WO2011050107A2 - Compositions and methods for delivery of therapeutic agents to the cns - Google Patents

Abstract

The present invention provides compositions and methods for sustained delivery of therapeutic agents to the CNS, using a novel implant material composed of fibrillar collagen coated with human laminin, allowing increased implant biocompatibility to sustain more efficient delivery of therapeutic agents to the CNS, which is an improvement in the field of sustained delivery of therapeutic agents to the CNS. This composition can be implanted in the CNS where needed and will persist for long times with minimal tissue reaction. The compositions are fibrillar collagen porous matrices coated with human laminin, to treat resection cavities of CNS tumors, which can be used to provide a sustained release delivery vehicle for therapeutic agents including chemotherapeutic drugs or tumor-targeting human cells from cultures, such as human neural progenitor cells.

Description

COMPOSITIONS AND METHODS FOR DELIVERY OF THERAPEUTIC AGENTS

TO THE CNS

Cross-Reference to Related Applications

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Serial No. 61/253,111, filed 20 October 2009, and U.S. Provisional Patent

Application Serial No. 61/306,405, filed 19 February 2010, and these applications are expressly incorporated herein by reference in their entirety.

Technical Field

[0002] This application pertains to compositions and methods in the field of body treatment. More particularly it concerns a collagen implant material as a pharmaceutical carrier or as a surgical prosthesis in the central nervous system (CNS).

Background Art

[0003] The CNS of mammals consists of the brain, spinal cord and associated structures. Neuronal and glial cells differentiate from neural stem/progenitor cells (NSC) which are responsible for producing the neurons, astrocytes and oligodendrocytes, which make up the bulk of the CNS along with endothelial and microglia cells (which are derived from hematopoietic stem cells and migrate into the CNS later in development) during embryonic development, and the tissues are essentially static after birth. Recently it has been appreciated that some rare NSC's still remain through adulthood although they are limited in number and location.

Dysfunction of the CNS due to disease, defect, or injury causes a wide variety of diseases in man and the CNS of the post-natal mammal has only limited capacity for repair or regeneration.

[0004] The CNS is an attractive target therefore for therapeutic agents to treat a wide variety of diseases, defects, or injuries but the delivery of therapeutic agents needed to treat these afflictions has proven challenging due to the low bioavailability of oral or injected drugs due to the blood-brain-barrier. This term represents the uniquely selective tight barrier to diffusion of many classes of drug entities through the endothelium to target CNS tissues for therapeutic benefit. Many drugs cannot be effectively delivered to the CNS unless directly injected or implanted, therefore compromising the blood-brain-barrier.

[0005] Therapeutic cells, such as but not limited to, cultured neural progenitor cells, and in general cells of the CNS which could be therapeutically effective, cannot be delivered through the blood stream as well, as these cells are not normally migratory there and do not seed into the brain when delivered intravenously with any significance.

[0006] The glial cells of the CNS, particularly astrocytes and microglia cells (which are macrophage-like) react to foreign objects and injuries through a process of inflammation and scar formation which eventually limit the efficacy of materials implanted in the CNS, and this leads to ineffective treatments. Regardless of whether these implants deliver a drug entity, (such as a chemotherapeutic agents, other small molecules, or peptides and proteins) or a therapeutic cell (such as a NSC or differentiated dopaminergic or motor neurons) glial cells react and form a scar which separates the implant from target CNS cells and tissues.

[0007] Implants in the CNS have been used, such as those composed of synthetic polymers which degrade over time and provide local delivery of therapeutic drugs, such as GLIADEL® which is GLIADEL® Wafers are small, dime-sized biodegradable polymer wafers that are designed to deliver BCNU or carmustine directly into the surgical cavity created when a brain tumor is resected. Immediately after a neurosurgeon operates to remove the high-grade malignant glioma, up to eight wafers are implanted along the walls and floor of the cavity that the tumor once occupied. Each wafer contains a precise amount of carmustine that dissolves slowly, delivering carmustine to the surrounding cells.

[0008] Although these implants are effective at delivering the drug, they have a number of drawbacks, including increased complications due in part to the tissue response to the synthetic implant materials chemical and physical properties. The incidence of brain abscess or meningitis was 4% in patients treated with GLIADEL® Wafer and 1% in patients receiving placebo. And both brain edema (4% increase from placebo) and intracranial hypertension (7% increase from placebo) are problems which limit the overall effectiveness of the therapeutic agent.

[0009] Previously others have attempted to use extracellular matrix (ECM) molecules such as collagen and laminin to enhance relatively low engraftment efficiency of cultured neural cells (Tate, et ah, J Tissue Eng Regen Med. 2009 Mar;3(3):208-17). Various collagen implants have been known and collagen has been used as a coating to improve device biocompatibility, however it was not appreciated that the use of laminin coated collagen could enhance biocompatibility and sustained delivery to CNS tissues. Similarly, collagen gels have been used to deliver therapeutic agents, but not generally to the CNS.

[0010] There is a clear need for new compositions for enhanced delivery of therapeutic agents to the CNS, and new methods of use for treating patients with disease or dysfunction of the CNS.

[0011] The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety.

Summary of the Invention

[0012] The present disclosure provides methods and compositions for delivering therapeutic agents to treat CNS disorders.

[0013] Provided herein is an implantable composition comprising a fibrillar collagen and a laminin. The composition may further comprise a nidogen-1. The collagen may be a human collagen, a bovine collagen or a rodent collagen. The collagen, e.g., human collagen, may be purified from a cultured cell or derived from a recombinant DNA process. The laminin may be a human laminin. The laminin, e.g. , human laminin, may be purified from an epithelial cell.

[0014] In one embodiment, the collagen, e.g. , human collagen, comprises about 90-95% type I collagen and about 5-10% type III and/or type V collagen. In another embodiment, the collagen, e.g. , human collagen, is derived from a partially-processed cadaveric tissue.

[0015] In one embodiment, the purified laminin, e.g. , human laminin, comprises a laminin with a beta-1 chain, a gamma- 1 chain, and an alpha chain selected from the group consisting of alpha-1, alpha-2, alpha-3, alpha-4 and alpha-8 chain. In another embodiment, the laminin, e.g. , human laminin, is selected from the group consisting of laminin-111 and laminin- 1.

[0016] In one embodiment, the implantable composition comprises about 10 to about 120 mg/ml collagen (weight/volume). In another embodiment, the implantable composition comprises about 60 to about 100 mg/ml collagen. In yet another embodiment, the implantable composition comprises less than about 10% water by weight/weight. In still another embodiment, the implantable composition comprises less than about 1% water by

weight/weight. [0017] The implantable composition may form a porous matrix or a paste-like material. In one embodiment, the implantable composition is a semi-solid material at ambient temperature. In another embodiment, the implantable composition is a solid material at ambient temperature. The solid implantable composition may have a tensile strength of about 0.6 MPa to about 16 MPa. In yet another embodiment, the solid implantable composition has a tensile strength of about 1 MPa to about 4 MPa. In still another embodiment, the solid implantable composition has a tensile strength of about 2 MPa to about 16 MPa.

