365] In some embodiments, the pulse or intermittent lithium treatment is administered in combination with a pain reliever, antibiotic and antibacterial use or other anti-infectives (such as, e.g., tea tree oil), debridement, drainage of wound fluid, mechanical removal of bacteria, removal of devitalized tissue (such as, e.g., by surgery or maggot therapy), irrigation (e.g., by pulsed lavage), vacuum-assisted closure (otherwise referred to as negative pressure wound therapy), warming, oxygenation (e.g., using hyperbaric oxygen therapy), antioxidant therapy, revascularization therapy, moist wound healing, removing mechanical stress, use of elastase inhibitors, or adding cells or other materials to secrete or enhance levels of healing factors.
[00366] In some embodiments, the pulse or intermittent lithium treatment is administered in combination with the upregulation of endogenous growth factors or exogenous application of growth factors, which may accelerate normal healing and improve healing efficacy. Such growth factors include, but are not limited to, vascular endothelial growth factor (VEGF), insulin-like growth factor 1-2 (IGF), PDGF, transforming growth factor-β (TGF-β), epidermal growth factor (EGF), EGF -receptor, members of the FGF family, and others described herein and listed in, e.g., Table 10.2 in Lorenz & Longaker, which is incorporated by reference herein in its entirety. Such growth factors can be applied exogenously or may be applied by spreading onto the wound a gel of the patient's own platelets, implanting cultured keratinocytes into the wound, or treating the wound with artificial skin substitutes that have fibroblasts and keratinocytes in a matrix of collagen.
[00367] In some embodiments, the pulse or intermittent lithium treatment is administered in combination with a treatment that reduces the time it takes for a wound to heal or that reduces the extent of the wound. Such treatments are known in the art and include, for example, periodic rotation of the patient or wounded tissue or use of an air mattress, use of a lower pressure cast or relieving excessive suture tension, cleansing of the wound, debridement of tissue, particularly necrotic tissue, improvement of circulation and oxygen delivery to the tissue by, e.g., hyperbaric oxygen therapy or other oxygen administration, whirlpool therapy, ultrasound therapy, electrical stimulation, magnetic therapy have been utilized to aid the body in healing wounds coverage of wound with vascularized tissue, revascularization of the wounded tissue, treatment of circulatory obstruction or other treatment that improves circulation, treatment of ischemia, edema, or hypoxia, or improvement of the hematocrit (e.g., to at least 15%). Other treatments to enhance wound healing that may be used in combination with the pulse or intermittent lithium treatments described herein include treatment of tissue necrosis, treatment or prevention of infection (e.g., with antibiotics such as povidone-iodine, chlorhexidine gluconate, hexachlorophene, or silver sulfadiazine and others described herein (particularly for burn wound care), irrigation (e.g., with saline), and/or debridement), improvement of nutrition {e.g., increasing intake of vitamins, e.g., vitamin A, C, Bl, B2, B5, or B6, or trace metals, such as, e.g., zinc and copper, amino acids such as arginine, glutamine, or Bromelain, Curcumin, etc.), herbal supplements (e.g., Aloe Vera, Centella), diabetes treatment (for example, to improve vascular conditions, or by administering glucose), skin graft, treatment with hormones (such as estrogen) or treatment with growth factors (e.g., epidermal growth factor, Insulin-like Growth Factor, human growth hormone, fibroblast growth factor, vascular endothelial growth factor, interleukin-6, and interleukin-10).
[00368] In another embodiment, the pulse or intermittent lithium treatment is administered in combination with a treatment that slows the natural adult wound healing process. In certain embodiments, such combination treatments are used in the presence of a sterile wound dressing that obviates the need to heal the wound quickly (for example, in natural wound healing, the wound heals quickly in order to avoid infection). In one embodiment, the pulse or intermittent lithium treatment is administered in combination with a treatment that causes the postnatal wound healing process to resemble the fetal wound healing process. In some embodiments, this is accomplished by placing the wounded skin into a womb-like environment, for example, using a dressing and/or heat.
[00369] In one embodiment, the pulse or intermittent lithium treatment is administered in combination with an agent that reduces or inhibits the inflammatory phase of wound healing, using, e.g. , an anti-inflammatory agent such as a NSAID or a topical glucocorticoids, an anti- androgen, or an antagonist of TNFa, TGF , NFkB, IL-1, IL-6, IL-8, IL-10, IL-18, or an antagonist of one or more other proinflammatory cytokines. In an alternative embodiment, the pulse or intermittent lithium treatment is administered in combination with an agent that slows the wound healing process by extending the inflammatory phase, e.g., an androgen (see, e.g., Gilliver et al, 2007, Clin. Dermatol. 25:56-62). In one embodiment, the treatment is administered in combination with an agent that suppresses the proliferative phase of wound healing, or the maturation and remodeling phase of wound healing. For example, in one embodiment, the treatment is administered in combination with an agent that slows or interferes with fibrin deposition, clotting caused by fibrin, or fibrin-induced immunity. In one embodiment, the treatment is administered in combination with a treatment that inhibits the activity of fibrinogen. In one embodiment, the treatment is administered in combination with an agent that decreases the activity of myofibroblasts. In particular embodiments, the treatment is administered in combination with a treatment that reduces collagen synthesis, deposition, or accumulation, for example, collagenases. In particular embodiments, the treatment is administered in combination with a treatment that maintains the wound in an open state for a longer than normal period of time. In another embodiment, a treatment is administered in combination with rapamycin or corticosteroids.
[00370] For example, in one embodiment, a biocompatible, synthetic skin substitute is placed on the wound, especially if the wound is deep, covers large area, and is bulk ablated. This process can help minimize or prevent the rapid wound contraction that occurs after loss of a large area of tissue, frequently culminating in scar tissue formation and loss of skin function. In one embodiment, the biocompatible synthetic skin substitute is impregnated with depots of a slow releasing lithium formulation described herein. This method of treatment may enable treating a large area in one session at the treatment clinic. In some embodiments, other molecules are also co-eluted at the site through the skin substitute, such as, e.g., anesthetics and antibiotics, to prevent further pain and minimization of infection, or any other compound described herein. The skin substitute, in the presence or absence of a lithium compound and/or other compounds described herein, may also be pre-cooled and applied to the wound to provide a feeling of comfort to the patient. This mode of lithium or other compound application may prevent the lithium or other compound from being cleared away from the wound site as the wound heals.
5.4.4 COMBINATION TREATMENTS FOR IMPROVEMENT OF SCARS AND SCAR REVISIONS
[00371] In some embodiments, a pulse or intermittent lithium treatment is administered in combination with a treatment that improves wound healing, in order to reduce the appearance or extent of scarring. In some embodiments, a pulse or intermittent lithium treatment is administered in combination with a treatment that improves the appearance and/or function of scarred skin, including any such treatment described herein or known in the art. For example, in one embodiment, a pulse or intermittent lithium treatment is administered in combination with scar revision, such as by skin graft, serial expansion of surrounding skin, or laser treatment as described in Section 5.4 above. In some embodiments, a pulse or intermittent lithium treatment is administered in combination with re-excision with subsequent healing by primary intention, treatment with steroids (e.g., corticosteroid injection), silicone scar treatments (e.g. , dimethicone silicone gel or silicone sheeting), use of porcine fillers or other cosmetic fillers (e.g., inserted under atrophic scars), ribosomal 6 kinase (RSK) antagonists, antagonists of pro-inflammatory cytokines, such as TGF 2 or TNF, osteopontin antagonists, the use of pressure garments, needling, dermabrasion, collagen injections, low-dose radiotherapy, or vitamins (e.g., vitamin E or vitamin C or its esters).
[00372] In some embodiments, a pulse or intermittent lithium treatment is administered in combination with a treatment that reduces surgical scarring, e.g. , by placement of elective incisions parallel to the natural lines of skin tension (Langer's lines) or by applying sutures in a "zigzag" pattern. In some embodiments, the pulse or intermittent lithium treatment is administered in combination with a treatment of wounds that minimizes scarring, by, for example, administering physical therapy to a subject (e.g., range-of-motion exercises), reducing infection, reducing separation of wound edges, minimizing collagen synthesis, deposition, or accumulation or otherwise causing the process of healing by secondary intention to better resemble healing by primary intention. Other interventions that reduce scarring and which may be used in combination with the methods described herein include meticulous hemostasis of wound healing (including control of bleeding by coagulation, desiccation, or ligation techniques), which decreases amount of hematoma to be cleared and thus decreases the inflammatory phase of wound healing, exercising care during dermal closure (e.g., avoiding forceps crush-injury of the epidermis and dermis), avoidance of necrotic tissue at the wound edge, which reduces inflammation, cleansing of the wound, and applying skin grafts where needed.
5.4.5 REGIMENS FOR COMBINATION TREATMENTS
[00373] In certain embodiments, intermittent lithium treatment or a pulse lithium treatment in combination with the aforementioned methods for enhancing scar revision or wound healing improves the effectiveness of these methods, making the treatment more effective, efficient, cost-effective, pain-free, and/or user friendly. For example, fewer treatments may be required. In certain embodiments, one of the previously described wound healing or scar revision treatments on its own is not cosmetically satisfactory, does not adequately restore function of the skin, or the benefits are too short-lived. When one of these treatments is combined with intermittent lithium treatment or a pulse lithium treatment, the wounded or scarred area may be more cosmetically satisfactory, the effects of the treatment longer lasting, or skin function is restored. [00374] For any of the combination treatments described above, in specific embodiments, the intermittent lithium treatment or a pulse lithium treatment can be administered prior to, concurrently with, or subsequent to the administration of a second (or third, or more) treatment.
[00375] In one embodiment, the intermittent lithium treatment or a pulse lithium treatment is administered to a subject at reasonably the same time as the other treatment. This method provides that the two administrations are performed within a time frame of less than one minute to about five minutes, or up to about sixty minutes from each other, for example, at the same doctor's visit.
[00376] In another embodiment, the intermittent lithium treatment or a pulse lithium treatment and other treatment are administered at exactly the same time.
[00377] In yet another embodiment, the intermittent lithium treatment or a pulse lithium treatment and the other treatment are administered in a sequence and within a time interval such that the intermittent lithium treatment or a pulse lithium treatment and the other treatment can act together to provide an increased benefit than if they were administered alone. In another embodiment, the intermittent lithium treatment or a pulse lithium treatment and other treatment are administered sufficiently close in time so as to provide the desired outcome. Each can be administered simultaneously or separately, in any appropriate form and by any suitable route. In one embodiment, the intermittent lithium treatment or a pulse lithium treatment and the other treatment are administered by different routes of
administration. In an alternate embodiment, each is administered by the same route of administration. The intermittent lithium treatment or a pulse lithium treatment and the other treatment can be administered at the same or different sites of the subject's body. When administered simultaneously, the intermittent lithium treatment or a pulse lithium treatment and the other treatment may or may not be administered in admixture or at the same site of administration by the same route of administration.
[00378] In various embodiments, the intermittent lithium treatment or a pulse lithium treatment and the other treatment are administered less than 1 hour apart, at about 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 1 1 hours apart, 11 hours to 12 hours apart, no more than 24 hours apart or no more than 48 hours apart. In other embodiments, the intermittent lithium treatment or a pulse lithium treatment and other treatment are administered 2 to 4 days apart, 4 to 6 days apart, 1 week a part, 1 to 2 weeks apart, 2 to 4 weeks apart, one month apart, 1 to 2 months apart, 2 to 3 months apart, 3 to 4 months apart, 6 months apart, or one year or more apart. In some embodiments, the intermittent lithium treatment or a pulse lithium treatment and the other treatment are administered in a time frame where both are still active. One skilled in the art would be able to determine such a time frame by determining the half life of each administered component.
[00379] In one embodiment, the intermittent lithium treatment or a pulse lithium treatment and the other treatment are administered within the same patient visit. In one embodiment, the intermittent lithium treatment or a pulse lithium treatment is administered prior to the administration of the other treatment. In an alternate embodiment, the intermittent lithium treatment or a pulse lithium treatment is administered subsequent to the administration of the other treatment.
[00380] In certain embodiments, the intermittent lithium treatment or a pulse lithium treatment and the other treatment are cyclically administered to a subject. Cycling treatment involves the administration of the intermittent lithium treatment or a pulse lithium treatment for a period of time, followed by the administration of the other treatment for a period of time and repeating this sequential administration. The first treatment may be with the intermittent lithium treatment or a pulse lithium treatment or with the other treatment, depending on the subject's prior treatment history and the intended outcome. Not only does such cycling treatment have the advantages described herein (attributable, at least in part, to the synchronization of the hair and/or Follicle Cycle), cycling treatment can also reduce the development of resistance to one or more of the treatments, avoid or reduce the side effects of one of the treatments, and/or improve the efficacy of the treatment. In such embodiments, alternating administration of the intermittent lithium treatment or a pulse lithium treatment may be followed by the administration of another treatment (or vice versa) 1 year later, 6 months later, 3 months later, 1 month later, 3 weeks later, 2 weeks later, 1 week later, 4 to 6 days later, 2 to 4 days later, or 1 to 2 days later, wherein such a cycle may be repeated as many times as desired. In certain embodiments, the intermittent lithium treatment or a pulse lithium treatment and the other treatment are alternately administered in a cycle of 3 weeks or less, once every two weeks, once every 10 days or once every week. Such time frames can be extended or reduced depending on whether a controlled release formulation of either the lithium compound or the other treatment formulation is used, and/or depending on the progress of the treatment course. See the examples in Section 7 and 10 for specific treatment variations. [00381] For embodiments in which the lithium treatment accompanies skin or hair transplantation {e.g., follicular unit extraction), an area of skin that was pre-treated with lithium (and optionally another treatment) is used as a source for transplanted follicles. After follicle implantation, treatment with lithium at the wounds(s) from which transplanted tissue was obtained and/or the site of implantation is initiated for one week, and then discontinued and optionally followed by another treatment.
[00382] Other regimens for combination treatments for use in the methods described herein include those described in Section 5.4 supra.
5.5 PATIENT POPULATIONS AND INDICATIONS
[00383] A candidate subject for intermittent lithium treatment (i.e., alternating lithium treatment with "vacation/holiday" periods) or a pulse lithium treatment for promoting hair growth is any subject at risk for, has, or has had a wound or scar.
[00384] The subject may be any subject, preferably a human subject, including male, female, intermediate/ambiguous {e.g., XO), and transsexual subjects. In certain
embodiments, the subject is a Caucasian subject. In certain embodiments, the subject is an African subject or an African-American subject. In certain embodiments, the subject is a human adolescent. In certain embodiments, the subject is undergoing puberty. In certain embodiments, the subject is a young adult. In certain embodiment, the subject is a middle- aged adult. In certain embodiments, the subject is a premenopausal adult. In certain embodiments, the subject is undergoing menopause. In certain embodiments, the subject is postmenopausal. In certain embodiments, the subject is elderly. In certain embodiments, the subject is a human of 1 year old or less, 2 years old or less, 2 years old, 5 years old, 5 to 10 years old, 10 to 15 years old, e.g., 12 years old, 15 to 20 years old, 20 to 25 years old, 25 to 30 years old, 30 years old or older, 30 to 35 years old, 35 years old or older, 35 to 40 years old, 40 years old or older, 40 to 45 years old, 45 to 50 years old, 50 years old or older, 50 to 55 years old, 55 to 60 years old, 60 years old or older, 60 to 65 years old, e.g., 65 years old, 65 to 70 years old, 70 to 75 years old, 75 to 80 years old, 80 to 85 years old, 85 to 90 years old, 90 to 95 years old or 95 years old or older. In some embodiments, the subject is a male 20 to 50 years old. In some embodiments, the subject is a male or female 12 to 40 years old. In some embodiments, the subject is not a female subject. In some embodiments, the subject is not pregnant or expecting to become pregnant. In some embodiments, the subject is not a pregnant female in the first trimester of pregnancy. In some embodiments, the subject is not breastfeeding. [00385] In one embodiment, the intermittent lithium treatment or a pulse lithium treatment is delivered to an area in which enhanced wound healing or scar revision is desired, for example, the scalp, face (e.g., the eyebrow, eyelashes, upper lip, lower lip, chin, cheeks, beard area, or mustache area) or neck, or another part of the body, such as, e.g., the chest, breasts, sternum, abdomen, arms, armpits (site of axillary hair), legs, hands, feet, or genitals. In some embodiments, a wounded or scarred part of the skin is treated. In some
embodiments, the wounded or scarred part of the skin is a flexion surface or involves the extremities, breasts, sternum, face, or neck.
[00386] Wounds treatable by the methods described herein include, but are not limited to, any form of wound known in the art or to be discovered. Non-limiting examples of wounds treatable by the methods described herein include acute wounds (surgical and non-surgical), chronic or non-healing wounds, pressure sores (also referred to as decubitus ulcers or bed sores), pressure necrosis, lower extremity ulcers, radiation injury (such as, e.g., caused by radiation overdose), an erythema, skin abrasion, or a non-healing wound caused by wounding (e.g., a surgical incision) of irradiated skin. In some embodiments, the methods described herein are used to enhance healing of wounds caused by blisters, cutaneous trauma, and surgery, such as described in Mulvaney & Harrington, 1994, Chapter 7, "Cutaneous trauma and its treatment," in Textbook of Military Medicine: Military Dermatology, Office of the Surgeon General, Department of the Army, Virtual Naval Hospital Project, which is incorporated by reference herein in its entirety. In some embodiments, the methods described herein are used to enhance (e.g., hasten, improve, minimize scarring, etc.) healing of wounds by primary intention. In some embodiments, the methods described herein are used to enhance healing of wounds by secondary intention. In some embodiments, the methods described herein are used to enhance healing of wounds by tertiary intention.
