CA2422657A1 - Method for nonsurgical treatment of the intervertebral disc and kit therefor - Google Patents

Abstract

This invention relates to a method anal compositions for treating pathological intervertebral discs comprising the seep of delivering an agent that causes chemical crosslinking of the native molecular components of the disc. Supplemental materials which are susceptible to crosslinking by the aforementioned agent are optionally delivered to the disc in order to increase and maintain disc height.

Description

pEP-OG~3 lVlethod for Noetsurgical Treatnnent of the Intervertebral Disc and Kit Therefor SACIf~CROUi~ID OF THIS INVENTION
The intervertebral disc contains three major components= a nucleus pulposus (a fluid-like component comprising proteoglycans and collagen), an annulus fibrosis (a t0 flexible, collagen-based ring surrounding the nucleus pulposus) and a pair of cartilaginous endplates which help enclose the nucleus pulposus within the annulus i'tbrosus. A normal, healthy nuc3eus pulposus acts much like a pressurized fluid by transferring and distributing compressive load to the annulus Fibrosis, thereby causing a slight expansion of the annulus iibrasus . Horwever, injury and/or degeneration of the l 5 intervertebral disc in the human spine can be caused by disc hermiation, rupture of the annulus, prolapse of the nucleus pulposus; meciauli,;~,~ ii~~~:~aiuy of the disc and/or dehydration of the disc, thereby leading to back pain. In addition, danxage or degeneratiotx of the annulus fibrosus in the form of a herniation, tear andlor crack reduces its ability to resist the tensile stresses conferred by the nucleus pulposus.
Thus, the disc 2o experiences excessive bulging that may result in spinal cord and/or nerve root impingement and subsequent back pain. Further, the nucleus pulposus can leak into the foramenai spaces, causing in-ication of nerve roots and fvraxnenal stenosis.
Treatments such as diseectorr~y, farainectorny, laminotomy andlor spine fusion procedures represent state of the art surgical treatment for disc problems.
Typically, the 25 goal of these treatments is to relieve pressure on the neural. elements by eliminating the material causaz~,g stenosis or irritation of the neural eterrjents. However, discectomy when performed alone can result in significant loss of disc height and frequently provides only temporary pain relief. Lanninectomy/laminotorny procedures also provide only temporary relief by opening up the spinal canal and decompressing the spinal cord, which 3o is susceptible to restenosis due to scar tissue formation at the operative site. Spine fusion is considered by some to be n last resort, ''most" invasive procedure that eliminates the flexibility of the motion segment and usually involves permanent hardware implantation.
Furthermore, fusing spinal segments has been linked to adjacent level disc degeneration_ A.I1 of these techniques have the disadvantage that they require surgical intervention eo carry out th~ treatment.
fercutaneous rnicrodisccetomy has been suggested as a minimally invasive discectomy procedure but stilt carries the disadvantage of causing disc height loss-Chemonueleolysis has been used clinically for decades and relieves pressure on a disc herniation by breaking down the nucleus pulposus. In essence, chemonueleolysis is a chemical discectom,y_ Because the goal of this treatment is to basically digest the nucleus, the consequent reduction in viscosity of the nucleus pulposus makes it more Susceptible to leakage. In addition. this proeeduz~e appears to be associated with a J
1 o incidence of anaphylaxis which has caused patient deaths. Furthermore, the procedure is known to also cause disc height loss.
Because of the drawbacks associated with. the conventional procedures, newer procedures have been developed with an airn towards relieving back pain without m~uiuing invasive surgery and without reducing disc height and proviuir~g~ a lora~m~
is lasting therapeutic effect.