[0018] The implantable composition may comprise an effective amount of a therapeutic agent for delivery of the therapeutic agent to the CNS in a mammal. In one embodiment, the implantable composition provides a sustained release of the therapeutic agent. In another embodiment, the implantable composition provides a sustained release of the therapeutic agent after surgical implantation or injection. In yet another embodiment, the implantable

composition further comprises a pharmaceutically acceptable carrier.

[0019] In one embodiment, the therapeutic agent may comprise a small molecule drug, a chemotherapy drug, or an antimicrobial drug. In another embodiment, the therapeutic agent may also comprise a stem cell such as a human neuronal progenitor cell, or a cell derived from the CNS of a mammal such as a mesenchymal cell. In yet another embodiment, the therapeutic agent may comprise an antibody, an antibody fragment, or a combination thereof. In still another embodiment, the therapeutic agent may comprise a nucleic acid or a combination thereof, or a viral particle for gene delivery.

[0020] In another aspect, provided herein is a method of making an implantable

composition, which method comprises incubating a fibrillar collagen with a laminin. The fibrillar collagen may be made by neutralizing a collagen followed by dehydration of the neutralized collagen, wherein the dehydration of the neutralized collagen may occur at about 10°C to about 50°C, preferably at about 37°C.

[0021] Also provided herein is a method of making an implantable composition, which method comprises: a) neutralizing a collagen; b) dehydrating the neutralized collagen into a fibrillar collagen; c) incubating the fibrillar collagen with a laminin; and d) dehydrating the fibrillar collagen-laminin mixture. Dehydration of the neutralized collagen may occur at about 37°C and the dehydration of the fibrillar collagen-laminin mixture may last about 48-72 hours. [0022] In one embodiment, the collagen is an atelopeptide collagen. In another embodiment, the atelopeptide collagen is neutralized with disodium phosphate. In yet another embodiment, the atelopeptide collagen is neutralized with disodium phosphate at about 0.02M. In still another embodiment, the atelopeptide collagen is neutralized to about pH 7-8.

[0023] The laminin may be adsorbed by the fibrillar collagen, which may be facilitated by calcium.

[0024] The neutralized collagen may be dehydrated by centrifugation, incubation or lyophilization. In one embodiment, the fibrillar collagen-laminin mixture is dehydrated into a semi-solid material or a solid material at ambient temperature. In another embodiment, the fibrillar collagen-laminin mixture is lyophilized in the presence of a therapeutic agent.

[0025] Further provided herein is an implantable composition made by the method comprising: a) neutralizing a collagen; b) dehydrating the neutralized collagen into a fibrillar collagen; c) incubating the fibrillar collagen with a laminin; and d) dehydrating the fibrillar collagen-laminin mixture.

[0026] In yet another aspect, provided herein is a method of making an implantable therapeutic composition, which method comprises mixing an implantable composition comprising a fibrillar collagen and a laminin with an effective amount of a therapeutic agent. The therapeutic agent may be seeded directly to the implantable composition. Alternatively, the implantable composition may be lyophilized in the presence of the therapeutic agent. In one embodiment, the therapeutic agent is a cell. In another embodiment, the therapeutic agent is selected from a group consisting of a small molecule drug, a chemotherapy drug, a

macromolecule drug, a polypeptide, a protein, a peptide, a growth factor, an antibody, a viral particle, a polynucleotide, a nucleic acid, a lipid, a carbohydrate, a sugar and a combination thereof.

[0027] In still another aspect, provided herein is a method of treating a disease in a subject, which method comprises administrating an effective amount of an implantable composition comprising a fibrillar collagen and a laminin to the CNS of the subject. The implantable composition may be surgically-implanted or injected into the subject. In one embodiment, the disease is a CNS disease. In another embodiment, the subject may be a mammal, preferably a human being. [0028] In one embodiment, the implantable composition is further contacted with a non- biological device. In another embodiment, the non-biological device is selected from a group consisting of an electrode lead, a wire and a surgical device used in the CNS. In yet another embodiment, the implantable composition is applied to the resection cavity of a tumor postsurgical removal of the tumor.

[0029] Further provided herein is a method to inhibit scarring and/or reduce pain in a subject, which method comprises administrating an effective amount of an implantable composition comprising a fibrillar collagen and a laminin to the CNS of the subject. In one embodiment, the scarring results from a surgery, such as a discectomy or a laminectomy.

[0030] Still further provided herein is a method of conducting experiments in an animal, which method comprises administrating an effective amount of an implantable composition comprising a fibrillar collagen and a laminin to the CNS of the animal. In one embodiment, the animal may be a rodent model of a CNS disease or condition for research use or clinical studies.

[0031] In a further aspect, provided herein is a kit comprising an implantable composition comprising a fibrillar collagen and a laminin, a container for the implantable composition, and/or an instruction on how to use the implantable composition.

Brief Description of the Drawings

[0032] Fig. 1 - Morphological and immunocytochemical characterization of neural stem cells derived from the brain of three week old C57BL/6 mice. (A) Phase contrast image of a typical neurosphere culture in presence of 20 ng/ml FGF-2 and EOF. (B) The majority of cells displayed immunoreactivity for nestin (red). (C) Multipotency was demonstrated by the differentiation into GFAP-positive astrocytes (green), MAP2 -positive neuronal cells (red) and (D) GalC-positive oligodendrocytes (green). Cells were counterstained with DAPI. Bar: (A) 100 μιη, (B and D) 50 μιη, (C) 20 μιη.

[0033] Fig. 2 - In vitro culture of 3DECM-embedded neural stem cells. Phase contrast images at the rim of a NSC-loaded 3DECM implant demonstrated the distribution of single NSC around collagen fibers within the 3DECM immediately after loading (A). (B) Seven days after in vitro culture under neural stem cell conditions numerous neurospheres and (C) cell planes between fibers were apparent (D) distributing throughout the 3DECM implant. H&E stained cryosections of 3DECM implants seven days after NSC loading demonstrated a growth pattern of NSC around collagen fibers as multiple cell layers or neurosphere-like clusters (E, F). (G) The expression of the proliferation marker MIB-5 (brown cell nuclei) indicated a high proliferation rate of NSC. (H) Nestin immunoreactivity (red) confirmed the uncommited state of the 3DECM-embedded NSC. (I) Astroglial differentiation as demonstrated by GFAP immunoreactivity (green) was a rare event (<1 ). Cells in H and I were counterstained with DAPI. Bar: (A, B, C, E, F) 100 μιη, (D) 1 mm, (G) 50 μιη.