[00387] In one embodiment, the wound to be treated by the methods described herein has wound dehiscence, which is the premature "bursting" open of a wound along surgical suture. In some embodiments, the patient is at risk for wound dehiscence, based on one or more of the following risk factors: age, diabetes, obesity, poor knotting or grabbing of stitches, and trauma to the wound after surgery, or inadequate ability to form scars.
[00388] In some embodiments, the methods described herein are used to treat a radiation scar, acne scar, curettage scar, spread scar, split-thickness scar, flap necrosis, scarring following infection, leg ulcer, burn scar, sternotomy scar, or as treatment to minimize scarring following curettage, following surgical excision, following follicular unit transplantation, or following Cesarean section, as exemplified in the examples of Section 7.
I l l [00389] In some embodiments, the methods described herein are used to enhance healing of transplanted skin at recipient sites {e.g., skin grafts or hair transplantation, such as long- term frontal hair scalp or eyebrow plugs), so that, for example, the skin blends in with the skin at the recipient site with regard to thickness, pigmentation, hair patterning, etc. In one exemplary embodiment, a scar that results from skin grafting where the graft edges join the host skin, common in battlefield wounds, is treated by the methods described herein. In general any "flap" surgery or "free flap" graft will result in these scars. In another embodiment, the methods described herein are used to enhance healing of a split thickness skin graft. In one embodiment, the split-thickness donor skin tissue for grafting of wound sites is taken from the scalp, as described in Weyandt, et al, 2009, Dermatol. Surg. 35: 1873- 1879, which is incorporated herein by reference in its entirety. Without being bound by any theory, lithium treatment may benefit this process by facilitating the "recipient dominance" phase (that temporally follows "donor dominance"). It is postulated that pulse or intermittent lithium treatment can make skin grafts (even pinch grafts) take on attributes of the recipient site by stimulating "local" tissue stem cells to form site-appropriate follicles. Such an intervention can help not only autologous grafts, but also allogeneic grafts, fetal cell grafts (like placenta stem cell "bandaids"), and also stem cell grafts {ex vivo expanded
mesenchymal stem cells).
[00390] Scars treatable by the methods described herein include, but are not limited to, any form of scar known in the art or to be discovered. Non-limiting examples of scars that can be revised or otherwise treated by the methods described herein include scars that form by secondary intention, atrophic scars, hypertrophic scars, keloid scars, hypopigmented scars, hyperpigmented scars, depressed scars (including ice-pick scars), and spread scars. Scars form following a variety of causes including, e.g., cosmetic procedures and skin transplants are not really clinical categories of scars. Also treatable by the methods described herein are scars caused by a disease or disorder such as scarring (cicatricial) alopecia, scars caused by excessive wound healing, scars caused by joint contracture, or scars caused by burns or wounds. The methods described herein may also be used to treat wounded skin, or skin that may become wounded, in order to prevent, minimize, or reduce scar formation. In one embodiment, the scar is caused by surgery, such as a open heart surgery, joint surgery, face lift, skin graft, or hair transplant, etc.
[00391] In a particular embodiment, the subject for whom pulse or intermittent lithium treatment is intended is a patient who has scarring (cicatricial) alopecia, a condition of permanent hair loss in which the hair follicle is destroyed by inflammation and replaced with scar tissue. In some embodiments, the scarring alopecia is moderate to severe.
[00392] There is primary cicatricial (scarring) alopecia and secondary cicatricial alopecia. In Primary, the follicle is the direct target. See Harries, M.J., Sinclair, R.D., Macdonald-Hull, S., Whiting, D.A., Griffiths, C.E., and Paus, R. 2008. Br. J. Dermatol. 159: 1-22. In secondary, the follicle is destroyed by events outside the follicle such as infection - trauma. The current aim of treatment is to reduce symptoms and to slow or stop PCA progression, namely the scarring process. See also Ross, 2007. Primary cicatricial alopecia: clinical features and management. Dermatol. Nurs. 19: 137-43
[00393] In some embodiments, the subject has wounding or scarring caused by, exacerbated by, or associated with medication, such as corticosteroid use, chemotherapy {e.g., anti-cancer therapy or cytotoxic drugs or other antiproliferative agents), thallium compounds, vitamins {e.g., vitamin A), retinoids, anti-viral therapy, or psychological therapy. In some embodiments, the subject has wounding or scarring caused by, exacerbated by, or associated with radiation (including therapeutic radiation treatment or radiation overdose), trauma (chronic or acute, mild or severe), physical trauma, endocrine dysfunction, surgery (including, for example, face lift, hair transplant, cosmetic surgery, and surgery of flexion surfaces, the extremities, breasts, sternum, and neck), sutures, x-ray atrophy, burning or other wound or injury, stress, aging, an inflammatory disease or condition (acute or chronic), an autoimmune disease or disorder, malnutrition (including, e.g., vitamin or trace metal deficiency, scurvy), anemia, diabetes, obesity, a circulatory disorder, such as, e.g., arterial or venous insufficiency, occlusive vascular disease, microvascular occlusive disease, vasoconstriction, hypovolemia, venous valvular disease, impaired oxygen delivery or tissue perfusion, caused by, e.g., ischemia, hypoxia, stroke, embolism or other circulatory obstruction, edema, sepsis, an infection (such as, e.g., a fungal, viral, or bacterial infection, including chronic deep bacterial, a biofilm, or fungal infections; of the wound itself or elsewhere, and which may cause weakening of the tissue), dehiscence, a disease associated with poor wound healing (e.g., Ehlers-Danlos), cellulites, dermatitis, psoriasis, acne, eczema, pregnancy, allergy, a severe illness {e.g., scarlet fever), myxedema, hypopituitarism, early syphilis, discoid lupus erythematosus, cutaneous lupus erythematosus, lichen planus, deep factitial ulcer, granuloma {e.g. , sarcoidosis, syphilitic gummas, TB), inflamed tinea capitis (kerion, favus), a slow-growing tumor of the scalp or other skin tumor, or any other condition, disease, or disorder associated with or that causes damage to the skin known in the art or described infra. 5.6 METHODS FOR EVALUATING TREATMENT
5.6.1 ANIMAL MODELS
[00394] Human skin and hair have features that are relatively unique among terrestrial mammals. For example, in mouse models involving human diseases associated with scarring or wounding, the mice appear deficient in scarring and heal their wounds rapidly.
[00395] Similarly, there are differences with regard to hair patterning in animals compared to humans. First, the great majority of human skin appears hairless to the naked eye, while the vast majority of other terrestrial mammals are essentially covered with visible hair. Second, visible human hair appears and disappears in patterns that have spatial and temporal components. Third, the patterns of visible human hair are distinct in typical male and females (exhibit gender dimorphism). Accordingly, it is evident that relative to other mammals, humans have distinct hair patterning and humans have correspondingly distinct molecular, cellular and tissue mechanisms that regulate hair growth and that control human hair, patterning. Modulating human hair follicle neogenesis, and, consequently, wound healing and scar revision as a result of such modulation, requires considerations that are unique to humans and for which other animals are insufficient models.
[00396] It should be noted that certain non-human primates share features of hair patterning with humans, but not to the degree or extent. Old World Apes (gorillas and chimpanzees) have areas of skin that lack visible hair; on the face surrounding the eyes, nose and mouth; on ears; and the plantar surfaces of hands and feet. In addition, Rhesus Macaque has patterned alopecia in males and females. Gorillas have hair patterning with respect to color on dominant males: i.e., the "Silverback". While certain of these mechanisms share similarities to humans, the extent and degree of hair patterning in human remains relatively unique.
5.6.1.1 HUMAN SKIN XENOGRAFT MODELS
[00397] Preliminary evidence of hair follicle neogenesis has been demonstrated in human skin (obtained from the hair line during a face lift procedure) grafted onto the back of an immunodeficient SCID mouse. Such human skin xenograft models are useful for testing the safety and efficacy of the intermittent lithium treatments or a pulse lithium treatment described herein, as well as the combination treatments described in Section 5.3 supra. Although any method for producing human skin xenografts known in the art may be used, an exemplary model is described in the example of Section 6 below. [00398] Alternatively, a human skin xenograft (without skin appendages) can be considered as similar to a scar, and can be wounded and then treated pharmacologically to induce hair follicles and/or monitor revision of the scar. Xenografts can also be combined with inducible genetically modified cells to activate pathways know to form hair follicles.
[00399] In some embodiments, the safety and efficacy of a pulse or intermittent lithium treatment, optionally as part of a combination treatment described in Section 5.4 supra, is tested in a full thickness or a split thickness human skin xenograft {e.g., obtained surgically from scar revisions; from foreskin; or cadaveric), or may be tested in a three-dimensional organotypic human skin culture on SCID mice.
[00400] Success of a pulse or intermittent lithium treatment can be measured by:
• improvement of pigmentation of the scarred or wounded area
• improved thickness of the scarred or wounded area
• improved surface contour of the scarred or wounded area
• improved texture of the scarred or wounded area
• improved overall cosmetic outcome
• hair follicle regeneration
• return of adnexal structures to the area
• increased proportion of hair follicles in anagen or decreased proportion of follicles in telogen
• increased numbers of follicular units with 3 or more hair follicles.
[00401] Any method known in the art may be used to evaluate the safety and efficacy of an intermittent lithium protocol or pulse lithium protocol, or of the combination treatments described in Section 5.4. Preferably, a human skin xenograft model is used. For example, an intermittent lithium treatment or pulse lithium treatment may be administered with a full thickness excision, laser, inflammatory stimulus, or dermabrasion procedure for integumental perturbation. A synergistic effect of an intermittent lithium treatment or pulse lithium treatment on another treatment for enhancing wound healing or scar revision may be measured as an improvement over a control subject receiving only one of the two treatments (i.e., the intermittent lithium treatment or pulse lithium treatment alone or the second treatment alone). 5.6.1.2 OTHER ANIMAL MODELS
[00402] Another animal model for use in evaluating treatment that may more closely mimic the biology of human skin and hair is a guinea pig model (see, Stenn & Paus, 2001, Physiol. Revs. 81 : 449-494). The methods for evaluating treatment in animals described elsewhere in this section and in the example in Section 16 below may be applied to guinea pigs according to methods known in the art. See also, e.g., Kramer et al, 1990, Dermatol Monatsschr. 176:417-20; and Simon et al, 1987, Ann Plast Surg 19:519-23. Other animal models that may be of use in evaluating the treatments described herein include pig, cat, or stumptailed macaque models.
5.6.2 METHODS FOR EVALUATING TREATMENT IN HUMANS
[00403] The safety and efficacy of the intermittent lithium treatments or pulse lithium treatment described herein may also be measured in human subjects according to methods known in the art.
[00404] For example, success of a pulse or intermittent lithium treatment can be measured by:
• improvement of pigmentation of the scarred or wounded area
• improved surface contour of the scarred or wounded area
• improved texture of the scarred or wounded area
• improved skin depth, i.e., thickness (if the scar started out as depressed relative to the plane of the skin) or thinness (if the scar started out as elevated relative to the plane of the skin) of the scarred or wounded area
• improved overall cosmetic outcome (e.g., using the Visual Analogue Scale
(VAS)
• subjective patient measures of improved outcome
• presence of elastin
• proper collagen orientation
• Improvement in viscoelasticity
• return of adnexal structures
• return of normal pore pattern
• increased number of hair germs
• hair follicle neogenesis or regeneration • increased proportion of hair follicles in anagen or decreased proportion of follicles in telogen
• increased numbers of follicular units with 3 or more hair follicles
• reduction in the size of the wound or appearance of the scar compared to a wound or scar not treated with lithium
• conversion of the dermal epidermal junction from a flat junction between the dermis and epidermis (typical of a scar) to rete pegs (epithelial extensions that project into the underlying connective tissue) with interdigitating dermis, as assessed by in vivo scanning laser microscopy
• normalization of blood vessels as assessed using laser Doppler analysis.
• normal values according to the Vancouver Scar Scale (VSS). The VSS has 4 separate domains: pigmentation (graded 0 = normal, to 2 = hyperpigmentation), vascularity (graded 0 = normal, to 3 = purple), pliability (graded 0 = normal, to 5 = contracture) and height (graded 0 = normal, 3 = > 5 mm):
PIGMENTATION
0. Normal;
1. Hypopigmented;
2. Hyperpigmentation;
VASCULARITY
0. Normal: resembles the color over the rest of the body area;
1. Pink;
2. Red;
3. Purple.
PLIABILITY
0. Normal
1. Supple: flexible with minimal resistance;
2. Yielding: giving way to pressure;
3. Firm: inflexible, not easily moved, resistant to manual pressure
4. Banding: rope-like tissue that blanches with extension of scar;
5. Contracture: permanent shortening of scar, producing deformity or distortion. HEIGHT
0. Normal: flat;
1. < 2 mm; 2. 2-5 mm;
3. > 5 mm.
[00405] In some embodiments, the intermittent lithium treatment or pulse lithium treatment improves one of the foregoing measures by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more. Such an improvement may be measured after 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or one year or longer after initiation of the intermittent lithium treatment or pulse lithium treatment.
[00406] A synergistic effect of an intermittent lithium treatment or pulse lithium treatment on another treatment described herein may be measured as an improvement over a control subject receiving only one of the two treatments (i.e., the intermittent lithium treatment or pulse lithium treatment alone or the second treatment alone).
5.6.3 IN VITRO MODELS
[00407] Skin explant model. The efficacy of the intermittent lithium treatments or pulse lithium treatment described herein may be tested using skin explants, for example, prepared from skin biopsies or other surgical procedures. See, e.g., Ballanger et al, supra.
[00408] Human skin equivalents can be grown and assembled in vitro, with the advantage that they can be grown to theoretically to any size/shape; can be comprised of different types of cells, including keratinocytes (hair follicle derived and non-hair follicle derived), dermal cells (hair follicle derived and non-hair follicle derived), other cell types {e.g. , mesenchymal stem cells); can contain cells that are genetically modified to include, e.g., markers or "inducible" signaling molecules; provide an unlimited and uniform source of human cells; from normal skin based on histology and marker studies; are generally devoid of skin appendages; and can be wounded and show similar wound healing events as in vivo.
5.7 DISCUSSION
[00409] Without being bound by any theory, the intermittent lithium treatments or pulse lithium treatments may facilitate wound healing and scar revision by:
• regulating the unique human processes that regulate hair follicles and, e.g., visible hair growth
• regulating the activity of specialized human hair follicles
• regulating specific activities of specialized human hair follicles • altering the activity of specialized human hair follicles, sometimes in conjunction with transplantation
• regulating donor dominance and recipient site effects
• altering, delaying or preventing programmed senescence of hair follicles
• recruiting bone marrow derived mesenchymal stem cells that differentiate into skin cells during healing (see, e.g., Sasaki et al, 2008, J. Immunol. 180:2581- 2587)
• resident dermal mesenchymal stem cells located in the dermal papilla and dermal (connective tissue) sheath of hair follicles contributing to new dermal fibroblasts and myofibroblasts
• resident and bone marrow-derived endothelial stem cells giving rise to new blood vessels in the healing wound
• epithelial stem cells, located in the epidermis and in the hair follicle, giving rise to keratinocytes that re-epithelialize the wound (see, e.g., Lau et al, 2009, Exp. Dermatol. 18:921-933).
[00410] A number of molecular targets have been proposed for lithium action, including but not limited to inositol monophosphatase, a family of structurally related
phosphomonoesterases, and the protein kinase glycogen synthase kinase-3 (GSK-3) (for a review see Phiel & Klein, 2001, Annu. Rev. Pharmacol. Toxicol. 41 :789-813). While not bound by any theory of how the invention works, the invention is based, in part, on the principle that the lithium ion (Li+) is an inhibitor of the polyphosphoinositide cycle that can reversibly arrest cells in cell cycle. In plant cells which demonstrate remarkable precision in the timing of mitotic events, the lithium ion has been shown to cause metaphase arrest that can be reversed by the addition of CaC12 or myo-inositol. (Wolniak, 1987, Eur. J. Cell Biol. 44: 286-293). The lithium ion has also been shown to arrest cancer cell lines at certain stages of the cell cycle (see, e.g., Wang JS, 2008, World J. Gastroenterol. 14:3982-3989).
[00411] As it relates to the contribution of hair follicles to improved wound healing, the invention is based in part on the inventors' recognition that the lithium ion can be used in a pulse or intermittent treatment regimen to synchronize groups of hair follicle cells or hair follicle stem cells that are in various stages of cell cycle (cycling asynchronously). For example, the lithium ion may cause hair follicle stem cells to stop dividing and more readily differentiate into hair follicles and thereby improve wound healing. Restarting the cell cycle at the termination of a pulse lithium treatment, or during the "holidays" between intermittent lithium treatments should restart cell cycle synchronously. The synchronization phenomenon can be described by analogy to traffic lights: periodically arresting the motion of individual cars generates synchronization because cars pile up behind stop lights. Similarly, by introducing a signal that periodically arrests cell division, synchronization is generated because when the "stop" signal is removed, cells initiate division at the same time. Such synchronization of cell cycles in the hair follicle cells results in relative synchronization of hair follicle cycle stage in groups of follicles that otherwise have a stochastic distribution of stages of follicle cycle (asynchronous follicle cycle).