Injecting curable or hardening materials into the dl5c follorwing diseectomy has been suggested in order to provide a filter material for the space left by removal of the nucleus andlor annular defect. US Patent No. 6,206,921 ("Cuagliano") discloses a method of first removing the nucleus pulposus andlor herniated portaon of the annulus 2a fibrosis, then injecting a heated, resilient, natural rubber material that hardens upon cooling. US Patent No. 6,187,048 ("Milner") discloses an ire situ polyrnerizable nucleus pulposus replacement material that may be enclosed by any impermeable container to prcvcnt leakage. I~owever, these materials may be susceptible to Ieaka~e our of the nucleus pulposus if no balloon or sheath has been formed around the material, 25 particularly if the annulus fibrosis has not been adequately repaired.
Furthermore, the implant may be subjected to repeated loads exceeding its strengttl-t over the lifetime it is expected to function in the patient. 'fhe potential consequence of failure is generation of foreign particulate, which has been linked to osteolysis and chronic foreign body reactions, 30 Other treatment options, which do not require discectomy or nucleotomy, have also been explored. US Patent 6,126,682 ~"Sharkey") discloses a method of treating annular fissures by delivering energy andlor materials to localized places near the inner wall of the annulus tibrosus, particuh;iy at the rc~~øPrior margins, using a functional element. Heating the disc has the potential to "weld" defects and/or s~u'ink disc tissues.
However, the mechanism of heating the disc to weld defects is not well understood and rrray cause secondary problems such as tissue necrosis and nerre root damage.
Sharkey also discloses delivering dissolved collagen, adhesives, cements or hydrogels to seal fissures in the annulus fibroses using the surgical instrument and functional element.
However, this delivery is restricted to local points along the inner wall of the annulus fibroses for treating annular fissures.
l o Sharkey does not disclose providing a crosslinking agent in an amount effective to cause the crosslinking of any native molecular proteins of t)he disc, nor does Sharkey disclose depositing a sealant material into the center of the nucleus pulposus portion of the dtsC_ PCT i~'ubiiLaiiox~ No, vvt~ X0/62832 ("I~aldimann") discloses an in situ curable t5 sealant material that repairs defects in the disc annulus to prevent leakage of nucleus pulposus material. In sorrre embodiments, Haldirnann discloses that the sealant material is made of two precursor components: a buffered protein solution (including collagen) and a bifunctional crosslinking agent (including PEGr with an activated terrninai group).
Typically, this injectable material adheres to the surrounding tissues by mecl~anieal interlocking. In some embodiments, Haldiman discloses that covalent bonds are formed between the preferred hydrogei bio-compatible material and the surrounding annulus fibroses tissue to further increase and secure the attachment of the sealant to the annulus fibroses tissue in the proximity to the defect in the annulus fihrosus. There is also disclosed a method of adding "artificial nucleus pulpostas material" to achieve a volume 25 eomp2~rabIe to a normal nucleus pulposus followed by sealing the annulus.
Haldiman does not disclose providing the erossli~nking agent in an amount effective to cause the erosslinking of any of the native n,~olecular proteins of the disc.
Haldirnan does not disclose depositing the sealant material into the nucleus pulposus.
PCT Patent Publication l~Io. Wp O1f70151 ("Aksan") discloses a method of 3o strengthening and stabilizing collagenous tissues comprising the steps of heating to shrink the collagen followed by crossiinking with a non-to~cie agent. The treatment is mainly focussed on glenohurneral instability and loose skin prolalems, but application of capsular shift proeedur~s used to repair injuries of the spine is also mentioned. I~owever, the crosslinking treatment always follows a thermal shrinkage step ixt the disclosed procedure. Thetrnal shrinkage is very difficult to achieve in the intervertebral disc beyond s a local region without inducing damaging effects arid is likely to cause a highly nonuniform tissue morphology. The crosslinking described is focused on collagen nnoleeules, which exist in relatively small proportians in the nucleus pulposus of the spine (~-5%).
Aksan does not specifically disclose injecting the crosslinking agent into the to intervertebral disc portion of the spine. Aksan does not disclose any method of crosslinking an untreated protein component oPthe disc.