[0034] Fig. 3 - Glioma targeted migration of 3DECM-embedded NSC after intracerebral transplantation. (A, B) Tumor tropism of NSC out of 3DECM implants was assessed by administration of implants into the contralateral cortex of growing GL261 or NCE-G55 glioma. (C) One week after surgery Dil-labeled NSC (red) migrated out of the 3DECM implant, enriched in the adjacent surrounding brain parenchyma and migrated towards glioma growing in the contralateral hemisphere. Dil-labeled (red) and GFP-expressing (green) NSC were observed throughout the tumor mass of GL261- (D) and NCE-G55 glioma (E). (F) In control animals with no tumor Dil-labeled stayed at the adjacent brain parenchyma surrounding the 3DECM implant and did not display any distant migration. (G, H) The plasticity of 3DECM implants ensured a close contact to the surrounding brain parenchyma as demonstrated by H&E counterstained cryosections at the transplantations site. Sections (B-F) were counterstained with DAPI. Bars: 100 μιη.

[0035] Fig. 4 - Transplantation of 3DECM-embedded NSC into the surgical resection cavity. (A) Microsurgical resection of human NCE-G55 glioblastoma xenografts was immediately followed by intracavitary administration of NSC within a 3DECM implant. (B) Residues of 3DECM containing GFP-expressing NSC (green) were found in the former tumor resection cavity. (C) GFP-expressing NSC (green) have migrated out of the 3DECM enriching in the border zone of the recurrent tumor and adjacent brain parenchyma. (D) Furthermore, GFP- expressing NSC were observed throughout the recurrent tumor mass regrown into the resection cavity. Sections were counterstained with DAPI. Bars: 100 μιη.

Detailed Description of the Invention

[0036] The present invention is based, in part, on our studies on a three-dimensional(3D) ECM as a delivery system for the transplantation of glioma targeting NSCs. Specifically, we developed a 3DECM which is based on ECM purified from tissue-engineered skin cultures containing laminin-coated collagen fibers. The 3DECM preparation enabled the in vitro expansion of NSC encased within the 3DECM while retaining their uncommited differentiation status. When implanted in intracerebral glioma models NSC were able to migrate out of the 3DECM to targeted glioma growing in the contralateral hemisphere or to recurrent glioma when implanted into a tumor resection cavity. In absence of tumor the 3DECM-embedded NSC did not display any distant migration. The semisolid consistency of the 3DECM implants allowed simple handling during the surgical procedure of intracerebral and intracavitary application and ensured a continuous contact with the surrounding brain parenchyma. We demonstrated proof - of-concept of a matrix supported intracerebral and intracavitary transplantation of glioma targeting NSC. In contrast to injections of cell suspensions intracerebral application of NSC within a semisolid 3DECM has the potential to increase transplantation efficiency by reducing metabolic stress and providing mechanical support especially when administered in a surgical resection cavity.

[0037] Accordingly, the invention provides methods and compositions for delivering therapeutic agents to treat CNS disorders.

[0038] In one aspect, there are provided implantable compositions comprising a fibrillar collagen and a laminin.

[0039] In another aspect, there are provided methods of making an implantable composition, which method comprises incubating a fibrillar collagen with a laminin.

[0040] In yet another aspect, there are provided methods method of making an implantable therapeutic composition, which method comprises mixing an implantable composition comprising a fibrillar collagen and a laminin with an effective amount of a therapeutic agent.

[0041] In a further aspect, there are provided methods of treating a disease in a subject, which method comprises administrating an effective amount of an implantable composition comprising a fibrillar collagen and a laminin to the CNS of the subject.

[0042] The invention further provides systems and kits useful for methods described herein.

Definitions

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, patent applications (published or unpublished), and other publications referred to herein are incorporated by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.

[0044] As used herein, "a", "an", and "the" can mean singular or plural (i.e., can mean one or more) unless indicated otherwise.

[0045] As used herein, "collagen" refers to a group of naturally occurring proteins. The tropocollagen or "collagen molecule" is a subunit of larger collagen aggregates such as fibrils. It is approximately 300 nm long and 1.5 nm in diameter, made up of three polypeptide strands (called alpha chains), each possessing the conformation of a left-handed helix. A distinctive feature of collagen is the regular arrangement of amino acids, called atelopeptides, in each of the three chains of these collagen subunits. The sequence often follows the pattern Gly-Pro-Y or Gly-X-Hyp, where X and Y may be any of various other amino acid residues. Proline or hydroxyproline constitute about 1/6 of the total sequence. The tropocollagen subunits spontaneously self-assemble, with regularly staggered ends, into even larger arrays in the extracellular spaces of tissues. There is some covalent crosslinking within the triple helices, and a variable amount of covalent crosslinking between tropocollagen helices forming well organized aggregates (such as fibrils). Collagen fibrils are semi-crystalline aggregates of collagen molecules. Collagen fibers are bundles of fibrils.

[0046] As used herein, the term "stem cell" refers to a cell that possess two properties: (1) the ability to self-renewal, or the ability to go through numerous cycles of cell division while maintaining the undifferentiated state, and (2) a high level of potency, or the capacity to differentiate into specialized cell types. Stem cells may have different levels of potency, which are described by different terms. Totipotent stem cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent. These cells can differentiate into embryonic and extraembryonic cell types. Pluripotent stem cells are the descendants of totipotent cells and can differentiate into cells derived from any of the three germ layers. Embryonic stem (ES) cells, cells that derive from the inner cell mass (ICML) of a blastocyst, are pluripotent stem cells. Multipotent stem cells can produce only cells within one particular lineage (e.g. , hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.). Unipotent cells can produce only one cell type, but have the property of self -renewal which distinguishes them from non-stem cells (e.g. , muscle stem cells). Multipotent stem cells and unipotent cells are also referred to as progenitor cells.

[0047] As used herein, a "pharmaceutically acceptable carrier" refers to any substance or vehicle suitable for delivering a yeast vaccine of the present invention to a suitable in vivo or ex vivo site. Such a carrier can include, but is not limited to, an adjuvant, an excipient, or any other type of delivery vehicle or carrier. Adjuvants are typically substances that generally enhance the immune response of an animal to a specific antigen. Suitable adjuvants include, but are not limited to, Freund's adjuvant; other bacterial cell wall components; aluminum-based salts;

calcium-based salts; silica; polynucleotides; toxoids; serum proteins; viral coat proteins; other bacterial-derived preparations; gamma interferon; block copolymer adjuvants, such as Hunter's Titermax adjuvant (CytRx™, Inc. Norcross, GA); Ribi adjuvants (available from Ribi

ImmunoChem Research, Inc., Hamilton, MT); and saponins and their derivatives, such as Quil A (available from Superfos Biosector A/S, Denmark). Adjuvants are not required in the yeast vaccine of the present invention, but their use is not excluded. Carriers are typically compounds that increase the half-life of a therapeutic composition in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release formulations, biodegradable implants, liposomes, oils, esters, and glycols.