[00412] The Follicle Stem Cells that are thought to be involved can be derived from (1) other Follicle Stem Cells (e.g., from the bulge or crypt), (2) from other tissue stem cells, termed "pre-Follicle Stem Cells" (from the interfollicular skin), (3) from bone marrow- derived stem cells ("BMST", such as hematopoietic stem cells), (4) uncommitted epithelial progenitor cells; and/or (5) from mesenchymal stem cells such as hair follicle dermal sheath cells and adipocyte stem cells. In the case of bone marrow derived stem cells (BMST), their differentiation into Follicle Stem Cells requires intact follicles, whose cells can play the role of "nurse cells" and provide appropriate signals to guide the differentiation of bone marrow derived stem cells into Follicle Stem Cells. Integumental perturbation (by wounding, e.g., during scar revision, or by the induction of inflammation) (1) provides signals for Follicle Stem Cells to divide symmetrically to begin the process of forming new follicles; (2) mobilizes tissue stem cells ("pre-Follicle Stem Cells") from interfollicular skin to
differentiate into stem cells and (3) increases the trafficking of bone marrow derived stem cells to wounded areas of skin and promotes their differentiation into Follicle Stem Cells by nurse cells in existing follicles. When used in combination with such procedures, intermittent or pulse lithium treatment organizes the normally asynchronous state of human hair follicle cells in Cell Cycle and human hair follicles in Follicle Cycle into relatively more
synchronous states of human hair follicle cells in Cell Cycle and human hair follicles in Follicle Cycle, leading to the increased deposition of hair follicles into the wound site and thereby improving wound healing.
6. EXAMPLE; HUMAN SKIN XENOGRAFT ANIMAL MODEL
[00413] This protocol is adapted from the IACUC VA protocol. Specifically, 4 week old male SCID mice are obtained from Charles River and allowed to acclimate for at least 1 week. In preparation for surgery, mice are anesthetized with ketamine (80 mg/kg)/xylazine (20 mg/kg) delivered i.p. in a volume < 100 μΐ, and monitored by toe pinch to determine the surgical plane of anesthesia. Full thickness adult human skin (measuring approximately 1.5 cm x 2 cm; removed during surgical procedures from the CHTN, NDRI or cadaver scalp skin from ABS) is sutured into a full thickness skin excision site on the dorsal surface of the mouse. The grafts are bandaged and allowed to heal for at least 5 weeks. After healing and successful "take," prior to wounding, the human skin is analyzed by histology and/or photography to determine the "control" or "pre- wounded" state of the skin graft. Prior to wounding, mice are anesthetized with ketamine (80 mg/kg)/xylazine (20 mg/kg) delivered i.p. in a volume of < 100 μΐ, and monitored by toe pinch to determine the surgical plane of anesthesia. The epidermis of the human skin is removed using a microdermabrasion device to dermabrade as described above. (Experiments with microdermabrasion on ex vivo human abdominal skin have established the initial parameters for removal of epidermis, however some testing in mice may be required to confirm and/or optimize these settings for human scalp xenografts. Additionally, some mice may be required to test the differences between full thickness and split thickness human scalp xenografts. Furthermore, reducing the overall thickness of the human skin may improve the "take" rate of the grafts, which is
approximately 50%). The wounds are allowed scab and heal naturally. The mice are observed and photographed daily in order to monitor the formation of the scab and the timing of its detachment (scab detachment should occur within 2 weeks of wounding). As soon as the scab detaches, mice receive vehicle alone or the lithium composition, delivered systemically or topically, or neither vehicle nor lithium composition, for 5 consecutive days, (the lithium composition chosen is the one determined to be most efficacious in the
C57BL/6J model, with efficacy determined to be increased number and/or size of neogenic hair follicles). One dose of the lithium composition is delivered, using the most efficacious dose as described above, systemically and, in a separate experiment, a dose is delivered topically. Additionally, histology and/or photography is performed daily (until the end of the experiment) following scab detachment in order to monitor hair follicle neogenesis. An additional set of mice are treated with the lithium composition or vehicle or neither, with the exception that the xenografted mice are not wounded, in order to assess the effect of the lithium composition in the absence of wounding. At approximately 2 weeks post-scab detachment, all mice are anesthetized with ketamine (80 mg/kg)/xylazine (20 mg/kg) delivered i.p. in a volume of < 100 μΐ, and monitored by toe pinch to determine the surgical plane of anesthesia. Subsequently, they have a terminal blood draw (to detect drug in the plasma), and are euthanized. The wound is then removed, which is trisected with one-third taken for biochemistry, one third for determination of lithium levels in the skin using mass spectrometry, and one third for histology/immunohistochemistry. For the experimental design in which the human (xenograft) skin is wounded, there are 3 treatment groups (lithium compositions, vehicle, no lithium composition or vehicle) with 2 different delivery methods (IP and topical). With 10 mice per group, this requires 50 mice (only 1 group of "no lithium composition or vehicle"). The most efficacious combination of lithium composition and mode of delivery is repeated in 3 more independent experiments (with only lithium composition-treated and vehicle groups), thus adding 60 more mice, giving a total of 1 10 mice. An identical experiment is carried out, but without dermabrasion wounding (epidermal removal), requiring an additional 110 mice. This yields a total of 220 mice for the wounding and lithium composition portion of this experiment. An additional 20 mice are needed for the optimization of microdermabrasion settings and split thickness versus full thickness xenografts. Considering that the "take" rate of human skin xenografts is approximately 50%, the total number of mice to optimally receive human skin grafts is approximately 500.
7. EXAMPLE; TREATMENT PROTOCOLS
7.1 TREATMENT OF RADIATION SCAR
[00414] A female human subject, 75 years old, underwent treatment of a basal cell carcinoma of the nose five years prior to presentation. The resulting scar is atrophic, hypo- pigmented and lacking normal pore pattern. The scar is mechanically disrupted by excision, dermatome planing, dermabrasion, laser abrasion, or Fraxel, and treatment with topical lithium gluconate 8% is initiated.
[00415] Response to treatment is determined by measuring skin thickness, return of pigmentation and re-establishment of adnexal structures.
7.2 TREATMENT OF ACNE SCARS
[00416] A male human subject, 28 years old, presents with extensive, broad, shallow, acne scars. The scars are atrophic, hypo-pigmented and lack normal pore pattern. The scars are dermabraded using a moderate grit diamond fraise and treatment with 8% topical lithium is initiated.
[00417] Response to treatment is determined by measuring skin thickness of the scar, return of pigmentation and re-establishment of adnexal structures.
7.3 TREATMENT OF CURETTAGE SCAR [00418] A female human subject, 45 years old, was treated for a basal cell carcinoma of the forehead by electrodessication and curettage. The resulting scar was depressed, atrophic, hypo-pigmented and lacked normal pore pattern. The scar is mechanically disrupted by excision, dermabrasion, laser abrasion, or Fraxel, and treatment with 8% topical lithium gluconate is initiated.
[00419] Response to treatment is determined by measuring skin thickness, return of pigmentation and re-establishment of adnexal structures.
7.4 TREATMENT OF A SPLIT THICKNESS SKIN GRAFT
[00420] A male human subject, 75 years old, underwent excision of a large malignant melanoma of the forehead with subsequent reconstruction using a split-thickness skin graft. After one year, the resulting graft demonstrates depression, skin atrophy, hyper-pigmentation and loss of normal pore pattern. The graft is mechanically disrupted by excision, dermabrasion, laser abrasion, or Fraxel, and treatment with 8% topical lithium gluconate is initiated.
[00421] Response to treatment is determined by measuring skin thickness, establishment of normal pigmentation and re-establishment of adnexal structures.
7.5 TREATMENT OF A SPREAD SCAR
[00422] A female human subject, 30 years old, undergoes excision of a large congenital nevus of the cheek. Due to the tension of closure, the incision line spreads and widens over time resulting in a scar that is atrophic, hypo-pigmented and lacking normal pore pattern. The scar is mechanically disrupted by excision, dermabrasion, laser abrasion, or Fraxel, and treatment with 8% topical lithium gluconate is initiated.
[00423] Response to treatment is determined by measuring skin thickness, return of pigmentation and re-establishment of adnexal structures.
7.6 TREATMENT OF FLAP NECROSIS
[00424] A female human subject, 65 years old, underwent a face-lift procedure. A portion of the cheek flap subsequently became necrotic and healed by secondary intent. The resulting scar was atrophic, hypo-pigmented and lacked normal pore pattern. The scar is mechanically disrupted by excision, dermabrasion, laser abrasion, or Fraxel, and treatment with topical 8% lithium gluconate is initiated. [00425] Response to treatment is determined by measuring skin thickness, return of pigmentation and re-establishment of adnexal structures.
7.7 TREATMENT OF SCARRING FOLLOWING INFECTION
[00426] A female human subject, 37 years old, underwent a phenol chemical peel of her lips to decrease rhytides. During the healing phase, the patient developed a staphylococcal (herpetic) infection of the treated area. The infected area healed with an atrophic, hypo- pigmented scar that lacked the normal pore pattern. The scar is mechanically disrupted by excision, dermabrasion, laser abrasion, or Fraxel, and treatment with 8% topical lithium gluconate is initiated.
[00427] Response to treatment is determined by measuring skin thickness, return of pigmentation and re-establishment of adnexal structures.
7.8 TREATMENT OF LEG ULCERS
[00428] A male human subject, 70 years old, developed non-healing leg ulcers involving the tibial aspects of both legs. The affected area was pre-treated with 8% topical lithium carbonate and subsequently grafted using small pinch grafts harvested from the thighs.
[00429] Response to treatment is determined by measuring skin thickness and re- establishment of adnexal structures over the grafted area following graft take.
7.9 TREATMENT OF SPLIT THICKNESS GRAFT DONOR SITES
[00430] A female human subject, 40 years old, underwent split thickness grafting to reconstruct a facial defect following excision of a squamous cell carcinoma. The graft donor site healed with a hypo-pigmented scar that lacked the normal pore pattern. The scar is mechanically disrupted by excision, dermabrasion, laser abrasion, or Fraxel, and treatment with 8% topical lithium gluconate is initiated.
[00431] Response to treatment is determined by measuring skin thickness, return of pigmentation and re-establishment of adnexal structures.
7.10 TREATMENT OF A BURN SCAR
[00432] A male human subject, 30 years old, suffered a burn of the left cheek that healed with a contracted, hypo-pigmented scar that lacked normal pore pattern. The scar is mechanically disrupted by excision, dermabrasion, laser abrasion, or Fraxel, and treatment with 8% topical lithium gluconate is initiated. [00433] Response to treatment is determined by measuring skin thickness, return of pigmentation and re-establishment of adnexal structures.
7.11 IMMEDIATE TREATMENT FOLLOWING CURETTAGE
[00434] A 76 year old fair skin male with a history of multiple basal cell carcinomas presents with 2 new pigmented nodular pearly lesions of 1 cm on his left (A) and right (B) scapula. Shave biopsies reveal both lesions to be nodular BCCs. Lesion (A) is treated with aggressive 3 pass curettage then application of aluminum chloride. Lesion (B) is treated with aggressive 3 pass curettage then application of aluminum chloride. Lesion B is post-treated with topical 8% lithium gluconate daily for 5 days post-procedure (in addition to routine wound care). In 3 and 6 week follow-up, the lesions are compared with respect to skin thickness, return of pigmentation, re-establishment of adnexal structures and global assessment the physician and patient's satisfaction regarding the cosmetic result of the 2 distinct lesions using a Visual Analogue Scale (VAS).
7.12 TREATMENT FOLLOWING SURGICAL EXCISION
[00435] A 76 year old fair skin male with a history of multiple basal cell carcinomas presents with 2 biopsy proven BCC lesions of 1 cm on his left scapula (A) and 1 cm on his right scapula(B). Conservative surgical excision is performed on both lesions . Lesion (A) is treated with then treated with routine wound care. Lesion (B) is treated with pre-treated with 5 days topical 8% lithium gluconate daily prior to surgery followed by application for 5 days post-procedure (in addition to routine wound care). In 3 and 6 week follow-up, the lesions are compared with respect to skin thickness, return of pigmentation, re-establishment of adnexal structures and global assessment the physician and patient's satisfaction regarding the cosmetic result of the 2 distinct lesions using a Visual Analogue Scale (VAS).
7.13 TREATMENT FOLLOWING STERNOTOMY SCAR
[00436] A 74 year old black male with history CAD undergoes CABG surgery requiring median sternotomy. Following the procedure the scar is found to measure 14 cm in diameter. Starting Day 1 thru 7 he applies topical 8% lithium gluconate to the 7 cm superior portion of the scar (in addition to routine wound care). In 3 and 6 week follow-up, the superior and inferior aspects of the sternotomy scar are compared with respect to skin thickness, return of pigmentation, re-establishment of adnexal structures and global assessment the physician and patient's satisfaction regarding the cosmetic result of the 2 distinct lesions using a Visual Analogue Scale (VAS).
7.14 TREATMENT FOLLOWING FOLLICULAR UNIT
TRANSPLANTATION
[00437] A 51 year old white male with history Androgenic Alopecia undergoes Hair Transplant with the "Strip Harvesting Method" with donor area located at the posterior scalp along the occipital protuberance. Surgery requires a long scar on the posterior scalp measuring 28 cm. Starting Day 1 thru 7 topical 8% lithium gluconate is applied only to the 14 cm "left" portion of the scar (in addition to routine wound care). In 3 and 6 week follow-up, the right and left aspects of the scar are compared with respect to skin thickness, return of pigmentation, re-establishment of adnexal structures and global assessment the physician and patient's satisfaction regarding the cosmetic result of the 2 distinct lesions using a Visual Analogue Scale (VAS).
[00438] In a variation, hair that is regenerated in the treated donor area may be used as a source of future, repeated hair transplants in accordance with the foregoing method.
7.15 TREATMENT FOLLOWING FOLLICULAR UNIT EXTRACTION
[00439] A 51 year old white male with history Androgenic Alopecia undergoes Hair Transplant with the "Follicular Unit Extraction" with donor area located at the posterior scalp above and below the occipital protuberance. In total 1000 punch graft were taken from the posterior scalp and left to heal with secondary intention. Starting Day 1 thru 7 topical 8% lithium gluconate is applied only to the "left" portion of the donor area (in addition to routine wound care). In 3 and 6 week follow-up, the right and left aspects of the scar are compared with respect to skin thickness, return of pigmentation, re-establishment of adnexal structures and global assessment the physician and patient's satisfaction regarding the cosmetic result of the 2 distinct lesions using a Visual Analogue Scale (VAS).
[00440] In a variation, hair that is regenerated in the treated donor area may be used as a source of future, repeated hair transplants in accordance with the foregoing method.
7.16 TREATMENT FOLLOWING CESAREAN SECTION
[00441] A 27 year old G1P1 white female, undergoes Cesarean section surgery secondary to failure for labor to progress. Surgery requires a long scar on the lower abdomen measuring 24 cm (patient has decided not to breast feed post-delivery due to prior breast augmentation surgery). Starting Day 1 thru 7 topical 8% lithium gluconate is applied only to the 12 cm "left" portion of the scar (in addition to routine wound care). In 3 and 6 week follow-up, the right and left aspects of the scar are compared with respect to skin thickness, return of pigmentation, re-establishment of adnexal structures and global assessment the physician and patient's satisfaction regarding the cosmetic result of the 2 distinct lesions using a Visual Analogue Scale (VAS).
7.17 EXAMPLE; CLINICAL PROTOCOL FOR TESTING EFFICACY OF COMBINATION LITHIUM AND LASER TREATMENT OF SURGICAL SCARS
[00442] A patient has a 5 cm scar resulting from surgery. Two to three months after surgery, the scar is treated with fractional laser. Half the scar is treated with lithium and the whole scar is treated with laser. In another variation, a patient has two surgical scars, one of which is treated with the lithium and laser combination and the other of which is treated with laser alone.
8. EXAMPLES OF LASER TREATMENT VARIATIONS
[00443] Alternatives to the laser treatments provided in the examples of Section 7.1, 7.3- 7.7, 7.9, 7.10, and 7.17 follow.
8.1 FRACTIONAL. NON-ABLATIVE LASER TREATMENT
[00444] The subject is administered a fractional and non-ablative laser therapy using an Erbium- YAG laser with an emission at 1540-1550 nm (set to 50-70 J/cm2, treatment level of 8-10 (density of the "dots"), and 8 passes) and the subject is provided with a topical preparation of Lithium gluconate 8% gel (Lithioderm 8% gel) and instructed to apply the Lithium gluconate 8% gel to the treated area of the ear for one week. After one week, treatment with lithium gluconate is discontinued and he is evaluated after three weeks.
[00445] The treatments may alternatively be accomplished by applying an ablative laser treatment in place of the non-ablative laser treatment. In such ablative laser treatments, the application of Lithium gluconate is sterile and, optionally, the treatment area is covered by a bandage. For example, ablative laser treatment may accomplished using an Erbium- YAG laser at 2940 nm or a CO2 laser at 10,600 nm.
8.2 ULTRAPULSE CO? FRACTIONAL LASER
[00446] After shaving/clipping any existing hair in the area to be treated, and followed by cleaning with antiseptic, Lidocaine HCL 2% with Epinephrine 1 : 100,000 are injected to anesthetize the surface of the area to be treated. An Ultrapulse (fractional mode) CO2 laser is used to disrupt the epidermis and dermis to approximately 100 to 500 μΜ in depth. The Ultrapulse laser produces an effect that is similar to that of dermabrasion yet the disruption produced delivers a greater amount of energy deeper into the skin in a non-scaring fractional ablation. The treated area is a 1.5 cm x 1.5 cm square. The Ultrapulse is set to deliver up to 350 mJ, up to 52.5 Watts, using pattern size #8, density #4, and fill the square treatment site with up to 5 passes.
8.3 ULTRAPULSE CO? ABLATION LASER
[00447] After shaving/clipping any existing hair in the area to be treated, and followed by cleaning with antiseptic, Lidocaine HCL 2% with Epinephrine 1 : 100,000 are injected to anesthetize the surface of the area to be treated. An Ultrapulse CO2 laser (ablative mode) is used to disrupt the epidermis and dermis to approximately 100 to 500 μΜ in depth. The Ultrapulse laser produces an effect that is similar to that of dermabrasion yet the disruption produced delivers a greater amount of energy deeper into the skin in a non-scaring ablation that resembles the dermabrasion. The treated area is a 1.5 cm x 1.5 cm square. The
Ultrapulse is set to deliver up to 500 mJ in 1 msec, 1 Watts, using a spot size of 3 mm at 2 Hz to fill the square treatment site, which may require up to 15 passes.