US Patent No. 4,931,546 (°~Tardy") discloses a method. for crossli.nking collagen comprising exposing the eollagerZ to a solution of periodic acid or a periodate, then allowing spontaneous crosslinking tc~ .xr4ur~ imrn the ~sci~,iny~e groups formed during IS exposure. Similarly, US Patent No_ 5,972,3$5 ("Liu") discloses a method of oxidizing polysaccharides, then reacting the oxidized product with collagen and adding a growth factor, and discloses application of material so formed in spinal fusion augtrientation..
Neither Tardy nor Liu describes in situ crosslinking of native laving tissues, particularly in the spine or i.ntervertebral disc.
2o Accordingly, there is a need for a minimally invasive method of treating pathological intervertebral discs that relieves back pain and encourages long-term disc Stability and pain prevention by maintaining disc height, preventing nucleus leakage posteriorly, relieving pressure on a disc herniation, and inducing less alteration of the normal spine biomechanics.
Zs SiJMMARY OF THE >tNYENTION
In accordance with the present invention, there is provided a method of treating a pathologic intervertebral disc wherein a crosslinking agent is delivered to the disc in an amount sufficient to cause the chemical crcasslinking of at least a portion of the unmodified native proteins present in the disc. The crossli,nkiztg stabilizes and stiffens 3o the disc structure, which has the therapeutic effect of relieving pressure on neural elements such as the spinal cord and the exiting nerve roots. The stabilization also prevents proIapse of the nucleus material, thereby preventing foramenal stenosis and loss of disc height. T h;,. inventiar further pros~ides .~.. least-invasive medical treatment for injured andlor degenerated inten~ertebral discs that will hopefully maintain most of the flexibility and functional biornecharaics of the normal disc and prolong the need for s surgical intervention.
The present invention involves crosslinking the native molecular components of the intervertebral disc, rather than removing or dissolving them. The potential effects of crosslinking are mufti-fold, including altering the biomechanical nature of the nucleus pulposus from a viscous gel to a viscoelastic solid, thereby preventing prolapse into the to spinal canal and reducing the tendency for excessive bulging of the annulus fibrosis, which are primary suspects for causing low hack pain and sciatica.
CrosslinPCing also inhibits the degradation of the disc by providing stable, durable chemical bonds, which can help maintain disc height and prevent excessive motion of the disc level, again ' preventing painful pinching of the nerves and rupture of the annular ilbe~~_ Tlic prcsecrr t S invention can be performed as an outpatient procedure and does not require surgical intervention, and so is particularly useful as a minimally invasive, early intervention strategy.
Therefore, in accordance with the present invention, there as provided A
method of treating in a living being an intervertebral disc Having a nucleus pulposus, comprising 2o the step of a)injecting a crosslinking agent into the nucleus puiposus.
Also in accordance with the present invention, there is provided method of treating in a livir~g being an intervereebral disc havimg a nucleus pulposus, an annulus fibrosus, and native proteins, comprising the step of 2s a) injecting an effective amount of a crosslinking agent into the intervertebral disc t0 Cause in 5ltu crosslinking of at least a portion of the native proteins present in the disc DETAILED DESCRIPTION t?F "fHE I1VVEN'>fI~N
For the purposes of the present invention, a "native protein" is any protein 3a possessing any native eleaxlents. Accordingly, a native protein may have a synthetic functional group produced by reaction with a foreign agent and still be considered a native protein. .An "unmodified protein'" is a protein which has not been subjected to heat in an amount sufficient to shrink tt~c colta;en Elic:ei.n.
The intervertebral disc comprises three major cvmpanents: 1 ) ehe nucleus pulposus, 2) the annulus fibroses. and 3) a pair of cartilaginous endplates.
The present s invention may be practiced upon any of these sites, alone or in any combination.
Preferably, the nucleus pulpost~s portion of the intervertebral disc is selected as the target site for the chemical crosalinking of the proteins therein.