[0048] The term "about" as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0049] It is understood that aspects and embodiments of the invention described herein include "consisting" and/or "consisting essentially of aspects and embodiments.

[0050] Other objects, advantages and features of the present invention will become apparent from the following specification taken in conjunction with the accompanying drawings.

Implantable compositions made of fibrillar collagen and laminin

[0051] Provided herein are implantable compositions comprising a fibrillar collagen and a laminin. The implantable composition may form a porous matrix or a paste-like material.

Collagen is thermally reversible and becomes more viscous with increasing temperatures. [0052] Accordingly, in some embodiments, there is provided an implantable composition that is a semi-solid material or a solid material at ambient temperature. In some embodiments, the solid implantable composition has a tensile strength of about 0.6 MPa to about 16 MPa, a tensile strength of about 1 MPa to about 4 MPa, or a tensile strength of about 2 MPa to about 16 MPa.

[0053] The collagen used in the implantable composition may be derived from a number of mammalian sources, including, but not limited to, human, bovine and rodent. The collagen may also be purified from a cultured cell line or from a partially-processed cadaveric tissue, or using recombinant DNA techniques.

[0054] Purified human laminin may be used in the implantable composition. As disclosed in Tate, et al. J Tissue Eng Regen Med. 2009 Mar;3(3):208-17, collagen and laminin based scaffolds enhance neural stem cell transplantation into the injured brain. In some embodiments, human laminin is purified from human epithelial cells, and comprises a laminin with a beta-1 chain, a gamma- 1 chain, and an alpha chain selected from the group consisting of alpha- 1, alpha-2, alpha-3, alpha-4 and alpha-8 chain. In other embodiments, the human laminin is selected from the group consisting of laminin- 111 and laminin- 1.

[0055] In some embodiments, there is provided an implantable composition comprising a fibrillar collagen and a laminin, wherein the implantable composition further comprises a nidogen-1. As disclosed in Pujuguet, et al. J. Cell Sci. 2000 113:849-858, nidogen-1 regulates laminin- 1 dependent mammary-specific gene expression by epithelial cells.

[0056] Also provided are implantable compositions that further comprise an effective amount of a therapeutic agent for delivery of the therapeutic agent to the CNS in a mammal. Accordingly, in some embodiments, the implantable composition provides a sustained release of the therapeutic agent. In some embodiments, the implantable composition provides a sustained release of the therapeutic agent after surgical implantation or injection.

[0057] In some embodiments, there is provided an implantable composition for delivery of cultured cells, such as transgenic human neural progenitor cells which can home to any remaining tumor elements missed by the surgeon, or to metastases which remain after the bulk of the tumor is removed, or for any reason thereafter, to allow a more efficient delivery and persistence of the cells for an enhanced therapeutic benefit, including delay of mortality or decreased morbidity of a patient. See, e.g. , Tate, et al. J Tissue Eng Regen Med. 2009

Mar;3(3):208-17.

[0058] The therapeutic agent used herein may be a small molecule drug, a chemotherapy drug, or an antimicrobial drug. The therapeutic agent used herein may also be a stem cell or a cell derived from the CNS of a mammal. The porosity of the implantable composition can be optimized for the particular therapeutic agent, such as, by way of non-limiting example, a small molecule drug such as carmustine which will require smaller pores (approximately lOnm up to lOOOnm) than a cultured cell (approximately 1 micron to 1000 microns or 1mm). Persons skilled in the art can appreciate the adjustment of pore size to fit the physico-chemical properties of the therapeutic agent that is to be delivered.

Methods for of making an implantable composition

[0059] The present invention in another aspect provides a method of making an implantable composition, which method comprises: a) neutralizing a collagen; b) dehydrating the neutralized collagen into a fibrillar collagen; c) incubating the fibrillar collagen with a laminin; and d) dehydrating the fibrillar collagen-laminin mixture.

[0060] Accordingly, in some embodiments, there are provided methods of making an implantable composition by neutralizing an atelopeptide collagen followed by dehydration to form a porous matrix of fibrillar collagen. The fibrillar collagen is dehydrated to obtain the desired porosity by, as non-limiting examples, centrifugation, heat drying at temperatures of about 50°C to about 37°C, lyophilization, or freeze-drying. It is an important aspect of the current invention to remove most of the water bound to the collagen fibrils, and increase the percentage of fibrillar collagen to provide a material with physical properties which allow for both the required manufacturing processes to bind high amounts of therapeutic agents, while maintaining a tissue-like consistency, that facilitates surgical implantation and long-term persistence in a patient's CNS. In some embodiments, the implantable composition comprises about 10 to about 120 mg/ml, preferably about 60 to about 100 mg/ml, collagen

(weight/ volume). In other embodiments, the implantable composition comprises less than about 10%, preferably less than about 1%, water (weight/weight).

[0061] Methods of making fibrillar collagen are known in the art, such as disclosed in U.S. Patent No. 4,424,208 and U.S. Patent No. 6,551,618. As a non-limiting example, suitable starting materials can be atelopeptede collagen at 1-3 mg/ml in 0.012N HC1 solution at pH ~2. Once the collagen fibers form after neutralization of pH with disodium phosphate and incubation at 37°C, dehydration can be accomplished by centrifugation of the fibre mass at 10,000g until a concentration of 10-50mg/ml is reached. Buffer and aqueous phase supernatant is removed by aspiration or "dumping" where the pelleted collagen fibres now form a paste-like material.

[0062] Laminin is incubated with the fibrillar collagen to achieve a fibrillar collagen-laminin mixture. In some embodiments, the laminin is adsorbed by the fibrillar collagen. In other embodiments, the adsorption is facilitated by calcium.

Methods of treating a disease in a subject

[0063] The present invention in another aspect provides methods of treating a disease in a subject, which method comprises administrating an effective amount of an implantable composition comprising a fibrillar collagen and a laminin to the CNS of the subject.

[0064] Suitable diseases that can be treated in the present invention include a variety of disorders or conditions of the CNS, including but not limited to cancer, Parkinson's disease, Alzheimer's disease, Huntington disease, stroke, retinal degeneration, spinal cord injury, ALS, and age-related CNS dysfunction.