8.4 CANDELA SMOOTH PEEL FULL-ABLATION ERBIUM LASER
[00448] After shaving/clipping any existing hair in the area to be treated, and followed by cleaning with antiseptic, Lidocaine HCL 2% with Epinephrine 1 : 100,000 are injected to anesthetize the surface of the area to be treated. The ablative erbium laser is set to deliver up to 5 Joules 240 msec in of energy at level 3 so that in up to 15 passes it will produce a disruption up to 500 μΜ deep. The treated area is a 1.5 cm x 1.5 cm square.
9. EXAMPLE OF DERMABRASION TREATMENT VARIATIONS
[00449] The dermabrasion treatments provided in the examples of Sections 7.1-1.7, 7.9, and 7.10 may alternatively be accomplished by applying one of the following dermabrasion treatments.
[00450] After shaving/clipping any existing hair in the area to be treated, followed by cleaning with antiseptic, Lidocaine HCL 2% with Epinephrine 1 : 100,000 is injected to anesthetize the surface of the area to be treated. Standard dermabrasion, using the Aseptico Econo-Dermabrader from Tiemann and Company, is performed to a depth of approximately 150 μΜ, that includes removal the entire epidermis and disruption of the papillary dermis (detectable by a shiny, whitish appearance) inducing the formation of small pools of blood in the treated area. Each dermabraded area is a 1.5 cm x 1.5 cm square. In an alternative example, a Bell Hand dermabrasion device may be used.
[00451] Variations of this protocol are found in the example of Section 11-16, which present mouse studies using dermabrasion, and the protocols for use in humans in the examples of Sections 7, 9 and 10. In the mouse studies, dermabrasion was carried out using a microdermabrasion device. While dermabrasion in humans may also be carried out using a microdermabrasion device, where sterile conditions are preferential, a dermabrasion device is preferably used.
10. EXAMPLE: CLINICAL EVALUATION OF NEOFOLLICULAR
DEVELOPMENT RESULTING FROM APPLICATION OF LITHIUM GLUCONATE IN COMBINATION WITH DERMABRASION
[00452] The following example provides a protocol for demonstrating the importance of timing of lithium gluconate treatment for the optimization of follicular neogenesis and wound healing/scar revision after integumental perturbation. In this protocol, patients are treated with a pulse lithium of 8% lithium gluconate (topical gel) in combination with dermabrasion. As controls, patients may be treated with the intermittent lithium treatment or a pulse lithium treatment alone (as described infra), dermabrasion alone (or with a vehicle, e.g., petrolatum), or may not receive any treatment.
[00453] Although any patient population may be treated, preferred patients are Caucasian males 20-50 years of age. Patients for whom treatment may be contraindicated (particularly at the clinical trial stage) are those who are currently participating in or have participated in any clinical study with an investigational drug within the thirty (30) days immediately preceding treatment, with current or recent use (<1 y) of isotretinoin (Accutane), currently taking hormone therapy, or steroids or other immunomodulators or have taken these medications within the past thirty (30) days (inhaled steroids are acceptable), currently using Rogaine or Propecia or used them in the past forty- five (45) days, immune compromised or undergoing therapy to treat an immune disorder, have a clinically significant medical condition that may interfere with the protocol described herein, have other active skin diseases (such as actinic keratosis or psoriasis) or skin infections (bacterial, fungal or viral, esp. HSV infection) in the area to be treated, have a history of keloids or hypertrophic scarring, hypersensitivity to lidocaine, poor wound healing, diabetes, or coagulopathy, undergoing current drug or alcohol abuse, psychiatric dysfunction, or other factors that would limit compliance, have sunburned skin, or who are currently taking anti-platelet agents other than aspirin.
[00454] Dermabrasion using alumina particles is performed on Day 0. Dermabrasion is performed to a depth of approximately 100 μΜ, which includes removal of the entire epidermis and disruption of the papillary dermis (detectable by a shiny, whitish appearance) inducing the formation of small pinpoints of blood in the treated area. Dermabrasion is performed in two sites of the skin. The area is then allowed to heal without manipulation. A 4 mm punch biopsy is performed on days 1 1 and 14, and the presence of new hair follicles is examined in these subjects based on histological assessment. A third biopsy is optionally performed on Day 14 on an untreated area 1 cm away from the treated area to serve as histologic control. In the event that limited follicle neogenesis is observed on day 14, another biopsy may be performed on day 17. Subjects scheduled for day 1 1 biopsies for whom the scab in the wound detaches before day 8, will have the biopsy rescheduled for 3 days afterwards. Conversely, subjects for whom the scab has not detached by day 10 will have the biopsy visit rescheduled for 3 days after the scab detaches. It is expected that the scab will detach around days 6-10. Subjects return on day 46-50 for follow up photography of the treated area.
[00455] The protocol may be amended in accordance with the findings. For example, if dermabrasion causes presence of neogenic hairs in a 4 mm punch biopsy in, for example, at least three of the first 15 patients, then additional patients will be treated.
10.1 DERMABRASION
[00456] The procedure begins with shaving/clipping of the existing hair in the area to be treated followed by a thorough cleaning with antiseptic cleansing agent. Numbing agents, such as lidocaine HCL 2% and Epinephrine 1 : 100,000, are injected to anesthetize the surface to be treated. Standard dermabrasion is performed to a depth of approximately 100 μΜ, which includes removal the entire epidermis and disruption of the papillary dermis
(detectable by a shiny, whitish appearance) inducing the formation of small pinpoints of blood in the treated area. Each dermabraded area is approximately a 1.5 cm x 1.5 cm square. Suitable dermabrasion devices are the ASEPTICO ECONO-DERMABRADER from
Tiemann and Company, the DX system from Advanced Microderm (see, e.g. ,
http://www.advancedmicroderm.com/products/tech_specs.html), or the M2-T system from Genesis Biosystems. Alternatively, sterilized sandpaper may be used for dermabrasion. Adhesive ocular shields are worn by the patient during the procedure to avoid complications due to aluminum crystals entering the eye (chemosis, photofobia, punctuate keratitis) and the doctor should wear safety goggles. The dermabrasion tool is carefully maneuvered over the area to carefully remove layers of skin until the desired level is reached. The procedure usually takes only a few minutes.
[00457] Pre-dermabrasion, patients should be asked to: not wear contact lenses during the procedure, discontinue use of over the counter exfoliation products such as Retinol, Glycolic or other hydroxy acids, Salicylic acid, Beta hydroxyl acids 3 days prior to treatment, discontinue use of retinoids 30 days prior to treatment, not receive Botox or collagen injections for 2 weeks prior to treatment.
[00458] Following the procedure the treated skin will be red, swollen and tender, and the wound should be cared for as follows until new skin starts to grow; this usually takes 7-10 days: 1) Keep the area clean and dry for today. The area should either be covered with Vaseline and bandaged after or covered with duoderm or a similar covering. Alternatively, it may be preferable to not cover, bandage, or otherwise manipulate the treated area; 2) Avoid touching the area when washing hair; 3) Pat the area dry. Do not cover, bandage, or otherwise manipulate the treated area.
[00459] The treated are may itch as the new skin grows and may be slightly swollen, sensitive, and bright pink for several weeks after dermabrasion.
[00460] The following measures are taken to prevent any complications.
[00461] · Inform your doctor of any yellow crusting or scabs - this may be the start of an infection.
[00462] · Swelling and redness should subside after a few days to a month. Persistent redness of an area could be the sign of a scar forming so contact your doctor immediately.
[00463] · No swimming is permitted for the first 7 days following dermabrasion.
[00464] · To avoid pigmentation, once the new skin is healed, keep out of the sun and apply a broad spectrum sunscreen daily for at least 3 months after microdermabrasion. Even the sun through window-glass can promote unwanted pigmentation.
10.2 PUNCH BIOPSY
[00465] The procedure begins with thoroughly cleaning the area to be biopsied with antiseptic cleansing agent. Lidocaine HCL 2% and Epinephrine 1 : 100,000 (approximately 0.5 cc to each site) are injected to anesthetize the site that will be biopsied. 4 mm punch biopsy is performed. Biopsied site is closed with 2 4.0 Ethilon sutures. Vaseline and band-aid are applied. Tissue samples are stored in formalin for histological analysis.
10.3 PRIMARY ENDPOINTS
[00466] Histologic analysis of hair follicle neogenesis following dermabrasion. The null hypothesis is that no (0) neogenic follicles will form, since that is the current dogma in humans. A positive response to treatment is characterized as the appearance of 3 or more neogenic follicles in a 4 mm punch biopsy.
[00467] Among the factors to be evaluated when determining success of treatment are: scarring; re-epithelialization; crusting/scabbing; comedones; infection; pigmentary changes (e.g., absent, hypopigmentation (mild, moderate or severe), or hyperpigmentation (mild, moderate or severe)); or presence of hair follicles by gross observation.
10.4 SECONDARY ENDPOINTS
[00468] 1) To quantify the number and morphological developmental stage of follicles in each biopsy.
[00469] 2) Clinical characteristics of dermabraded areas.
11. EXAMPLE: MODULATION OF RELEASE PROFILES OF
LITHIUM IONS AS A FUNCTION OF FORMULATION COMPOSITION
[00470] This example provides a protocol for characterizing and comparing the percutaneous absorption pharmacokinetics of four formulations containing a lithium salt, in human cadaver skin, using the in vitro skin finite dose model. This model is a well- established tool for the study of percutaneous absorption and the determination of the pharmacokinetics of topically applied drugs. The model uses human cadaver skin mounted in specially designed diffusion chambers allowing the skin to be maintained at a temperature and humidity that match typical in vivo conditions. A dose (e.g., 0.1 gram) of formulation is applied to the top of the partial thickness skin or dermis and drug absorption is measured by monitoring its rate of appearance in the reservoir solution bathing the other surface of the skin.
11.1 TEST FORMULATIONS
[00471] The compositions of the formulations are provided in Table 2 below. The formulations were tested initially for stability in solution at 4 °C, 25 °C and 40 °C. All formulations were stable solutions or emulsions at the temperatures tested. The excipients selected for the formulations were based on levels approved for topical drug formulations and each excipient was selected for its viscosity-enhancing properties or its ability to enhance permeation through tissues. Methylparaben was added to the formulations for its preservative activity.
Table 2. Lithium Chloride Compositions
Figure imgf000134_0001
Lithium Lithium Gluconate Gel, Lithium gluconate = 8% gluconate 8% Glycerol = 10%
hydrogel Carbopol 980 = 1.5%
Methyl Paraben = 0.10%
Propyl Paraben=0.05%
NaOH = Q.S.P. to pH 6.8
Purified Water = Q.S.P. to 100%
11.2 METHODS
[00472] Skin Preparation. Percutaneous absorption was measured using the in vitro cadaver skin finite dose technique. Cryopreserved, split-thickness human cadaver trunk skin was obtained from a skin bank and stored in water-impermeable plastic bags at < -20° C until use. Prior to the experiment, skin was removed from the bag, thawed at room temperature for 20 minutes and then cut into sections large enough to fit on 0.81 cm2 Franz diffusion chambers. The dermal chamber was filled with phosphate buffered saline, pH 7.4 ± 0.1, and the epidermal chamber (chimney) left open to ambient laboratory conditions. Skin samples were comprised of both full thickness (epidermis plus dermis) as well as dermal tissue. Dermal tissue was prepared by heating the full thickness skin at 40 °C for 20 minutes in de- ionized water and removing the epidermis using sterile forceps. All cells were mounted in a diffusion apparatus in which the dermal bathing solution was stirred magnetically at approximately 600 RPM and its skin surface temperature maintained at 32.0° ± 1.0 °C.
Franz Cell Testing Conditions:
Temperature: 32°C
Receptor Solution: PBS (Spectrum, USP grade).
[00473] Dosing and Sample Collection. All formulations were applied to the skin sections using a positive displacement pipette set to deliver 0.1 g. The dose was spread throughout the surface of the skin. At pre-selected time intervals after test formulation application, aliquots of the reservoir solution were removed and replaced with an equivalent amount of the fresh solution (phosphate buffered saline), and an aliquot taken for analysis.
[00474] Analytical Methods. Quantification of Lithium Chloride was done using the Infinity (tm) Lithium test kit from Thermo Fisher (Middletown, Virginia). The concentration of lithium in each aliquot was calculated. A calibration curve was first generated and subsequently used to derive the concentration of lithium in each aliquot collected.
11.3 RESULTS AND DISCUSSION [00475] The results for the percutaneous absorption of Lithium Chloride are shown in Figures 5-9. The water/oil (w/o) emulsion 28A displayed the lowest percent permeated through the dermis, with 60% released over a 12 hour time period. This emulsion was significantly different than the other prototypes, in that the drug was entrapped within lanolin alcohol lipid spheres and dispersed in a non-aqueous continuous medium (mineral oil). The emulsion 35A' demonstrated relatively rapid permeation through the dermis, with 100% released in approximately 5 hours at 32 °C. The formulation BX is a neutral hydrogel, with its gel-like consistency produced by the presence of high molecular weight hydroxyethyl cellulose (HEC). The diffusion of lithium through the hydrogel and through the dermis is slower than 35A', with 80% released in approximately 8 hours. Use of an anionic hydrogel (formulation BV-001-003A) slowed down release even further, with 80% released in 12 hours. It is possible that complexation of lithium ions with the anionic polymer Carbopol 980 slows down the release of lithium ions from the hydrogel.
12. EXAMPLE; IN VIVO TIME-COURSE ASSESSMENT OF PERMEATION AND RESIDENCE TIME OF TOPICALLY ADMINISTERED LITHIUM IONS (AS A FUNCTION OF FORMULATION TYPE) THROUGH MOUSE SKIN TREATED WITH DERMABRASION AND FULL-THICKNESS EXCISION
[00476] This example provides an assessment of the rate of permeation and residence time of lithium ions provided in various formulations in an in vivo mouse model developed for follicle neogenesis. Based on the data, appropriate formulations are selected for an in vivo mouse experiment to assess neogenesis. Formulations that have an adequate rate of permeation through the dermis and longest residence time are selected as formulations to enroll in an in vivo model for neogenesis. It is postulated that lithium ions can induce differentiation of stem cells into neogenic hair follicles.
[00477] Formulations selected in this experiment were : 35A', 35BX and BV-001-003A with their respective compositions as shown in Table 2 supra.
12.1 EXPERIMENTAL DESIGN
[00478] 24 C57/BL 6 mice were enrolled in each group. There were 6 groups in total, with 3 groups enrolled for dermabrasion (DA) and 3 for FTE treated skin. A different formulation was enrolled in each of the three groups for DA and FTE.
[00479] Dosing for the DA groups was started at day 0, immediately after debriding the mouse skin with dermabrasion, and continued to day 5. Scab formation on the wound occurs approximately at day 1 and thus the formulations are delivered on top of scabbed wounds. [00480] Dosing for the FTE groups was started at approximately day 7, or when the scab detached from the wound. The formulations were delivered to the re-epithelialized skin for five days.
[00481] Each wound was dosed with a formulation volume of 0.1 ml, or 0.1 g since the density of each formulation was determined to be approximately 1 g/ml. Dosing was accomplished with a 100 microliter Wiretrol device. Post-dosing, the wound was covered with a non-stick Tegaderm bandage.
[00482] Skin and Plasma samples were taken every day at 2 hours, 4 hours and 8 hours post dosing to establish the skin permeation and residence time, correlated with lithium ion plasma concentrations in mM.
12.2 RESULTS AND DISCUSSION
[00483] The data (Figure 10) show that Li ions can be delivered through skin that has been perturbed by a standard method of integumental perturbation, such as dermabrasion. Significant levels of Li can be delivered through the dermis, with peak levels at
approximately 8 mM Li and trough levels at approximately 0.03 to 0.09 mM. Multiple dosing is preferred in order to achieve significant levels of Li in the skin. The pharmacokinetic profile shows that "pulsed" Li delivery can be accomplished.
[00484] Blood levels were an order of magnitude lower than in skin, possibly because the formulation used, in which the Li ion is complexed with CarboPol 980 to form a polymer, enhances its residence in the skin, in contrast to Li ion in, for example, saline, which is expected to be highly water soluble.
[00485] These data demonstrate that Li ions can be delivered adequately through skin that has been perturbed by dermabrasion. In this dosing format, a once or twice daily
administration of lithium for a short period of time is envisioned.
[00486] Dermabrasion by any other means, such as full-thickness or partial-thickness excision, micro-needle roller perturbation, laser fractional, non-fractional or ablative, are alternate means of integumental perturbation, prior to administration of lithium.
13. EXAMPLE; IN VIVO SKIN AND PLASMA DISTRIBUTION OF LI WITH SUBCUTANEOUSLY DELIVERED LITHIUM CHLORIDE IN A DOSE- ESCALATING FASHION IN C57/BLK MICE. WITH THEIR SKIN TREATED WITH DERMABRASION OR FULL THICKNESS SKIN EXCISION
[00487] In this example, the skin and the corresponding plasma concentrations of Li ions were determined following subcutaneous administration of lithium chloride at increasing dose concentrations. This protocol can also be adapted to determine follicular neogenesis as a function of increasing dose concentrations of lithium.
13.1 EXPERIMENTAL DESIGN
[00488] All lithium-containing formulations used lithium chloride dissolved in isotonic saline.
[00489] Mice were treated with either DA or FTE or unwounded (see Table 3 below), and dosed subcutaneously with 0.1 ml of a formulation containing increasing concentrations of lithium chloride in isotonic saline.
[00490] Lithium treatment of the dermabraded mice started on the day of dermabrasion (= Day 1). DA mice received 42 mg/kg, 127 mg/kg, or 381 mg/kg subcutaneously, twice daily for 4 days, and one dose on the 5th day.
Table 3. DA
# Animals /
FTE Group Grp Dose (mg/kg)
1 15 42
2 15 127
3 15 381*
Wound + Vehicle
4 15 (saline)
5 15 Wound Only
No Wound, No
Figure imgf000138_0001
6 15 Treatment
* In this experiment, there was a 600 mg/kg group in which all the animals died after the first injection.
[00491] Lithium treatment of FTE mice started the day of scab detachment (at day 10-11). FTE mice received 64 mg/kg, 150 mg/kg, or 240 mg/kg subcutaneously, twice daily for 4 days, and one dose on the 5th day.
[00492] At the 5th day, either one hour before intended dosing (for trough levels) or one hour after dosing (for peak levels), the mice were sacrificed, and the entire wound area of skin was analyzed for Li concentration and blood was drawn and centrifuged into red blood cells (RBC) and plasma. Then, at the 21st day, a section of skin was biopsied and analyzed for Li concentration, along with the corresponding plasma and RBC concentrations.
[00493] Mice that received FTE treatment as a mode of wounding were dosed on the day of scab detachment (e.g., on day 10-15 post-wounding) with a single dose of lithium chloride for 5 days. At the 5th day of dosing, either one hour before intended dosing (for trough levels) or one hour after dosing (for peak levels), the mice were sacrificed and the wound was biopsied and analyzed for Li concentration. Correspondingly, blood was drawn and centrifuged into RBC and plasma and assayed for lithium levels.
[00494] Lithium concentrations were measured by the validated bioanalytical ICP method provided below. LOQ for Li in the assay = 50 mM.
13.2 VALIDATED BIOANALYTICAL ICP METHOD FOR MEASURING SKIN LITHIUM CONCENTRATION
13.2.1 SCOPE AND APPLICATION
[00495] This method was developed to quantify lithium in murine skin, plasma and pellet of red blood cells (RBC). Known quantities of lithium were added to matrices collected from control mice that were not exposed to any lithium as part of preclinical testing. Processing of samples is described below and involved digestion with hot nitric acid to reduce interference by organic matter and to convert particulate-associated metals to a form that could be measured by ICP/MS. Acid digests were cooled and then filtered prior to injection into the ICP /MS.
[00496] The calibration process involved using lithium standards that were dissolved in purified water. The primary lithium standard was diluted to make a set of lithium working standards. One set of these working standards was used to generate the calibration curve. A separate set was used to prepare the QC samples described in the section entitled "Accuracy." The method was validated for all three murine matrices. The final calibration curve covered the concentration range from 0.05-50 μg/L.
[00497] Processing of plasma samples. A known amount of lithium working standard was added to measured volume of plasma. Concentrated nitric acid (1 mL) was added, and the mixture was heated in a microwave for approximately fifteen minutes. After digestion was complete, digest was cooled and purified water was added to achieve a total volume of 25 mL. Once the solution was mixed and passed through a 0.45-μιη nylon filter, it was ready for injection into the ICP/MS.
[00498] Processing of skin samples. Each skin sample was diced into smaller pieces. A known amount of lithium working standard was added to measured skin mass. Concentrated nitric acid (1 mL) was added and the mixture heated in a microwave for approximately fifteen minutes. After digestion was complete, digest was cooled and purified water was added to achieve a total volume of 50 mL. Once the solution was mixed and passed through a 0.45-μιη nylon filter, it was ready for injection into the ICP/MS. [00499] Processing of pellet of red blood cells. To a vial containing pellet of erythrocytes, 0.5 niL of 0.125% Igepal® CA-630 (dissolved in purified water) was added. Igepal-70 is a non-ionic detergent and was added to dissociate erythrocyte membranes. The solution was triturated with a pipettor and then transferred to a microwave vessel. The mass of transferred solution was measured before adding known amounts of lithium working standard and 1.0 mL concentrated nitric acid. Mixture was heated in a microwave for approximately fifteen minutes. After digestion was complete, the digest was cooled and purified water was added to achieve a total volume of 25 mL. Once the solution was mixed and passed through a 0.45- μιη nylon filter, it was ready for injection into the ICP/MS.
13.2.2 METHOD VALIDATION AND RESULTS
[00500] Prior to performing any quantitative assays on unknown test samples, method validation was performed to ensure that the methodology was sensitive, accurate, precise, and reproducible for the analyte of interest. The parameters to be characterized during method validation were system suitability, Lower Limit of Quantitation (LLOQ), precision, and accuracy (recovery).
[00501] System Suitability. The method assay was shown to work with sample matrices collected from many animals. Biological samples were obtained from at least six different mice that experimentally were not exposed to lithium, and replicates of these control samples were individually spiked with known amounts of the lithium-standard. Samples were processed for analysis as described above. In the absence of any interference, the amount of lithium measured with the analytical assay was expected to be identical to the spiked amount. This was the case, as shown in Table 4 for each of the three murine matrices: plasma, skin and pellet of red blood cells. Percent Recovery, which is the ratio of amount measured to amount spiked, was near 100% for all three matrices, varying between 98 - 110%. Matrix variability between replicate samples had a %CV value of less than 3%, which met acceptance criterion. (Acceptance Criterion: %CV < 15.0)
[00502] This included running six replicates of the mid range standard of the calibration curve. The Standard Deviation (SD) and Coefficient of Variation (CV) for the plasma, skin and RBCs were calculated. All three matrices met the criterion for %CV; for plasma it was 2.78, for skin it was 1.54 and for RBCs it was 1.18. The results are presented in Table 4. Table 4.
Figure imgf000141_0001
[00503] Specificity. This test was performed to demonstrate that none of the components in test samples, either originally present or added to them during processing, contained significant amounts of test analyte. Because Igepal® CA-630 solution was used for the processing of RBC pellets, its lithium content was also tested. No interference was found in either water or Igepal-70 even when they were directly injected into the analytical instrument. The results in Table 5 indicate the level of lithium that was measured for blank unincurred matrix samples. These results include contribution from all solutions used in processing and indicate that lithium content was about 0.050, 0.034 and 0.035 μg/L for control plasma, skin and RBC pellets, respectively. (Acceptance Criterion: No significant interference from water and matrix blanks)
[00504] Water, plasma, skin, RBCs and Igepal® CA-630 blanks were run to show the specificity of the system. The baseline noise of lithium in plasma, skin, and pellet of red blood cells was 0.050, 0.034, and 0.035. The results are presented in Table 5. Table 5.
Specificity
Figure imgf000142_0001
[00505] Linearity. Calibration curves for lithium assay are shown in Figure 11 for the various matrices runs. Each of these curves was obtained by spiking purified water with various amounts of lithium. The calibration curve was linear over the range of 0.05-50 μg/L when run with all matrices. The linearity of the curve was based on the coefficient of correlation associated with the scatter of the data points around the calculated straight line. In all cases, the r2 value for the coefficient of correlation exceeded 0.99. (Acceptance Criterion: The coefficient of correlation for each standard will have an r2 = 0.99 or greater)
[00506] The coefficient of correlation for the calibration curve was greater than 0.99
(Figure 11).
[00507] Precision at Lower Limit of Quantitation (LLOQ). LLOQ was measured at a concentration of 0.1 μg/L. The lowest point in the calibration curve was not selected due to the noise level of the blank matrices. Six replicate unincurred blank matrix samples were spiked with lithium. Each of the six replicates was injected three times into the IPC. The results are presented in Table 6. In terms of %CV, precision for all six replicates was less than 9%, for all matrices. (Acceptance Criterion %CV for six replicates will not exceed 20.0)
[00508] This was calculated from the results of six replicates of a calibration standard at 0.1 μg/L of the calibration curve. The %CV for plasma was 5.83, for skin 8.83 and for RBC 8.46. The results are presented in Table 6. Table 6.
Lower Limit of Quantitation
Figure imgf000143_0001
[00509] Accuracy. Accuracy was measured as % recovery of measured analyte when blank matrix samples were spiked with known amounts of lithium. This test differs from that described in the section entitled "System Suitability" in using fewer replicate samples, making no attempt to insure replicate matrices have originated from separate animals and in extending the measurement to three concentrations, which correspond to the low, mid, and upper end of the calibration range. Such spiked samples were also prepared and run routinely with unknown test samples— and so are called QC samples. They were the basis for demonstrating assay accuracy every time unknown samples were run. Table 7 presents the results obtained during validation. Except near LLOQ, percent recovery of measured analyte was found to be within 10% of its known amount. (Acceptance Criterion: Recovery of QC samples will be 80-120% for plasma and 70-130% for skin or RBCs)
[00510] Accuracy or recovery was measured with QC standards that were run in triplicate. Concentration of the three QC samples corresponded to the low, mid, and upper points of the calibration range. The recoveries for the plasma standards were from 87.2-109%, for skin standards 70.6-102% and for RBC 104-123%. The results are presented in Table 7.
[00511] Precision. Precision was measured as inter-injection variability, by analyzing six separate injections of the same QC sample, removed from the same vial. Analyte concentration for this test was near the mid-point of the calibration range. Results are presented in Table 8. In terms of %CV, precision was less than 1% for all matrices.
(Acceptance Criterion: %CV will not exceed 15.0.) Table 7.
Accuracy
Figure imgf000144_0001
Table 8.
Precision
Figure imgf000144_0002
[00512] Precision was based on six replicate injections of the QC standard, the concentration of which corresponds to the mid-point of the calibration range. The %CV for plasma was 0.642 for skin 0.631 and for RBC 0.540. The results are presented in Table 8.
[00513] Intermediate Precision. The procedure used for intermediate precision was essentially the same as for the section entitled "Precision," but in this case the QC sample was prepared on a different day. Specifically, the plasma, pellet of red blood cells and skin were collected from the same mouse species as used for preclinical testing of the Sponsor's lithium-based drug. Results are presented in Table 9. In terms of %CV, precision was less than 1.16%. (Acceptance Criterion: %CV will not exceed 15.0.)
Table 9.
Intermediate rreasion
Figure imgf000145_0002
[00514] Intermediate precision measurement was conducted according to the procedure in the section entitled "Precision," using a freshly prepared QC sample from the plasma, skin and the RBCs of the mice. The plasma, skin and the RBCs were from the same species of mice which were used for the preclinical mouse experiments described herein. The %CV for plasma was 1.03, for skin 1.15 and for pellet of RBCs 0.530.
13.2.3 CALCULATIONS
% Recovery
[00515] % Recovery = Calculated Concentration x 100
True Concentration
Coefficient of Correlation
[00516] r
Figure imgf000145_0001
where z is the standard score
n-1 Standard Deviation
Y [individual data (X) - Mean Value (X)]2
[00517] Standard Deviation (SD)
n - 1
% Coefficient of Variation
[00518] % CV = SD x 100
Mean
13.2.4 CONCLUSION
[00519] In this study, a method for the quantification of lithium in murine plasma, skin and red blood cells (RBC) was developed and validated.
[00520] The coefficient of correlation exceeded 0.99 for each of the three sample matrices over the calibration range from 0.1 μg/L to 50 μg/L. Plasma, skin, and RBC blank samples were spiked with known quantities of lithium to make standards. Standards for each matrix were prepared separately for each replicate of each parameter tested. The method was validated using 0.1 μg/L as the lower limit of quantitation (LLOQ). All criteria for the System Suitability, LLOQ, Accuracy, Precision, and Intermediate Precision were met. It is noted that for LLOQ, all three matrices were measured with 0.1 μg/L standard instead of 0.05 μg/L (which was the lowest point in the calibration curve) due to matrix interference. This did not affect the outcome of the study.
13.3 RESULTS AND DISCUSSION
[00521] In the DA experiments, the Li concentrations in skin and blood increased in a dose-related fashion. The concentrations of Li in RBC were negligible (data not shown). Li concentrations in skin, at peak, ranged from 0.00035 mM-0.0029 mM (Figures 12 and 13). Li concentrations in blood, at peak, ranged from 0.2 mM-3.4 mM (Figures 13, 14A and 14B).
[00522] When lithium is administered subcutaneously in the FTE experiment, as for the DA experiment, the Li concentrations in skin and blood increased in a dose-related fashion. Like for the DA experiments, little Li ion extraction into RBC (on average, regardless of the dosage, RBC had 0.004-0.005 mM Li) and skin (Figure 15) was observed. Most of the Li was in the plasma (Figure 16). Li concentrations in skin at peak (1 hour post dosing, i.e., on day 5 (wherein day 1 = day of scab detachment and first day of dosing)) were 0.00022-0.0015 mM (see Figures 15 and 17). Li concentrations in skin at trough (24 h post dosing on day 5) were 0.0001-0.0009 mM Li. However, Li concentrations in blood at peak (1 hr post dosing on day 5) were between 0.695 mM - 1.059 mM Li (see Figures 16 and 17), and trough concentrations in blood were 0.02-0.09 mM Li. Animals injected with > 300 mg/kg had toxicity effects.
[00523] Thus, the data show that when lithium chloride is delivered subcutaneously, lithium ions extract to the skin and the plasma in a linear dose-related fashion, although plasma concentrations were many-fold higher than in skin. The lithium concentration in skin at 5 days correlates in a linear dose-related fashion, with no difference observed between wounded skin and non-wounded skin (see Figure 14B). The skin samples were obtained 1 hour post-dosing, in other words at "peak" skin concentrations. The data demonstrate that lithium does distribute to the skin, where it may play a role in stem cell modulation toward differentiation into de novo hair follicles.
14. EXAMPLE; IN VIVO DISTRIBUTION OF LI WITH ONCE DAILY
TOPICALLY ADMINISTERED LITHIUM FORMULATIONS IN C57/BLK MICE, WITH THEIR SKIN TREATED WITH DERMABRASION OR FULL THICKNESS EXCISION
[00524] The purpose of the experiment in this example was to evaluate the absorption of Li ions into the skin and blood compartments in an in vivo mouse model developed for follicle neogenesis, with once/day topical administration of two lithium formulations, a Lithium Gluconate Hydrogel and a Lithium Chloride Hydrogel (see Table 10 below). This example provides a protocol for and assessment of the rate of permeation and residence time of lithium ions provided in the formulations, which can be adapted for an in vivo experiment to assess HF neogenesis.
Table 10
Formulation # 1 (Lithium Gluconate Formulation # 2 (Lithium Chloride
Hydrogel, "HC730"; also referred to Hydrogel, "BV-001-003A"; also
herein as "lithium gluconate") referred to herein as "lithium chloride")
Lithium Gluconate, 8% Lithium Chloride, 8%
CarboPol 980 CarboPol 980
Glycerol Glycerol
Methyl Paraben Methyl Paraben
Propyl Paraben Propyl Paraben
Distilled Water Distilled Water
Sodium hydroxide (for pH adjustment) Sodium hydroxide (for pH adjustment)
Formula Weight: 202 g/mol Formula Weight : 42.39 g/mol
14.1 EXPERIMENTAL DESIGN
[00525] Twenty-four (24) C57/BL 6 mice were enrolled in each group. There were 4 groups in total, with 2 groups enrolled for DA and 2 for FTE treatment. Dosing for the DA groups was started at day 1 , immediately after debriding the mouse skin with dermabrasion, and continued once daily to day 4 (i.e., single dose administered at Oh, 24 h, 48 h, 72 h). A thick scab forms on the wound after day 1 , and thus the formulations are delivered prior to scabbing of the wounds. Dosing for the FTE groups was started at approximately day 10-15 post-FTE wound, when the scab detached from the wound (this is referred to herein as "Day 1"), and continued to day 4 post-scab detachment (i.e., single dose administered at Oh, 24 h, 48 h, 72 h post scab detachment). Each day, animals were sacrificed at 1 h post-dose (for measurement of peak levels), 4-6 h post-dose, and 22-24 h post-dose (for measurement of trough levels) (i.e., tissue samples (skin and blood) were taken at t=l, 4-6h, 24h, 25h, 28h, 48h, 49h, 52h, 72h, 73h, 76h, 96h). See Table 11 below for a schematic of the experimental design. Table 11
Figure imgf000149_0002
Figure imgf000149_0001
[00526] Each wound was dosed with a formulation volume of 0.1 ml, or 0.1 g since the density of each formulation was determined to be approximately 1 g/ml. Dosing was accomplished with a 100 microliter Wiretrol device. Post-dosing, the wound was covered with a non-stick Tegaderm bandage.
[00527] Lithium ion concentrations were measured by ICP/MS/MS, using the validated method provided in Section 13.2 supra.
14.2 RESULTS AND DISCUSSION
[00528] The data show that Li ions are delivered into skin that has been perturbed by DA or FTE and into the blood when administered topically (see top and bottom graphs, respectively, of Figures 10 and 18, respectively). Lithium Chloride hydrogel and Lithium Gluconate hydrogel have similar pharmacokinetic profiles (compare light gray and dark gray curves, respectively). Significant levels of Li were delivered through the dermis, with initial peak levels in skin at approximately 8 mM Li and initial trough levels at approximately 0.3 to 0.09 mM (see top graphs of Figures 10 and 18). The pharmacokinetic profile shows that "pulsed" Li delivery can be accomplished, i.e., there is not a sustained release profile and repeated dosings are required to maintain peak or near-peak levels.
[00529] Lithium blood levels (see bottom graphs of Figures 10 and 18) were generally an order of magnitude lower than in skin, possibly because the formulation used - in which the Li ions are complexed with CarboPol 980 to form a polymer - enhances its residence in the skin, in contrast to Li ions in, for example, saline, which is expected to be highly water soluble. [00530] These data demonstrate that with topical administration of lithium chloride hydrogel or lithium gluconate hydrogel, Li ions are delivered through skin that has been perturbed by DA or FTE, but enter the blood at a much lower level. Delivery occurs in a pulsatile fashion.