[0065] Accordingly, in some embodiments, there are provided methods of treating a disease in a subject, which method comprises surgically-implanting or injecting the implantable composition into the subject. In some embodiments, the implantable composition is further contacted with a non-biological device. As a non-limiting example, electrodes for

neurostimulation can be coated with the implantable composition to improve electroconduction and biocompatibility. As another non-limiting example, essentially any non-biological material which is to be implanted for more than 1 day in the CNS of a patient can be improved by coating with the implantable composition. Anyone skilled in the art can appreciate that a wide variety of devices which contact the CNS of a patient would be improved by the present invention, and that many embodiments exist which could be used regarding the size, shape, and weight required to combine or coat these surgical and medical devices with the present invention.

[0066] The implantable composition can also by lyophilized to obtain a solid material with mechanical characteristics for surgical uses, such as post-surgical dural replacement or spinal discectomy or laminectomy forming a mechanical barrier to cover the peridural space to block the migration of cells from superficial layers to the epidural space, which can prevent or decrease scar formation. The lyophilized implant material can be sutured in place, and may have the desired strength characteristics to act as prosthesis for dural defects with a tensile strength of about 0.6 MPa to about 16 MPa. A preferred embodiment of the invention may have a tensile strength of about 1 MPa to about 4 MPa for the onlay materials, and about 2 MPa to about 16 MPa is preferred for the sutureable version of the invention. In addition, the elongation for these particular materials may be about 17% to about 40%. This elongation value seems to provide sufficient extensibility when subjected to a suturing under tension. The material has also been found to have minimal leakage of fluid when pressure is applied thereto. These implants have also been show to have an average burst strength value of about 20N to about 30N.

[0067] Further provided in the invention are methods of conducting experiments in an animal, which method comprises administrating an effective amount of an implantable composition comprising a fibrillar collagen and a laminin to the CNS of the animal. The animal may be a rodent model of a CNS disease or condition for research use or clinical studies.

Kits

[0068] The invention also provides kits for various methods described herein.

[0069] For example, in some embodiments, there is provided a kit containing an implantable composition comprising a fibrillar collagen and a laminin, and a container for the composition. The kit may further comprise an instruction or user manual detailing preferred methods of performing the methods of the invention, and/or a reference to a site on the Internet where such instructions may be obtained.

Materials and Methods

Cell Culture

[0070] The human glioblastoma cell line NCE-G55 (Kunkel P, Ulbricht U, Bohlen P, et al. Cancer Res. 2001 ;61:6624-6628) and the murine glioma cell line GL261 (DCTDC Tumor Repository, MD, USA) were cultured in Dulbecco modified Eagle medium (DMEM)

(Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal calf serum (FCS, Invitrogen). Cells were maintained in T-75 tissue culture flasks in 5% C02/95% air at 37 °C in a humidified incubator and were routinely passaged at confluency. For the intracranial implantation experiments, glioma cells were dispersed with a 0.05% solution of trypsin/EDTA (Invitrogen), washed with phosphate -buffered saline (PBS) and adjusted to the final concentration in PBS.

Culture, Labeling and Differentiation of Neural Stem Cells

[0071] Neural stem cells (NSC) were harvested from the frontoparietal brain of 3 week old C57BL/6 mice as described previously (Chojnacki A, Weiss S. Nat Protoc. 2008;3:935-940). The cells were grown as neurospheres in complete NSC growth medium containing neurobasal medium (Invitrogen) with B27 supplement (20 μΐ/ml; Invitrogen), Glutamax (10 μΐ/ml, Invitrogen), fibroblast growth factor-2 (20 ng/ml, FGF-2, Peprotech, Rocky Hill, NJ, USA), epidermal growth factor (20 ng/ml, EGF, Peprotech) and heparin (32 IE/ml, Ratiopharm, Ulm, Germany). Growth factors and heparin were refreshed twice weekly. Neurospheres were routinely split by mechanical dissociation when reaching a size of 200-500 μιη. EGFP- expressing NSC were established by retroviral transduction with pMSCV-EGFP using a commercially available kit (MSCV Retroviral Expression System, BD Biosciences, Heidelberg, Germany). NSC labeling using the lipophilic tracer Dil (Molecular Probes, Eugene, OR, USA) was performed for 30 minutes according to the manufacturer's protocol. Freshly dissociated NSC were differentiated in eight-chamber Lab Tec-slides (Nalge Nunc International, Rochester, NY, USA) at 5000 cells per well. Differentiation medium consisted of neurobasal medium with B27 supplement (20 μΐ/ml), Glutamax (10 μΐ/ml), 10% FCS (all from Invitrogen), ImM retinoic acid and 10 mg/ml cAMP (both from Sigma- Aldrich).

Preparation of 3-Dimensional Extracellular Matrix (3DECM)

[0072] Human extracellular matrix purified from tissue-engineered skin cultures (Contard P, Jacobs L, II, Perlish JS, Fleischmajer R. Cell Tissue Res. 1993;273:571-575; Slivka SR, Landeen LK, Zeigler F, Zimber MP, Bartel RL. / Invest Dermatol. 1993;100:40-46) was used to produce 3DECM implants. Implants were fabricated starting with atelopeptide collagens 0.012N HC1 pH -2.0 at a final concentration of 3.0 mg/ml. After neutralization to physiological pH (-7.5) by the addition of sodium phosphate buffer, collagen fibrils were allowed to form after incubation at 37°C for 16 to 24 hours. The resulting collagen fibers were centrifuged at approximately 10,000g for 10 minutes, then the supernatant was removed, and the pellet was washed once in 1ml of DMEM/F12 (Invitrogen) with N2 supplement (Invitrogen), and 0.1% (w/v) bovine serum albumin (Sigma-Aldrich) by centrifugation again at 10,000g for 10 minutes. The resulting pellet was then allowed to partially dry overnight by incubation in a 37°C oven. Depending on the grade of hydration the 3DECM could be prepared in various degrees of consistency ranging from semi-fluid to hard. In order to enhance cellular adhesion, the 3DECM was then coated with 200 μg/ml purified laminin from EHS sarcoma (Sigma Aldrish, St. Louis, MO, Catalogue #L2020) at 37°C overnight, which consists primarily of laminin-1 and nidogen-1 complexes.