15. EXAMPLE; IN VIVO DISTRIBUTION OF LI WITH TWICE DAILY
TOPICALLY ADMINISTERED LITHIUM FORMULATIONS IN C57/BLK MICE. WITH THEIR SKIN TREATED WITH DERMABRASION OR FULL THICKNESS EXCISION AND CORRELATION TO HAIR FOLLICLE NEOGENESIS
[00531] The purpose of the experiment in this example was to evaluate the absorption of Li ions into the skin and blood compartments as a function of dosage (% Li) and dosing frequency in an in vivo mouse model developed for follicle neogenesis, with twice/day topical administration of two lithium formulations, a Lithium Chloride Hydrogel and a Lithium Gluconate Hydrogel (8%) (also referred to as "lithium gluconate, 8%") (and see Table 10 above), as well as a 1% Lithium Gluconate Hydrogel (also referred to as "lithium gluconate, 1%") and a 16% Lithium Gluconate Hydrogel (also referred to as "lithium gluconate, 16%"). This example provides an assessment of the rate of permeation and residence time of lithium ions provided in the formulations in conjunction with in vivo mouse experiments to assess hair follicle neogenesis. This experiment shows that topically administered lithium can induce formation of new hair follicles. It is postulated that lithium induces differentiation of stem cells into neogenic hair follicles.
15.1 EXPERIMENTAL DESIGN. RESULTS. AND DISCUSSION
[00532] 24 C57/BL 6 mice were enrolled in each group. There were 3 groups in total. The mice were dermabraded as discussed above and dosing was started at day 1, immediately after debriding the mouse skin with dermabrasion, and continued twice daily to day 4. Scab formation on the wound occurs approximately at day 1 , and thus the formulations are delivered prior to scabbing of the wounds. Each day, animals were sacrificed at 1 h post-dose (for measurement of peak levels), 4-6 h post-dose, and 22-24 h post-dose (for measurement of trough levels). See Table 12 below for a schematic of the experimental design.
[00533] Each wound was dosed with a formulation volume of 0.1 ml, or 0.1 g since the density of each formulation was determined to be approximately 1 g/ml. Dosing was accomplished with a 100 microliter Wiretrol device. Post-dosing, the wound was covered with a non-stick Tegaderm bandage. Table 12
Figure imgf000151_0001
t
Figure imgf000151_0002
1
[00534] Lithium ion concentrations were measured by ICP/MS/MS, using the validated method provided in Section 13.2 supra.
[00535] The data show that topical administration of Lithium Gluconate hydrogel delivers Li in a dose-correlated fashion into skin that has been perturbed by DA and, to a much lesser extent, into the blood (see Figure 19). Significant levels of Li were delivered through the dermis, and the trough concentrations were significantly higher with the twice daily dose than with once daily dosing. The pharmacokinetic profile shows that "pulsed" Li delivery is still maintained with twice daily dosing, i.e., there is not a sustained release profile and repeated dosings are required to maintain peak or near-peak levels.
16. EXAMPLE; MOUSE MODEL OF LITHIUM TREATMENT FOLLOWING INTEGUMENTAL PERTURBATION USING FULL THICKNESS EXCISION OR DERMABRASION
[00536] The example in this section provides exemplary protocols for integumental perturbation by full thickness excision (FTE) and dermabrasion, which resemble wounding and/or scar revision, in mice and the results of studies conducted using these protocols that demonstrate the efficacy of combination therapies comprising lithium and integumental perturbation in hair follicle neogenesis and improvement of wound healing.
[00537] Specifically, this example provides protocols using four different combinations of lithium and integumental perturbation to induce hair follicle neogenesis: 1) full thickness excision + subcutaneously delivered lithium, 2) full thickness excision + topically delivered lithium, 3) dermabrasion + subcutaneously delivered lithium, and 4) dermabrasion + topically delivered lithium. 16.1 FULL THICKNESS EXCISION PROTOCOL
[00538] 1. Twelve (12)-day old C57BL6/J mice pups are used. They are fed high fat food from the day they arrive to the day of surgery (10 days).
[00539] 2. When the mice are 21 days old, full thickness excision (FTE) surgery is carried out.
[00540] 3. Mothers, domes, and high fat food is removed from the cages. Food is replaced with normal food.
[00541] 4. All pups for the experiment are placed into a large container to randomize.
[00542] 5. The pups are weighed one at a time. If under 7 g, they are placed into a separate "runts" cage and FTE is not performed on these mice. If weight is made, mice are injected with calculated buprenorphine ("BUP"; 0.05 mg/kg). A stock BUP solution may be used that works out to 0.009 mg/ml, so 50 μΐ per 9 g mouse is injected. Place 6 mice per cage until all mice are weighed and given buprenorphine. During the administration of the anesthesia and for the duration of the time that the mice were anesthetized, the cages are placed on heating pads which are set to low heat.
[00543] a. ALTERNATE: If working with many mice (50+), it may be preferable to stagger the BUP dosing in order to avoid the effect of the analgesic wearing off before surgery. It is preferable to stay as close as possible to BUP administration at 1 -2 hours pre- surgery.
[00544] 6. One hour after giving the analgesic, the mice from one cage are injected with ketamine (70 mg/kg) / xylazine (8 mg/kg). Again, a stock that works out to 50 μΐ per 9 g mouse may be used.
[00545] a. ALTERNATE: Giving an additional 10 μΐ KX above the calculated dose (based on weight) seems to put the mice out to toe pinch quicker without increased fatality. However, it is only suggested to do this if few mice are not fully going out to toe pinch.
[00546] 7. Once mice are anesthetized, the proper number is ear-punched, their weight recorded, their back hair shaved with clippers, and a 1.5 cm x 1.5 cm box is marked on the rear dorsum.
[00547] 8. Eye ointment is applied to the mice, to keep their eyes from drying out during their immobilization, and the cages are pre-warmed on low-setting heating pads.
[00548] 10. The surgery site on the rear dorsum is sprayed/wiped with alcohol (70% ethanol) to prevent infections. [00549] 11. A full thickness excision (FTE) (1.5 cm2) along the marked lines is cut out from the skin (cut inside the vertical lines and directly on the horizontal lines) using a pair of blunt-tip scissors and curved-tip forceps.
[00550] a. ALTERNATE: If letting mice live past day-5 post scab detachment, India ink should be used to mark the corners and sides of the wound (8 "dots").
[00551] 12. Finished mice are placed back in the pre-warmed cages and the cages are left on the pads until all mice awake. Eye ointment is reapplied if/when necessary.
[00552] 13. Each cage is supplied with a dish of wet food (moistened regular chow), regular dry chow, water, a dish of flavored JELL-O®, and a water bottle with flavored Prang (bio-serv.com, F2351-S; also avail. From Fisher) mixed in to aid in hydration. Saline is administered to mice that exhibit signs of pain, dehydration, malnourishment, or stress.
Optionally, all cages can be provided with flavored Prang for the duration of the experiment in addition to their water supply. The mice may also be given flavored JELL-O® for the 2 days post surgery (JELL-O® when introduced in earlier experiments for rehydration purposes had a positive effect - mice eagerly ate the JELL-O® and looked healthier on days following the procedure).
[00553] 14. The mice are monitored and weighed over the next two days, as well as 2-3 times per week thereafter. The FTE wounds are allowed to heal by secondary intention. Dose mice AM and PM (approximately 10 AM and 5 PM) with BUP on day 1 after FTE. Dose AM and if necessary in PM on day 2. Replace JELL-O® or water as needed.
[00554] 15. Mice are monitored daily for scab detachment (occurs 11-18 days post FTE).
[00555] 16. On the day of scab detachment, mice are placed into treatment groups:
[00556] 16a. For topical lithium experiment, animals are assigned to groups as follows: the 1 st mouse to lose its scab is placed in Lithium gluconate hydrogel (also abbreviated in this section as "lithium gluconate"), 8%; the 2nd - lithium gluconate, 16%; the 3rd - Lithium Chloride hydrogel (also abbreviated in this section as "lithium chloride"), 8%; the 4th - lithium gluconate 1 %; the 5th - Placebo; and the 6th - No Treatment. This order is repeated as scabs detach until all mice are distributed evenly into treatment groups. After all mice are allotted to treatment groups, the sample sizes are as follows: No treatment with mock handling (mock handling = Pick up and handle mice as if being dosed. An empty capillary tube is used to mock spread out drug onto their back under the bandage. This is done to ensure all mice undergo the same stresses during dosing): No treatment (n=18); Placebo (n=18); Lithium Chloride 8% (n=19); lithium gluconate 1% (n=19); lithium gluconate 8% (n=19); lithium gluconate 16% (n=19) (numbers correspond to the mice in the actual experiment summarized in Section 16, in which 1 12 mice remained alive at the time of scab detachment).
[00557] For topical lithium experiment, bandages (to prevent mice from licking off the drug) are applied as follows: On the day of scab detachment, the mice are administered 75% of a normal dose of ketamine-xylazine according to weight. While the mice are anesthetized, the posterior dorsal, posterior lateral, and posterior ventral sides are shaved and treated with Nair. Care is taken to ensure that Nair did not cover the wounded area. Mice are then wrapped in Tegaderm & Telfa pad bandages. The Tegaderm is tightly wrapped around the mouse with the Telfa pad fixed over the wound / treatment area. The first treatment is administered once the bandages are in place. The mice are placed back into their cages before the anesthesia wears off. If at any point during the treatment period the mice escape from their bandage, the bandage is replaced. Mice are checked for bandages at every dosing interval.
[00558] 16 b. For subcutaneous lithium experiment, animals are assigned to groups as follows: On the day of scab detachment, mice are placed into five treatment groups: Saline (n=15), LiCl 64 mg/kg (n=15), LiCl 150 mg/kg (n=15), LiCl 240 mg/kg (n=15), No treatment (n=15) (numbers correspond to the mice in the actual experiment summarized in Section 16). Note that there are no bandages applied for the subcutaneous lithium experiment.
[00559] 17 a. For the topical lithium experiment, dosing is as follows: topical lithium doses (100 ul per 20 g mouse) are administered twice per day (for each mouse respectively), with the morning dose at ~ 1 1 AM and the afternoon dose at ~ 5 PM. The day of scab detachment is denoted as Scab Detachment Day 1 (SD1). Mice in treatment groups receive an AM and PM dose on SD1, SD2, SD3, and SD4 (8 total doses). Drummond wiretrols and accompanying ΙΟΟμΙ capillary tubes are used to dispense ΙΟΟμΙ onto the wound site of each mouse. The tube is then used to spread out the drug to encompass the entire wound. One capillary tube is used per mouse. Drug treatment vials are replaced when necessary.
[00560] 17 b. For the subcutaneous lithium experiment, dosing is as follows: mice are dosed once their scab detaches (SD1 = Scab Detachment - 1). Doses are given twice per day (am and pm) subcutaneously (100 μΐ for a 20 gram mouse).
[00561] 18. All mice are harvested on SD5, including mice used for determination of "peak" and "trough" lithium levels (see below). The mice are not treated on their respective harvest day (SD5), unless that mouse is used for a peak dosage sample, i.e., selected as a peak value point for testing lithium compound levels in blood and skin. Some mice from each group were selected for evaluation of lithium levels in the skin and blood. Per group, three mice are used for "peak" levels and three mice for "trough" levels. Mice selected for the peak levels have blood drawn 1 hour post AM dosing on SD5 (9 total doses). Mice selected for "trough" levels have blood drawn 18-24 hours post SD4 PM dose (i.e., prior to harvest without treatment). On day 5 post scab detachment, approximately 0.5 ml of blood is collected per mouse, using the cheek lancet technique, and the mice are sacrificed. Wound skin is then analyzed by confocal microscopy and collected as described below.
[00562] 19. Processing of mice for data collection: In preparation for in vivo scanning laser microscopy, the hair surrounding the healed wound is trimmed to reveal the wound surface. The surface is imaged with a confocal microscope; this includes 1 -3 Vivablocks (8 mm x 8 mm) at depths between 40-100μιη, and 1-3 Vivastacks (500 uM x 500 uM) of areas with many neogenic hair follicles (NHF's) or interesting features. The mice are sacrificed, and the entire wound is excised (excluding normal surrounding tissue as much as possible), and tissue allocated in a procedure that lasts ~ 3-5 minutes per mouse after the time of death. Following excision, the wound is bisected. Half of the wound is placed on a section of a 3x5 inch index card and stored in cold 4% Paraformaldehyde for histology. The other half is frozen on dry ice for biochemical assays, including lithium levels. The frozen samples are placed on dry ice for the duration of the harvest period and then transferred to a -80° Celsius freezer for long term storage. The histology samples are taken out of the 4% Para- Formaldehyde solution following overnight fixation and moved to 30% sucrose/lX PBS. Within 24 hours the samples are taken from the sucrose solution and dabbed dry, then embedded in OCT. The OCT is frozen in a slurry of crushed dry ice and 2-O-methyl-butane. Cryosections are generated for histology. In the actual experiment the results of which are summarized in Section 16, for quantification of histology, approximately 10 slides were generated from the cut edge (midline) of the sample, and the first slide was stained with hematoxylin and eosin and was used for quantification. In the minority of cases, the tissue sections on the first slide were damaged, which then necessitated use of the next slide in the sequence. Only samples in which the wound was clearly demarcated from the adjacent normal were used for quantification of neogenic hair follicles.
[00563] Section 16.3 provides the results of an experiment in which the above FTE protocol was used.
16.2 DERMABRASION PROTOCOL
[00564] In the following protocol, a microdermabrasion device is used to perform dermabrasion. [00565] 1. 7 week old C57BL6/J female mice are obtained. The mice are allowed to acclimate for 2 weeks + 5 days before the day of surgery. Mice are 9 weeks + 5 days old on the day of procedure.
[00566] 2. Mice are weighed and treated with buprenorphine ("BUP"; single IP injection, 0.05 mg/kg, 50 μΐ per 20 g mouse using a dosing solution of 0.02 mg/ml) 60 minutes before the procedure, on the day following dermabrasion (two doses, ~ 8 hours apart), and as needed the second day after dermabrasion.
[00567] 3. After one hour has passed mice are re-weighed (because they are not ear punched before ketamine/xylazine dose), anesthetized with ketamine (80 mg/kg) / xylazine (8 mg/kg), and ear punched for identification. Mice are given an eye ointment to keep their eyes from drying out during their immobilization.
[00568] 4. Once the mice have ceased being mobile both the left and right sides of the dorsal rear back skin are clipped.
[00569] a. Nair® is applied for 1 minute to the right and left flank, the hair wiped off with a wet paper towel, and dried with paper towel.
[00570] 5. The mice are dermabraded once they do not react to a toe-pinch.
[00571] a. The microdermabrasion device settings (Advanced Microderm, DX model) are set to max vacuum, large tip, and max mixture.
[00572] b. aluminum oxide crystals are used for dermabrasion.
[00573] c. 10 passes on the right dorsal rear flank are carried out (each pass is a single movement from cranial to caudal direction; skin is held taught).
[00574] d. The left side of the body is not dermabraded.
[00575] 6. After dermabrasion, the 4 corners and the midpoints along the edge of the wound are tattooed with an injection of India ink (using a tuberculin syringe); 8 total tattoo marks made.
[00576] 7. Prior to waking up, the mice are bandaged as in the FTE protocol (only topical lithium experiment is bandaged), and dosing initiated (day 1) as follows:
[00577] A. For the topical lithium experiment: 1) Lithium gluconate hydrogel (also referred to in this section as "lithium gluconate") 1% (n=15); 2) lithium gluconate 8% (n=15); 3) lithium chloride hydrogel (also referred to in this section as "lithium chloride") 8% (n= 15); 4) lithium gluconate 16% (n=15); 5) placebo (n=15); and 6) no treatment (with mock handling)(n=15) (described elsewhere in this section).
[00578] B. For the subcutaneous lithium experiment: lithium chloride 42 mg/kg (n=15), lithium chloride 127 mg/kg (n=15), lithium chloride 381 mg/kg (n=15), lithium chloride 600 mg/kg (n=19), Saline (n=15), Wound-No Treatment (n=15), and No Wound-No Treatment (n=5). (No Wound-No Treatment is anesthetized and depilated with Nair, but not dermabraded) (for the experiment described in Section 16.3).
[00579] 8. The mice are then placed back into their respective cages which were pre- warmed on low-heat heating pads prior to surgery and kept on heating pads (under cage) until they wake up.
[00580] 9. Upon waking up (in the afternoon), the mice are treated again with the specified treatment, according to the label on their cage. Twice-a-day dosing continued until day 4 post-MDA / day 5 total
[00581] 10. During the dosing period, weights and observations of mice are recorded daily. The time of delivery of dose is recorded. For the subcutaneous lithium experiment, water consumption is monitored, as well as each cage provided with a salt water source (450 mM NaCl).
[00582] 11. Tissue is harvested from 1 cage (2-5 mice) of each group on day 5 post-DA for i) bioassay of target, ii) assay for concentration of compound in skin, and iii) histology.
[00583] a. This is done by resecting the skin from the right side and observing the ink spots from the dermal side of the skin. The wound area is cut out and divided into three pieces for assays as indicated above.
[00584] 12. Blood samples are also collected into potassium-EDTA vacutainers (2 ml capacity, lavender cap) from these same animals via cheek puncture lancets. The blood is centrifuged (maximum speed in non-refrigerated microfuge), the supernatant (plasma) is removed to a separate microfuge tube, labeled, and then frozen (along with the RBC cell pellet) for storage and shipment. Blood is taken within 1 -2 hours of the last dose to determine peak levels of lithium compound in skin, or alternatively prior to harvest without dosing if trough levels are desired.
[00585] 13. Remaining animals (~10 per group) are allowed to survive until 21-23 days post dermabrasion at which time they were clipped and razored (disposable razor) on both the wound and non-wounded skin. A full thickness excision of skin from both treatment sites is excised. The skin samples are separately placed on 3x5 note cards and the surface of the excised skin (wound - right side; non-wounded - left side) analyzed by confocal microscopy in order to quantify the thickness of the hair shafts, density of hair shafts, the density of hair pores, and the number of shafts per pore.
[00586] 14. Following confocal microscopy, the skin is divided into three pieces as above. [00587] a. For biochemical assays, samples are immediately placed in Eppendorf tubes and frozen on dry ice, after which they are transferred to -80 °C freezer.
[00588] b. For histology, a piece of tissue is immersed in 4% PF A/PBS and stored at 4 °C until processing for paraffin histology, or for cryosectioning, then changed to 30% sucrose/PBS (after overnight fixation) for between 12-24 hours. Samples are then embedded in optimal cutting temperature (OCT) and stored at -80 °C.
[00589] Section 16.3 provides the results of an experiment in which the above DA protocol was used.
16.3 SUMMARY OF EXPERIMENTAL DESIGN AND RESULTS
[00590] Based on results from the experiments described in the examples of Sections 11 to 15, an experiment to assess hair follicle neogenesis in mice was undertaken using the protocols described in Section 16.1 and 16.2 supra, as follows: One set of mice was enrolled for DA with 8% lithium chloride hydrogel, placebo and sham (untreated) (as described in Table 7). A second set of mice was enrolled for FTE (as described in Table 13). Dosing for the FTE groups was started at approximately day 10 - 15 post wounding, when the scab detached from the wound (this is referred to herein as "Day 1 "; each mouse was monitored for scab detachment and when this occurred, this was set as "Day 1 " of treatment), and continued twice daily to day 4 post-scab detachment. Dosing for DA starts on the day of the DA procedure.
Table 13.
Figure imgf000158_0001
[00591] Samples were taken from the mice administered topical and subcutaneous lithium at day 5 (skin and blood) to assess skin and blood concentrations at peak and trough. For the dermabrasion experiment, 5 of 15 mice are sacrificed to take samples at day 5 post DA. For the FTE experiment, all mice are sacrificed at day 5 post scab detachment (first day of treatment), some of which are used for measuring lithium levels in blood and skin. The results for the topical experiment are shown in Table 14. The blood levels at "peak" were at least 9-fold lower than the corresponding skin levels.
Table 14.
FTE / Skin (values in mM Li)
Peak Stdev Trough Stdev
Placebo 0.00 0.00 0.00 0.00
Lithium Gluconate, 1 % 0.92 0.93 0.15 0.21
Lithium Gluconate, 8% 2.67 2.55 0.70 0.12
Lithium Gluconate, 16% 11.20 0.77 2.65 2.41
Lithium Chloride, 8% 3.77 2.29 0.25 0.15
FTE / Blood (values in mM Li)
Peak Stdev Trough Stdev
Placebo 0.00 0.00 0.00 0.000
Lithium Gluconate, 1 % 0.04 0.04 0.03 0.043
Lithium Gluconate, 8% 0.30 0.24 0.29 0.38
Lithium Gluconate, 16% 0.19 0.17 0.18 0.13
Lithium Chloride, 8% 0.30 0.045 0.12 0.015
DA / Skin (values in mM Li)
Peak Stdev Trough Stdev
Placebo 0.00 0.00 0.00 0.00
Lithium Gluconate, 1 % 0.73 0.47 0.01 0.01
Lithium Gluconate, 8% 3.08 1.43 0.28 0.42
Lithium Gluconate, 16% 8.26 2.31 0.75 0.57
Lithium Chloride, 8% 1.12 0.30 0.22 0.06
DA / Blood (values in mM Li)
Peak Stdev Trough Stdev
Placebo 0.00 0.000 0.00 0.000
Lithium Gluconate, 1 % 0.03 0.02 0.01 0.004
Lithium Gluconate, 8% 0.29 0.07 0.09 0.02
Lithium Gluconate, 16% 0.91 0.14 0.11 0.02
Lithium Chloride, 8% 0.27 0.022 0.10 0.035
[00592] Parts of the same tissue samples shown in Table 14 were also used to assess hair growth.
16.3.1 FTE RESULTS
[00593] In the FTE study using topically administered lithium, quantification of confocal images showed that, compared to placebo, treatments containing 8% lithium (lithium gluconate 8% or lithium chloride 8%) had lower mean and median total wound areas (TWA) and higher mean and median numbers of germs, germ densities within the germ forming region (GFR), and germ densities within the TWA. The 8% lithium chloride treatment also had higher mean and median GFR areas and percent coverages of TWA by GFR, while the 8% lithium gluconate treatment had higher mean, but not median, GFR areas and percent coverages of TWA by GFR. Although results for the lowest strength lithium treatment (1% lithium gluconate) were essentially indistinguishable and in some cases slightly less favorable than those of the placebo group, there was not an overall dose response because the results for lithium gluconate 16% were less favorable that those for the two 8% strengths. Except for TWA, results from the group of mice that did not receive any treatment were generally superior to placebo, 1% lithium gluconate, and occasionally rivaled those from the 8% and 16% strength treatments.
[00594] As shown in Figures 20-22, in the FTE experiment using topically administered lithium, relative to placebo and lithium gluconate 1% and 16%, lithium chloride 8% and lithium gluconate 8% promoted increased differentiation of neogenic hair follicles as detected histologically. Compared to placebo, the lithium chloride 8% treatment was statistically significantly superior with respect to both the total number of neogenic hair follicles and the proportion of mature stages (Stages > 5). The lithium gluconate 8% treatment also appeared to be superior to placebo, but the differences did not reach statistical significance after adjusting for multiple comparisons. The lithium gluconate 16% treatment had somewhat better results than placebo, but none of the comparisons approached statistical significance. The results for the lithium gluconate 1% group were similar to those for placebo.
[00595] It is noted that mice from all groups treated with topical lithium had
approximately the same weight gain profile throughout the entire experiment (see Figure 23), indicating that topical lithium (at all concentrations) did not have any negative effects, such as systemic effects that negatively impact overall health of the mice, and then which may indirectly affect hair growth.
[00596] Insignificant effects were seen in the experiments in which lithium was delivered subcutaneously, despite achieving maximal tolerated doses in the mice. It is postulated that lithium administered subcutaneously at non-toxic dosages did not achieve high enough concentrations in the skin to effect hair follicle neogenesis.
16.3.2 DA RESULTS
[00597] In the dermabrasion experiment (using dermabrasion + topical lithium), it was intended to analyze four variables using confocal imaging: hair shaft density, hair pore density, number of hair shafts per pore, and hair shaft diameter. Since the number of pores and hair shafts was the same, the density analysis was done on hair shafts. No formal histological analysis was made in this experiment as hair follicle density was unchanged among all treatment groups.
[00598] Compared to placebo, the mean hair shaft diameter was statistically significantly larger (16%) for mice receiving lithium gluconate 8% (see Figures 24 and 25). The lithium chloride 8% and lithium gluconate 16% treatments also had higher mean hair shaft diameters, but unlike the lithium gluconate 8% groups, the differences did not reach statistical significance. With respect to hair shaft density, there were no statistically significant differences between placebo and any of the lithium-containing treatments. Results from the group of mice that did not receive any treatment were comparable to those of the placebo group.
[00599] The histological hallmark of male and female pattern hair loss is miniaturization of hair follicles with a progressive transformation of terminal hair follicles into vellus-like follicles: terminal hair follicles have a shaft diameter of greater than 0.06 mm, whereas vellus-like follicles are defined as hairs with a hair shaft diameter of 0.03 mm or less and are thinner than the hair's inner root sheath (Dinh and Sinclair, 2007). The findings of an increase in hair shaft diameter in the 8% and 16% lithium groups and the observation histologically that the hair follicles appeared to disassemble and later reassemble during wound healing is suggestive that hair follicle neogenesis occurs also following more superficial wounding in association with topical application of lithium.
[00600] It is noted that mice from all groups treated with topical lithium had
approximately the same weight gain profile throughout the entire experiment (data not shown), indicating that topical lithium (at all concentrations) did not have any negative effects, such as systemic effects that negatively impact overall health of the mice, and then which may indirectly affect hair growth.
[00601] Insignificant effects were seen in the experiments in which lithium was delivered subcutaneously, despite achieving maximal tolerated doses in the mice. It is postulated that lithium administered subcutaneously at non-toxic dosages did not achieve high enough concentrations in the skin to effect hair follicle neogenesis.
16.3.3 SUMMARY
[00602] Topical delivery of lithium achieved positive effects on hair follicle neogenesis in both the full thickness excision and dermabrasion models. Both the 8% lithium formulations (lithium chloride and lithium gluconate) used were found to improve growth of hair in the mouse skin. Compared to placebo, the animals treated with either topical formulation of 8% lithium had a higher percentage of neogenic hair follicles at a more mature stage of development (see Figures 20 to 22), increased thickness of hair shafts (see Figures 24 and 25), an increased number of neogenic hair follicles in the wound site (Table 15 and Figures 26 to 30), and normal patterning of hair follicles (as determined by density of neogenic hair follicles; see Table 15 and Figures 31 and 32).
Table 15.
Figure imgf000162_0001
[00603] Specifically, for the FTE + topical lithium experiment, in addition to an increased number of neogenic hair follicles observed histologically, twice-daily lithium 8% treatments increased the maturation of neogenic hair follicles (the numerical difference from the lithium chloride 8% group achieved statistical significance). For the dermabrasion + topical lithium experiment, twice-daily lithium 8% treatments increased the shaft diameters of hair follicles formed after dermabrasion (the numerical difference from the lithium gluconate 8% group achieved statistical significance). The effect of lithium 8% for hair follicle neogenesis and in increasing hair shaft diameters in these experiments was greater than that associated with the lithium 1% and 16% concentrations. Although 16% lithium gluconate administration resulted in significant delivery of Li to the skin, new hair follicles were not observed in mice treated with this concentration.
[00604] In summary, twice-daily topical application of lithium 8% induces hair follicle neogenesis following integumental perturbation by either full thickness excision or dermabrasion. The results of these experiments provide evidence of efficacy of pulsatile treatment of lithium in combination with integumental perturbation.
16.4 WOUND DATA
[00605] As shown in Figures 33 and 34, formulations of topical lithium 8% and 16% decrease the area of healed wounds in the FTE experiments. An increased number of neogenic hair follicles is correlated to the decrease in wound area in 8% lithium treated animals, but while decreased wound area was observed in the 16% lithium treated animals, this was not correlated to an increase in hair follicles.
17. EXAMPLE: COMPARISON OF RELEASE RATES OF COMPLEXED AND NON-COMPLEXED LITHIUM CHLORIDE SOLUTIONS
[00606] One approach to slowing down release of Li ions from a matrix is to "complex" or bind the positively charged ions to a negatively charged bioadhesive polymer. This experiment presents a correlation of release rate to the extent of binding of Lithium to the anionic polymer. The anionic polymer was selected to be partially crosslinked polyacrylic acid, or Carbopol 980, with ionizable carboxylic acid groups with a pKa -5-6.
17.1 EXPERIMENTAL DESIGN
[00607] Solutions of lithium chloride were prepared to 1, 5 and 8% in saline. These solutions are hypothesized to have instant release into the medium. Solutions of lithium chloride in 1% Carbopol were prepared in strengths 0.5%, 1%, 4%, 8%, 10% and 20% w/w. To prepare these solutions, lithium chloride solutions were added to a solution of Carbopol 980, dissolved previously with distilled water and pH to 7. The shear flow rheology of each of these formulations was tested in a cone-plate Brookfield Viscometer at temperature of 37 °C. In vitro release experiments were carried out using dialysis cassettes immersed in phosphate buffered saline at pH 7.4, with slow stirring.
17.2 RESULTS
[00608] Viscosity. The shear flow viscosity of lithium chloride was very low, as in water (< 10 cp). The viscosity of placebo Carbopol 980 at a 1% solids content was 3800 cp at 37 °C. The viscosity dropped as higher concentrations of Lithium Chloride were added to the Carbopol due to complexation and neutralization of the negative charge. The solution precipitated at 20% w/w Lithium Chloride, demonstrating the complexation had been completed and the resulting complex had exceed the theta point. The viscosity of Lithium Chloride as a function of concentration was as follows: Placebo : 3800 cp > 0.5% Li Carbopol : 2800 cp > 1% Li Carbopol : 2000 cp > 4% Li Carbopol : 1500 cp > 8% Li Carbopol : 1015 cp > 10% Li Carbopol : 430 cp > 20% Li Carbopol : precipitate.
[00609] Release Rates. The release rate of complexed Li was slowest for the precipitate and highest for uncomplexed Lithium Chloride in Saline. Since the concentration of Carbopol was identical in all of the complexation solutions, this suggests that complexed Li can be utilized as a technique to retard release of the highly water soluble Li ion from a matrix.
[00610] At pH > pKa, aqueous solutions of CarboPol increase dramatically in viscosity, due to repulsion of ionized carboxylates. Positively charged Li ions readily bind to the anionic polymer. Thus, the release kinetics of Li+ from a gel of CarboPol is due to its de- binding kinetics. The release of Li from this matrix is slower than its release from a matrix such as saline.
18. EXAMPLE: MICROENCAPSULATION OF LITHIUM
GLUCONATE IN BIODEGRADABLE POLY (D,L-LACTIDE-CO- GLYCOLIDEXPLG) MICROSPHERES
[00611] Lithium gluconate was encapsulated in PLG microspheres for the purpose of developing a sustained release Li Gluconate formulation that can be embedded into the dermis.
18.1 EXPERIMENTAL DETAILS
[00612] Lithium Gluconate was dissolved in water at a concentration of 8% and complexed with Carbopol 980. This is referred to as the "complexed Lithium Gluconate solution." Separately, Lithium Gluconate was dissolved in water at a concentration of 8%.
[00613] 1 ml of the Lithium Gluconate complex was added to 4 ml of 28 mg/ml PLG solution in methylene chloride and homogenized for 50 s at 7500 RPM. The resulting milky white dispersion was immediately poured into a aqueous solution containing 1% PVA and homogenized for another 1 minute at 7500 RPM. The dispersion was then poured into a large excess of 0.5% PVA and stirred for two hours at ambient temperature to evaporate the methylene chloride. The spheres were then washed three times with water and lyophilized to produce a free-flowing powder.
[00614] The encapsulation efficiency of complexed Lithium Gluconate was measured by a colorimetric Li assay.
[00615] In another experiment, uncomplexed Li was encapsulated and encapsulation efficiency measured by a colorimetric Li assay.
18.2 RESULTS AND DISCUSSION
[00616] The encapsulation efficiency of Li when using complexed Li was approximately 55%. In contrast, the encapsulation efficiency of Li using uncomplexed Li Gluconate was 7.74%. Spheres obtained by this method were approximately 1-4 microns in diameter, as shown in Figures 35 and 36.
[00617] This experiment demonstrates that encapsulation efficiency in microspheres can be enhanced with complexation of Li with an anionic polymer, prior to encapsulating in the PLG matrix.
19. EXAMPLE: PREPARATION OF SYNTHETIC BIODEGRADABLE
PLA:PLG SCAFFOLDS AND MODULATION OF LI+ RELEASE BY
VARYING THE POLYMER COMPOSITION
[00618] The objective of this experiment was to develop prototypes of biodegradable scaffold patches that could be placed on wounded tissue. The "scaffold" is a three- dimensional structure that can provide a high surface area for cell attachment. The cell signaling agent incorporated in the scaffold matrix is lithium gluconate, which in water, ionizes into gluconate anions and Li+ ions. By varying the polymer composition of the scaffold matrix, the release properties of Li+ can be modulated from 3 days to 14 days.
19.1 PREPARATION AND CHARACTERIZATION OF BIODEGRADABLE DRUG LOADED MESH PATCHES
[00619] Solutions of lithium gluconate were prepared in distilled water at a concentration of 50 mg/ml. Poly(lactide-co-glycolide) (PLG), MW 12000 g/mole, poly(lactic acid) (PLA), MW 30,000 g/mole and blends thereof, were used to prepare fibrous scaffolds. The blends of polymers were 100/0 PLA/PLG, 50/50 PLA/PLG, 25/75 PLA/PLG and 0/100 PLA/PLG, respectively. PLA and PLG were purchased from Purac, Inc.
[00620] A cotton candy machine (Gold Medal Floss, Cat# 3024) was set at a setting at 3 (there are five settings in total, ranging from temperatures of 40 degrees C to 200 degrees C).
[00621] 1 g of a blend of 100/0 PLA and 1 ml of the lithium gluconate solution was fed into the hopper, which resulted in fine fibers collecting (much like spider web) in the collection chamber. The fibers with incorporated drug were collected and pressed into patches of lg each, a low pressure Carver press. The patches were then punched out into 1 square inch squares.
[00622] A similar procedure was followed for the other blends of 50/50 PLA/PLG, 25/75 PLA/PLG and 0/100 PLA/PLG.
[00623] Scanning electron micrographs (SEM) (see Figure 37A and B)were taken of the patches. By SEM, the mesh size, or open-cell size was estimated to be around 100-200 microns. Estimated thickness of the fabricated patches was in the range of 500-1000 microns. [00624] The patches were placed into mesh buckets in dissolution baths containing phosphate buffered saline at 37 degrees C and pH 7.4, to simulate physiological conditions. Aliquots of the dissolution media were retrieved at predetermined time-points and analyzed for Li+ content by flame-emission atomic adsorption spectroscopy (AA).
[00625] Figure 37C is a plot of Percent Cumulative Release of Li+ as a Function of Time in Days, overlaid with release profiles of four different blends.
19.2 RESULTS
[00626] SEM. Scanning electron micrographs of 100/0 PLA and 0/100 PLG is shown in Figure 37A and B. The micrographs demonstrated a fibrous texture.