In vivo Studies

[0073] Orthotopic glioblastoma xenografts were established in 4-6 week old NMRI-nu/nu or in C57BL/6 mice for the syngenic glioma model (both Charles River, Sulzfeld, Germany). Mice were anesthetized (100 mg/kg ketamine and 5 mg/kg xylazine) and received a

stereotactically-guided injection of 4.5 x 104 human NCE-G55 glioblastoma cells or of 1.4 x 105 murine GL261 glioma cells into the left forebrain (2 mm lateral and 1 mm anterior to bregma, at a 2.5 mm depth from the skull surface). Eight days after tumor cell injection mice were anesthetized and a ~2 x 2 mm cortical incision into the contralateral right forebrain was performed. One NSC-loaded 3DECM implant was placed into the cortical incision. Control groups received NSC-loaded 3DECM implants in absence of a glioblastoma xenograft. In order to mimic the clinical scenario of a glioma resection we also used the recently developed glioma surgical resection model as described previously (Schmidt NO, Ziu M, Carrabba G, et al. Clin Cancer Res. 2004; 10: 1255-1262). Briefly, 12 days after NCE-G55 cell injection, established human glioblastoma xenografts were surgically removed using a microsurgical technique. At that time, some degree of tumor cell invasion had occurred resulting in the formation of small tumor extensions and satellites distant from the main tumor mass. Microsurgical removal was pursued until clear resection margins were visible. After achieving hemostasis, one NSC-loaded 3DECM was placed into the resection cavity. Skin incisions were closed by suturing. All animals were anesthetized and perfused with 4% paraformaldehyde 16 days after tumor cell injection. Brains were removed, embedded in OCT, and stored at -80°C until further processed for histological analysis. All animal studies were performed in accordance with institutional guidelines.

Immunohistochemistry

[0074] Frozen brains and 3DECM embedded in OCT were cut in serial 10 μιη sections and counterstained with hematoxylin and eosin (H&E) or DAPI for histological evaluation. Cells or frozen sections were fixed with 4% paraformaldehyde and permeabilized with 3% Triton X-100 in PBS (except for GalC staining) and blocked with 5% horse serum. Primary antibodies were mouse anti-nestin (1 :500; BD Biosciences), mouse anti-MAP2 (1:50; Chemicon, Temecula, CA, USA), mouse anti-NF (1:50; Dako, Glostrup, Denmark), rabbit anti-GFAP (1 :40; Dako), mouse anti-GalC (1 : 100; Chemicon) and mouse anti-MIB5 (1:50; Dako). After incubation for 90 min slides were washed with 5% horse serum. Secondary antibodies, donkey anti-mouse IgG rhodamine (1 :50; Chemikon) and donkey anti-rabbit IgG fluorescein (1 :50; Chemicon) were added for 30 min. For MIB5 staining we used the DAKO EnVision™+ System HRP kit. Slides were mounted using Vectashield Hard Set mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). For double staining, mouse antibodies were added first for 90 min, followed by the addition of the rabbit antibody for 60 min and simultaneous detection with secondary antibodies. Negative control slides were obtained by omitting the primary antibody. The proliferation index was quantified by counting the number of positively MIB-5 stained cells of 100 nuclei in five randomly chosen high-power fields.

[0075] The following examples are offered to illustrate but not to limit the invention.

Example 1

Process for producing implants from collagen

[0076] Human collagen purified from tissue-engineered skin cultures (Advnanced

Biomatrix, San Diego, CA) was used to produce 3DECM implants. Implants were fabricated starting with atelopeptide collagens 0.012N HC1 pH 2.0 at a final concentration of 3.0 mg/ml. After neutralization to physiological pH (7.5) by the addition of sodium phosphate buffer, collagen fibrils were allowed to form after incubation at 37°C for 16 to 24 hours. The resulting collagen fibers were centrifuged at approximately 10,000g for 10 minutes, then the supernatant was removed, and the pellet was washed once in 1ml of DMEM/F12 (Invitrogen) with N2 supplement (Invitrogen), and 0.1% (w/v) bovine serum albumin (Sigma- Aldrich) by

centrifugation again at 10,000g for 10 minutes. The resulting pellet was then allowed to partially dry overnight by incubation in a 37°C oven. Depending on the grade of hydration the 3DECM could be prepared in various degrees of consistency ranging from semi-fluid to hard. In order to enhance cellular adhesion, the 3DECM was then coated with 200 μg/ml purified laminin from EHS sarcoma (Sigma-aldrich Cat#L-2020) at 37°C overnight, which consists primarily of laminin- 1 and nidogen-1 complexes.

Example 2

In vitro Culture and Characterization of 3DECM-embedded NSC

[0077] Primary neural stem cells (NSC) used in this study were isolated from the frontoparietal brain of three week old C57BL/6 mice. Cells grew primarily as neurospheres (Fig. 1A) and cultures became expandable for more than 50 passages. The neurospheres expressed the stem cell marker musashi-1 (data not shown) and nestin (Fig. IB) while lacking the expression of mature markers of glial and neuronal lineage (data not shown). After one week under culture conditions favoring differentiation numerous cells were immunoreactive for the astoglial marker GFAP, the neuronal marker MAP2 (Fig. 1C) or the oligodendroglial marker GalC (Fig. ID) indicating multipotency.

[0078] We developed a 3DECM preparation soft enough to allow a simple cell loading procedure while still having a consistency hard enough to ensure practical handling during a surgical procedure. Loading of 3DECM for a final implant volume of 5-8 mm³ was performed by adding 3.5 x 105 NSC, followed by centrifugation at 1500g for 5 minutes to encase NSC with the 3DECM while simultaneously forming an implant which could be handled with forceps. Immediately after the loading procedure the majority of NSC were found as single cells adherent to fibers within the 3DECM (Fig. 2A). 3DECM implants containing NSC were cultured separately in 24- well plates with complete neural stem cell growth medium under the same conditions used for normal neurosphere cultures. Within 7 days NSC embedded in a 3DECM implant formed multiple neurospheres (Fig. 2B) and were spreading between the collagen fibers as cell planes (Fig. 2C) indicating that the 3DECM had no negative impact on the cells. The hole 3DECM implant was interspersed with NSC (Fig. 2D) while keeping a consistency which allows surgical handling necessary for transplantation procedures. The collagen fibers within the matrix were the origin for NSC growth and the formation of neurosphere-like clusters (Fig. 2E, F). The proliferation rate of NSC embedded in 3DECM implants was 39% + 4.9 (mean + standard deviation, n=4) indicating that diffusion of nutrients was possible throughout the 3DECM implant allowing the rapid expansion of cells (Fig. 1G). Since the 3DECM is meant as a delivery system of uncommitted and motile NSC we next asked whether the 3DECM is changing the differentiation status of the encased NSC. Like in the primary neurosphere culture the majority of NSC embedded in 3DECM displayed immunoreactivity for nestin (Fig. 1H) while being negative for mature neuronal (NF and MAP2) and oligodendroglial marker (GalC). Only occasionally (<1%) GFAP positive cells were found (Fig. II).

Example 3

Intracerebral Transplantation of 3DECM-embedded NSC

[0079] In order to assess if 3DECM-embedded NSC are able to migrate out of the 3DECM implant and to target a growing glioma we transplanted a NSC containing 3DECM into the right murine forebrain in presence of a glioma in the left contralateral hemisphere (Fig. 3A, B). 3.5 x 105 Dil-labeled or GFP-expressing NSC were loaded to an implant volume of ~5 mm³ and cultured in complete NSC growth medium overnight. NSC- containing 3DECM implants were transplanted the following day to the hemisphere contralateral to a growing glioma using the syngenic murine glioma cell line GL261 in C57BL/6 mice (n=3) or the human glioblastoma NCE-G55 cell line in nude mice (n=3). One week after intracerebral 3DECM application NSC migrated out of the implant (Fig. 3C) towards the GL261 or NCE-G55 glioma (Fig. 3D, E). Dil- labeled or GFP-expressing cells were dispersed throughout the whole tumor mass. Apart from the 3DECM transplantation site and the tumor mass no NSC were observed elsewhere indicating that the 3DECM-embedded NSC were able to receive chemotactic signals guiding the NSC towards a growing tumor mass. In control animals without any tumor (n=2) Dil-labeled NSC stayed at the transplantation site and enriched in the contact zone of brain parenchyma and 3DECM (Fig. 3F). No NSC were detected in the contralateral hemisphere. The consistency of the 3DECM implants enabled a close contact with the adjacent brain parenchyma and in the immuncompetent C57BL/6 mice no obvious inflammatory reaction was observed based on H&E stained cryosections (Fig. 3G, H).

Example 4

Intracavitary Transplantation of 3DECM-embedded NSC in the Glioma Resection Model

[0080] In order to mimic the clinical scenario of a stem cell application in the resection cavity directly after glioma surgery we tested the 3DECM as a delivery system for NSC in an orthotopic glioma resection model (Fig. 4A; n=3). 12 days after intracerebral NCE-G55 glioma cell injection, the established tumor mass was microsurgically removed. 3DECM implants of 5-8 mm³ size loaded with 3.5 x 105 GFP-expressing NSC were prepared the day before surgery. After achieving hemostasis one NSC-containing 3DECM implant was then

administered directly into a tumor resection cavity. The 3DECM implants could be cut to a size matching the proportions of the surgical resection cavity. As expected histological analysis a week later revealed that the invading glioma cells which remained in the infiltration zone after surgery have formed new tumor masses. Remnants of NSC-containing 3DECM were found at the former resection site which has been filled out by recurrent tumor (Fig. 4B). GFP-expressing NSC were observed at the rim of the recurrent tumor (Fig. 4C) and also dispersed throughout the glioma mass (Fig. 4D). No NSC were found elsewhere in the brain.

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[0081] Citation of the above publications or documents is not intended as an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

[0082] The invention is further illustrated by the following exemplary embodiments:

1. An implantable composition comprising a fibrillar collagen and a laminin.

2. The implantable composition of claim 1 , wherein the collagen is a human collagen.

3. The implantable composition of claim 1, wherein the collagen is a bovine collagen.

4. The implantable composition of claim 2, wherein the human collagen is purified from a cultured cell.

5. The implantable composition of claim 2, wherein the human collagen comprises about 90-95% type I collagen and about 5-10% type III and/or type V collagen.

6. The implantable composition of claim 2, wherein the human collagen is derived from a partially-processed cadaveric tissue.

7. The implantable composition of claim 2, wherein the human collagen is derived from a recombinant DNA process.

8 The implantable composition of claim 1 , wherein the laminin is a human laminin.

9. The implantable composition of claim 8, wherein the human laminin is purified.

10. The implantable composition of claim 9, wherein the purified human laminin is from an epithelial cell. 11. The implantable composition of claim 9, wherein the purified human laminin comprises a laminin with a beta-1 chain, a gamma- 1 chain, and an alpha chain selected from the group consisting of alpha-1, alpha-2, alpha-3, alpha-4 and alpha-8 chain.

12. The implantable composition of claim 8, wherein the human laminin is selected from the group consisting of laminin- 111 and laminin- 1.

13. The implantable composition of claim 1, wherein the implantable composition further comprises a nidogen-1.

14. The implantable composition of claim 1, wherein the implantable composition forms a porous matrix.

15. The implantable composition of claim 1, wherein the implantable composition forms a paste-like material.

16. The implantable composition of claim 1, wherein the implantable composition is a semi-solid material at ambient temperature.

17. The implantable composition of claim 1, wherein the implantable composition is a solid material at ambient temperature.

18. The implantable composition of claim 1, wherein the implantable composition comprises about 10 to about 120 mg/ml collagen (weight/volume).

19. The implantable composition of claim 18, wherein the implantable composition comprises about 60 to about 100 mg/ml collagen (weight/volume).

20. The implantable composition of claim 1 , wherein the implantable composition comprises less than about 10% water by weight/weight.

21. The implantable composition of claim 1, wherein the implantable composition comprises less than about 1 % water by weight/weight. 22. The implantable composition of claim 17, wherein the implantable composition has a tensile strength of about 0.6 MPa to about 16 MPa.

23. The implantable composition of claim 22, wherein the implantable composition has a tensile strength of about 1 MPa to about 4 MPa.

24. The implantable composition of claim 22, wherein the implantable composition has a tensile strength of about 2 MPa to about 16 MPa.

25. The implantable composition of claim 1, further comprising an effective amount of a therapeutic agent for delivery of the therapeutic agent to the central nervous system (CNS) in a mammal.

26. The implantable composition of claim 25, wherein the implantable composition provides a sustained release of the therapeutic agent.

27. The implantable composition of claim 25, wherein the implantable composition provides a sustained release of the therapeutic agent after surgical implantation or injection.

28. The implantable composition of claim 25, further comprising a pharmaceutically acceptable carrier.

29. The implantable composition of claim 25, wherein the therapeutic agent comprises a small molecule drug.

30. The implantable composition of claim 25, wherein the therapeutic agent comprises a chemotherapy drug.

31. The implantable composition of claim 25, wherein the therapeutic agent comprises an antimicrobial drug.

32. The implantable composition of claim 25, wherein the therapeutic agent comprises a stem cell. 33. The implantable composition of claim 32, wherein the therapeutic agent comprises a human neuronal progenitor cell.

34. The implantable composition of claim 25, wherein the therapeutic agent comprises a cell derived from the CNS of a mammal.

35. The implantable composition of claim 34, wherein the therapeutic agent comprises a mesenchymal cell.

36. The implantable composition of claim 25, wherein the therapeutic agent comprises an antibody, an antibody fragment, or a combination thereof.

37. The implantable composition of claim 25, wherein the therapeutic agent comprises a nucleic acid or a combination thereof.

38. The implantable composition of claim 25, wherein the therapeutic agent comprises a viral particle for gene delivery.

39. The implantable composition of claim 1, wherein the collagen is a human collagen and the laminin is a human laminin.

40. The implantable composition of claim 39, wherein the implantable composition is a semi-solid material or a solid material at ambient temperature.