[00627] Visual and Flexural Modulus. The pressed fiber patches were tested for flexural strength by a simple flex method of bending the patch between the thumb and the index finger. The patches could be bent, but they were brittle to the touch. Future patches should incorporate some plasticizing polymers such as PEGs, or silicones, to impart some flexibility to the patches. By SEM, the mesh size, or open-cell size was estimated to be around 100-200 microns. Estimated thickness of the fabricated patches was in the range of 500-1000 microns.
[00628] Release Rates. The release of Li+ could be modulated by varying the ratio of PLA to PLG. As a rule of thumb, the higher crystallinity of the poly(lactide) (PLA) slows down the release of Li+ from the matrix. The amorphous nature of poly (lactide-co-glycolide) (PLG) result in higher release rates of Li+. The approach of blending various ratios of PLA: PLG can be utilized effectively to modulate the release rate of Li+ from the matrix.
[00629] Biodegradability. The biodegradability of the patches can be tested in-vitro, by incubation of pre- weighed patches in phosphate buffer saline, pH 7.4 at 37 degrees C. Over time, the patches are removed from the bath and dried in a vacuum oven maintained at 30 degrees C. The weight of the patches at T=0 and t=t, provides biodegradation profile. Since the polymers degrade by hydrolysis and not by enzymolysis, the degradation buffer would not contain enzymes.
[00630] Bioadhesion. The bioadhesive-ness of the drug-loaded patches can be assessed by placing the patch of wet tissue, inverting the tissue and measuring the rate at which the patch detaches from the tissue.
[00631] Cell Adhesion. The propensity of the drug-loaded patches to adhere to cells is measured by in-vitro culture of COS cells or keratinocytes in the presence of the scaffolds. 20. REFERENCES CITED
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21. ILLUSTRATIVE EMBODIMENTS
[00652] The invention can be illustrated by the non-limiting, embodiments set forth in the following paragraphs.
1. A method for revising a scar in a human subject, comprising administering a pulse treatment or intermittent treatments of a lithium composition that delivers an effective amount of lithium ions in combination with integumental perturbation to a human subject in need thereof.
2. A method for treating a wound in a human subject, comprising administering a pulse treatment or intermittent treatments of a lithium composition that delivers an effective amount of lithium ions to a human subject in need thereof.
3. The method of paragraph 1 or 2, wherein the scar revision or wound heals by primary intention.
4. The method of paragraph 1 or 2, wherein the scar revision or wound heals by secondary intention.
5. The method of paragraph 1 or 2, wherein the scar or wound is caused by a burn.
6. The method of paragraph 1 or 2, wherein the scar or wound is caused by trauma.
7. The method of paragraph 1 or 2, wherein the scar or wound is caused by acne.
8. The method of paragraph 2, wherein the human subject has wound dehiscence.
9. The method of paragraph 2, wherein the wound is an ulcer.
10. The method of paragraph 1, wherein the treatment improves the appearance of the scar or restores function of the scarred tissue.
1 1. The method of paragraph 1 or 2, wherein the treatment has one or more of the following effects: no or reduced scarring or improves the function of the wounded tissue.
12. The method of paragraph 1 or 2 in which the lithium composition is added to freshly wounded skin. 13. The method of paragraph 1 or 2 in which the lithium composition is added to the skin around the wound.
14. The method of paragraph 1 or 2 in which the lithium composition is administered by a laser delivery device.
15. The method of paragraph 14, wherein the laser is a Smoothpeel laser, set at high, 20 passes.
16. The method of paragraph 14, wherein the laser is an ultrapulse laser, set at 350 mJ, 1.8 mm spot size, density 9, 2 passes.
17. The method of paragraph 14, wherein the laser is a Mixto laser, set at 84.8 J/cm, W=12, index=4, 4 passes.
18. The method of paragraph 1 or 2 in which the pulse treatment or intermittent treatment of the lithium composition is administered by topical administration to the skin.
19. The method of paragraph 1 or 2 in which the pulse treatment or intermittent treatment of the lithium composition is administered to the skin surface, transdermally, or intradermally.
20. The method of paragraph 1 or 2 in which the pulse treatment or intermittent treatment of the lithium composition is administered as part of a wound dressing.
21. The method of paragraph 1 or 2 in which the pulse treatment or intermittent treatment of the lithium composition is administered as part of a wound irrigation solution.
22. The method of paragraph 1 or 2 in which the pulse treatment or intermittent treatment of the lithium composition is administered via a scaffold that is applied to the skin.
23. The method of paragraph 22, wherein the scaffold is in the form of a gel, a spray, a dressing, or a wrap.
24. The method of paragraph 22, wherein the scaffold comprises a PLA:PLG scaffold.
25. The method of paragraph 1 or 2 in which the pulse treatment or intermittent treatment of the lithium composition is administered by subcutaneous, parenteral or oral administration. 26. The method of paragraph 1 or 2 in which the lithium composition comprises lithium gluconate, lithium carbonate, or lithium succinate.
27. The method of paragraph 1 or 2 in which the lithium composition does not comprise lithium chloride.
28. The method of paragraph 1 or 2 in which the lithium in the lithium composition is encapsulated in microspheres.
29. The method of paragraph 28, wherein the lithium in the lithium composition is encapsulated in microspheres of sizes between 0.10 microns and 200 microns.
30. The method of paragraph 29, wherein the microspheres are between 0.20 microns and 50 microns.
31. The method of paragraph 28, wherein the lithium in the lithium composition is encapsulated in liposomes of sizes between 10 nm and 50 microns.
32. The method of paragraph 31, wherein the liposomes are between 500 nm and 20 microns.
33. The method of paragraph 1 or 2 in which the lithium composition is administered as a cold liquid, which gels at a temperature of 32 °C - 37 °C.
34. The method of paragraph 1 or 2 in which the lithium composition is administered as a liquid, which then hardens into a depot that delivers lithium over time.
35. The method of paragraph 1 or 2 in which the lithium composition is administered as a hydrogel.
36. The method of paragraph 1 or 2 in which the lithium composition comprises one or more excipients that complex to lithium.
37. The method of paragraph 36, wherein the excipient comprises hyaluronic acid, polyacrylic acid or alginic acid.
38. The method of paragraph 1 or 2 in which the lithium composition comprises one or more permeation enhancing agents or carriers that solubilize the lithium in skin. 39. The method of paragraph 1 or 2 in which the lithium composition comprises propylene glycol, polyethylene glycol or ethanol.
40. The method of paragraph 1 or 2 in which the pulse treatment is a single dose of the lithium composition administered over a period of 1 day to 1 month.
41. The method of paragraph 1 in which the integumental perturbation treatments are administered during lithium treatment holidays.
42. The method of paragraph 1 in which the intermittent treatments are multiple courses of lithium and integumental perturbation treatment interrupted by treatment holidays.
43. The method of paragraph 2 in which the intermittent treatments are multiple courses of lithium treatments interrupted by lithium treatment holidays.
44. The method of paragraph 1, 41 or 42 in which an additional, third treatment is administered.
45. The method of paragraph 44 in which the third treatment is administered during a lithium treatment holiday.
46. The method of paragraph 2 or 43 in which a second treatment is administered.
47. The method of paragraph 46 in which the second treatment is administered during a lithium treatment holiday.
48. The method of paragraph 46 or 47 in which the second treatment is integumental perturbation.
49. The method of paragraph 1 in which the lithium treatment is administered before the integumental perturbation treatment.
50. The method of paragraph 1 in which the lithium treatment is administered concurrently with the integumental perturbation treatment.
51. The method of paragraph 1 in which the lithium treatment is administered after the integumental perturbation treatment. 52. The method of paragraph 2, wherein the lithium treatment is begun before the skin has been wounded.
53. The method of paragraph 2, wherein the lithium treatment is begun concurrently with wounding.
54. The method of paragraph 2, wherein the lithium treatment is begun after skin has been wounded.
55. The method of paragraph 2 in which a second treatment is administered to the human subject receiving the lithium treatment.
56. The method of paragraph 55 in which the second treatment is integumental perturbation.
57. The method of paragraph 55 in which the second treatment is administered before the lithium treatment.
58. The method of paragraph 55 in which the second treatment is administered concurrently with the lithium treatment.
59. The method of paragraph 55 in which the second treatment is administered after the lithium treatment.
60. The method of paragraph 1, 44, 45, 48, or 56 in which the integumental perturbation removes the epidermis partially or completely.
61. The method of paragraph 1, 44, 45, 48, or 56 in which the integumental perturbation removes all of the epidermis and part of the dermis.
62. The method of paragraph 1, 44, 45, 48, or 56 in which the integumental perturbation does not remove the epidermis.
63. The method of paragraph 1, 44, 45, 48, or 56 in which the integumental perturbation comprises surgical excision of skin, serial expansion of skin, a skin graft, or combination thereof.
64. The method of paragraph 1, 44, 45, 48, or 56 in which the integumental perturbation is accomplished by laser, light, heat, microneedle rollers, a felt wheel, ultrasound, iontophoresis, electrophoresis, dermabrasion with diamond fraise, or radiation treatment, or a combination thereof.
65. The method of paragraph 1, 44, 45, 48, or 56 in which the integumental perturbation is accomplished by laser.
66. The method of paragraph 65 wherein the laser is a pulsed dye laser.
67. The method of paragraph 65 wherein the integumental perturbation by laser is fractional and non-ablative.
68. The method of paragraph 67, wherein the fractional, non-ablative integumental perturbation by laser is performed by use of an Erbium- YAG laser at 1500-1590 nm.
69. The method of paragraph 65 wherein the integumental perturbation by laser is fractional and ablative.
70. The method of paragraph 69, wherein the fractional, ablative laser integumental perturbation results in fractional ablation of the skin at a depth between 100 microns and 4000 microns into the skin.
71. The method of paragraph 69, wherein the fractional, ablative laser integumental perturbation results in fractional ablation of the skin at a depth approximating the depth of a full-thickness excision wound.
72. The method of paragraph 69, wherein the fractional, ablative laser integumental perturbation results in fractional ablation of the skin over an area of 1.5 cm x 1.5 cm to 15 cm x 15 cm.
73. The method of paragraph 69, wherein the fractional, ablative laser integumental perturbation results in fractional ablation of the skin at a depth density of the micro-thermal zones of the fractional ablation approximates that of a full bulk ablation of the entire area of treatment.
74. The method of paragraph 69, wherein the fractional, ablative laser integumental perturbation is by full bulk ablation, wherein the tissue of the entire area of treatment is ablated. 75. The method of paragraph 74, wherein the fractional, ablative laser integumental perturbation by bulk ablation is over an area of 1.5 cm x 1.5 cm to 15 cm x 15 cm.
76. The method of paragraph 74, wherein the fractional, ablative laser integumental perturbation by bulk ablation is accomplished at 10,600 nm using a carbon dioxide laser.
77. The method of paragraph 74, wherein the fractional, ablative laser integumental perturbation by bulk ablation is accomplished at 2940 nm using a Erbium- YAG laser.
78. The method of paragraph 65 wherein the integumental perturbation by laser is non- fractional and ablative.
79. The method of paragraph 78, wherein the non- fractional, ablative laser integumental perturbation is by full bulk ablation, wherein the tissue of the entire area of treatment is ablated.
80. The method of paragraph 78, wherein the non- fractional, ablative laser integumental perturbation by bulk ablation is over an area of 1.5 cm x 1.5 cm to 15 cm x 15 cm.
81. The method of paragraph 78, wherein the non- fractional, ablative laser integumental perturbation by bulk ablation is accomplished at 10,600 nm using a carbon dioxide laser.
82. The method of paragraph 78, wherein the non- fractional, ablative laser integumental perturbation by bulk ablation is accomplished at 2940 nm using a Erbium- YAG laser.
83. The method of paragraph 1, 44, 45, 48, or 56 in which the integumental perturbation is accomplished using a microneedle array.
84. The method of paragraph 83, wherein the microneedle array is in the form of a roller or flat plate.
85. The method of paragraph 83, wherein the microneedle array can disrupt a skin area of 1.5 cm x 1.5 cm to 15 cm x 15 cm.
86. The method of paragraph 83, wherein the microneedle array can disrupt skin at a depth of 100 microns to 4000 microns.
87. The method of paragraph 83, wherein the microneedle array has hollow needles. 88. The method of paragraph 83, wherein the microneedle array top has a luer-lock fitting that can accommodate a syringe to deliver drug.
89. The method of paragraph 88, wherein the volume of the syringe is 1 ml to 3 ml.
90. The method of paragraph 1, 44, 45, 48, or 56 in which the integumental perturbation is accomplished by injury.
91. The method of paragraph 90, wherein the mode of injury is mechanical.
92. The method of paragraph 91, wherein the mechanical injury is accomplished by microdermabrasion, dermabrasion, tape stripping, or full thickness excision of tissue.
93. The method of paragraph 90, wherein the mode of injury is thermal injury.
94. The method of paragraph 91, wherein the thermal injury is accomplished by laser.
95. The method of paragraph 1, 44, 45, 48, or 56 in which the integumental perturbation is accomplished by inducing inflammation.
96. The method of paragraph 95 in which the method of inducing inflammation is by application of an adjuvant.
97. The method of paragraph 96, wherein the adjuvant is selected from the group of sodium dodecyl sulfate, aluminum salts, monophosphoryl lipid A, and cetyl triammonium bromide (CTAB).
98. The method of paragraph 95 in which the method of inducing inflammation is injury to tissue.
99. The method of paragraph 95 in which the method of inducing inflammation is by application of an cytokine {e.g., IL-1 beta).
100. The method of paragraph 95 in which of inducing inflammation is by application of an antigen {e.g. tetanus toxoid).
101. The method of paragraph 1, 44, 45, 48, or 56 in which the method of integumental perturbation is accompanied by stem cell mobilization {i.e., G-CSF). 102. The method of paragraph 44 or 45 in which the third treatment stimulates hair growth.
103. The method of paragraph 46 or 47 in which the second treatment stimulates hair growth.
104. The method of paragraph 44 or 45 in which the third treatment is the administration of an anti-senescence agent.
105. The method of paragraph 46 or 47 in which the second treatment is the administration of an anti-senescence agent.
106. The method of paragraph 44 or 45 in which the third treatment is a surgical transplantation of hair follicles.
107. The method of paragraph 46 or 47 in which the second treatment is a surgical transplantation of hair follicles.
108. The method of paragraph 44 or 45 in which the third treatment inhibits hair growth.
109. The method of paragraph 46 or 47 in which the second treatment inhibits hair growth.
1 10. The method of paragraph 44 or 45, wherein the third treatment is with an agent that modulates wound healing.
1 1 1. The method of paragraph 46 or 47, wherein the second treatment is with an agent that modulates wound healing.
1 12. The method of paragraph 110, wherein the agent is an agent that enhances wound healing.
1 13. The method of paragraph 1 1 1, wherein the agent is an agent that enhances wound healing.
1 14. The method of paragraph 1 10, wherein the agent is an agent that slows wound healing.
1 15. The method of paragraph 11 1, wherein the agent is an agent that slows wound healing. 1 16. The method of paragraph 114 or 1 15, wherein the agent that slows wound healing is rapamycin, a corticosteroid, a prostaglandin inhibitor, an inhibitor of fibrin, an inhibitor of collagen, an inhibitor of myofibroblast activity, an anti-inflammatory agent.
1 17. The method of paragraph 110, wherein the agent is an agent that reduces scarring.
1 18. The method of paragraph 11 1, wherein the agent is an agent that reduces scarring.
1 19. The method of paragraph 116, wherein the anti-inflammatory agent is an NSAID or an antagonist of TNFa, TGF , NFkB, IL-1, IL-6, IL-8, IL-10, or IL-18.

Claims

WHAT IS CLAIMED IS:
1. A method for revising a scar in a human subject, comprising administering to a human subject in need thereof a treatment of (i) integumental perturbation of a scarred area by fractional laser; (ii) followed by a pulse of a lithium composition that delivers an effective amount of lithium ions, wherein the treatment improves the appearance of the scar or restores function of the scarred tissue.
PCT/US2010/048439 2009-09-11 2010-09-10 Intermittent and pulse lithium treatments for scar revision and wound healing WO2011031977A1 (en)

Applications Claiming Priority (8)

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US24185709P 2009-09-11 2009-09-11
US61/241,857 2009-09-11
US33025010P 2010-04-30 2010-04-30
US61/330,250 2010-04-30
US35653410P 2010-06-18 2010-06-18
US35653110P 2010-06-18 2010-06-18
US61/356,534 2010-06-18
US61/356,531 2010-06-18

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EP3551111A4 (en) * 2016-12-07 2020-06-24 Sciton, Inc. Laser treatment of wounds
US20210322785A1 (en) * 2018-12-10 2021-10-21 C.P. Medical Corporation Compositions and methods for treating wounds
US11207511B2 (en) 2010-12-06 2021-12-28 Follica, Inc. Methods for treating baldness and promoting hair growth

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US20070002497A1 (en) * 2001-10-30 2007-01-04 Toshihiko Shimizu Carriage arm assembly for locating magnetic head, and magnetic disk apparatus using the same

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US20020013298A1 (en) * 1996-12-02 2002-01-31 William L. Hunter Compositions and methods for treating or preventing inflammatory diseases
US20070002497A1 (en) * 2001-10-30 2007-01-04 Toshihiko Shimizu Carriage arm assembly for locating magnetic head, and magnetic disk apparatus using the same

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11207511B2 (en) 2010-12-06 2021-12-28 Follica, Inc. Methods for treating baldness and promoting hair growth
EP3551111A4 (en) * 2016-12-07 2020-06-24 Sciton, Inc. Laser treatment of wounds
US20210322785A1 (en) * 2018-12-10 2021-10-21 C.P. Medical Corporation Compositions and methods for treating wounds

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