41. The implantable composition of claim 39, wherein the implantable composition comprises about 10 to about 120 mg/ml collagen (weight/volume).

42. The implantable composition of claim 40, wherein the implantable composition comprises about 60 to about 100 mg/ml collagen (weight/volume).

43. The implantable composition of claim 39, wherein the implantable composition comprises less than about 10% water by weight/weight.

44. The implantable composition of claim 43, wherein the implantable composition comprises less than about 1 % water by weight/weight. 45. A method of making an implantable composition, which method comprises incubating a fibrillar collagen with a laminin.

46. The method of claim 45, wherein the fibrillar collagen is made by neutralizing a collagen followed by dehydration of the neutralized collagen.

47. The method of claim 46, wherein the dehydration of the neutralized collagen occurs at about 10 °C to about 50 °C.

48. The method of claim 47, wherein the dehydration of the neutralized collagen occurs at about 37 °C.

49. The method of claim 46, wherein the collagen is an atelopeptide collagen.

50. The method of claim 45, further comprising dehydrating the fibrillar collagen- laminin mixture.

51. The method of claim 50, wherein the dehydration of the fibrillar collagen-laminin mixture lasts about 48-72 hours.

52. The method of claim 45, wherein the laminin is adsorbed by the fibrillar collagen.

53. The method of claim 52, wherein the adsorption is facilitated by calcium.

54. A method of making an implantable composition, which method comprises: a) neutralizing a collagen;

b) dehydrating the neutralized collagen into a fibrillar collagen;

c) incubating the fibrillar collagen with a laminin;

d) dehydrating the fibrillar collagen-laminin mixture.

55. The method of claim 54, wherein the collagen is an atelopeptide collagen.

56. The method of claim 54, wherein the dehydration of the neutralized collagen occurs at about 37 °C and the dehydration of the fibrillar collagen-laminin mixture lasts about 48-72 hours. 57. The method of claim 55, wherein the atelopeptide collagen is neutralized to about pH 7-8.

58. The method of claim 55, wherein the atelopeptide collagen is neutralized with disodium phosphate.

59. The method of claim 58, wherein the atelopeptide collagen is neutralized with disodium phosphate at about 0.02M.

60. The method of claim 54, wherein the laminin is adsorbed by the fibrillar collagen.

61. The method of claim 61, wherein the adsorption is facilitated by calcium.

62. The method of claim 54, wherein the collagen is a human collagen and the laminin is a human laminin.

63. The method of claim 54, wherein the neutralized collagen is dehydrated by centrifugation, incubation or lyophilization.

64. The method of claim 54, wherein the fibrillar collagen-laminin mixture is dehydrated into a semi-solid material or a solid material at ambient temperature.

65. The method of claim 64, wherein the fibrillar collagen-laminin mixture is lyophilized in the presence of a therapeutic agent.

66. An implantable composition made by the method of claim 45.

67. The implantable composition of claim 66, wherein the implantable composition forms a porous matrix.

68. The implantable composition of claim 66, wherein the implantable composition forms a paste-like material.

69. The implantable composition of claim 66, wherein the implantable composition is a semi-solid material at ambient temperature. 70. The implantable composition of claim 66, wherein the implantable composition is a solid material at ambient temperature.

71. A method of making an implantable therapeutic composition, which method comprises mixing an implantable composition comprising a fibrillar collagen and a laminin with an effective amount of a therapeutic agent.

72. The method of claim 71, wherein the therapeutic agent is a cell.

73. The method of claim 72, wherein the therapeutic agent is seeded directly to the implantable composition.

74. The method of claim 71, wherein the therapeutic agent is selected from a group consisting of a small molecule drug, a chemotherapy drug, a macromolecule drug, a polypeptide, a protein, a peptide, a growth factor, an antibody, a viral particle, a polynucleotide, a nucleic acid, a lipid, a carbohydrate, a sugar and a combination thereof.

75. The method of claim 74, wherein the implantable composition is lyophilized in the presence of the therapeutic agent.

76. A method of treating a disease in a subject, which method comprises

administrating an effective amount of an implantable composition comprising a fibrillar collagen and a laminin to the CNS of the subject.

77. The method of claim 76, which method comprises surgically-implanting or injecting the implantable composition into the subject.

78. The method of claim 77, wherein the implantable composition is further contacted with a non-biological device.

79. The method of claim 78, where in the non-biological device is selected from a group consisting of an electrode lead, a wire and a surgical device used in the CNS.

80. The method of claim 76, wherein the implantable composition is applied to the resection cavity of a tumor post-surgical removal of the tumor. 81. The method of claim 76, wherein the disease is a CNS disease.

82. The method of claim 76, wherein the subject is a mammal.

83. The method of claim 82, wherein the subject is a human being.

84. A method to inhibit scarring and/or reduce pain in a subject, which method comprises administrating an effective amount of an implantable composition comprising a fibrillar collagen and a laminin to the CNS of the subject.

85. The method of claim 78, wherein the scarring results from a surgery.

86. The method of claim 79, wherein the surgery is a discectomy or a laminectomy.

87. A method of conducting experiments in an animal, which method comprises administrating an effective amount of an implantable composition comprising a fibrillar collagen and a laminin to the CNS of the animal.

88. The method of claim 87, wherein the animal is a mouse.

89. A kit comprising an implantable composition comprising a fibrillar collagen and a laminin, and a container for the implantable composition.

90. The kit of claim 89, further comprising an instruction manual.

Claims

Claims

1. An implantable composition comprising a fibrillar collagen and a laminin.

2. A method of making an implantable composition, which method comprises incubating a fibrillar collagen with a laminin.

3. A method of making an implantable composition, which method comprises: a) neutralizing a collagen;

b) dehydrating the neutralized collagen into a fibrillar collagen;

c) incubating the fibrillar collagen with a laminin;

d) dehydrating the fibrillar collagen-laminin mixture.

4. An implantable composition made by a method which comprises incubating a fibrillar collagen with a laminin.

5. A method of making an implantable therapeutic composition, which method comprises mixing an implantable composition comprising a fibrillar collagen and a laminin with an effective amount of a therapeutic agent.

6. A method of treating a disease in a subject, which method comprises

administrating an effective amount of an implantable composition comprising a fibrillar collagen and a laminin to the CNS of the subject.

7. A method to inhibit scarring and/or reduce pain in a subject, which method comprises administrating an effective amount of an implantable composition comprising a fibrillar collagen and a laminin to the CNS of the subject.

8. A method of conducting experiments in an animal, which method comprises administrating an effective amount of an implantable composition comprising a fibrillar collagen and a laminin to the CNS of the animal.

9. A kit comprising an implantable composition comprising a fibrillar collagen and a laminin, and a container for the implantable composition.