Treating the nucleus pulposus with the method of the present invention can stiffen the nucleus pulposus (thereby reducing undesired mobility)" and prevent native material within the nucleus to pulposus from leaking out_ In some embodiments, the crosslinking agent is injected only into the nucleus pulposus. t~Vhcn the crosslir~lcitzg agent is injected into the nucleus pulposus, it is preferred that the agc;nt be injected into the center of the nucleus pulposus.
In same prefetTed embodiments, both the nucleus pulposus and the annulus fibrosis tray be treatea with the same injection of crosslinking agent. Still more t5 preferably, the injection causes not only the crosslinlCang of substantially the entire bulk of the nucleus pulposus, but also the crosslinking of the periphery of the nucleus pulposus to the inner wall of the annulus fibroses. When this embodiment is pz-acticed, it is preferred that the agent be injected only into (anal preferably into the center of) the nucleus pulposus, and that light exercise be performed by the patient in order to disperse za the agent to the periphery of the nucleus pulposus.
In other preferred embodiments, only the annulus fibrosis is treated. Treating the annulus fibroses with the method of the present invention can have the effect of binding a tear in the annulus fibroses. When this embodiment is practiced, it is preferred that the agent be injected into the defect produced by the tear.
25 In some procedures, compositions of the present in~entian array be first injected into a first site in the disc, and then injected into a second site in the same disc. For example, a probe can be first rnanuevered into the disc and positioned so that a composition of the present invention may be first delivered only into the nucleus pulposus portion of the disc. The probe can then be manuevered again within the disc and 3o positioned sa that the same (ac a different) composition Qf the present invention may be delivered to a defect in the annulus fibroses.

In some patients having a disc characterized by advanced. degeneration, the line of .. demarcation betv~een the annulus fibroses and khe nucleus pulposus becrat.:es unclear.
Accordingly, in these cases. the crosslinking agent is preferably injected into the center of the disc.
The nucleus pulposus typically contains more than 80 volume percent (vol%) water (depending on age and condftion). The protein content of the nucleus pulposus typically comprises approximately 50 weight percent (wt%) proteoglycans, 20 wt%
collagen (mainly Type II collagen>, and other small proteins such as fibronectin, thromospondin, and elastin. The water and proteoglycan content of khe nucleus pulposus generally deCFease5 with age and onset of pathological changes. JHence, they are expected to be present in lower amounts in the intervertebral discs in patients that are candidates for the raethod of this invention.
The annulus fibrosis is generally slightly less hydrated than the nucleus pulposus and its protein content comprises anour 13 wt°i° proteoglycan and 70 wt% collagen ~s (mainly Type I collagen). The annulus fibrosis may also loss: water with age and disease, but generally experiences more structural chan;es, such as tearing and formation of thick br~ndles, than biochemical changes.
The cartilaginous endplate is a thin layer of hyaline cartilage similar to articular cartilage and dry weight is composed of mainly Type II eollagera.
2fl "When the nucleus pulposus site is so selected, it is preferred that the erosslinking agent be selected so that it effectively causes the crosslinking of at least the proteoglycan protein component thereof (as it is the predoaninant protein in nucleus pulposus). It is more preferred that the crosslinking agent be selected so that it effectively causes the crosslinking of both the proteoglycan and collagen protein components thereof (as 25 collagen is the second most predominant protein in nucleus pulposus).
However, in some embodiments, the crosslinking agent is selected so that it effectively causes the cross linking of the collagen protein component thereof. In other embodiments, the crosslinking agent be selected so that it effectively causes the crosslinking of essentially all the protein components of the nucleus puiposus. When the annulus fibroses site is so 30 selected, it is preferred that the crosslinking agent tae selected so that it effectively causes the cross linking of at least the collagen protein component thereof (as it is the predominant protein in annulus fibrosus). It is more preferred that the crasslinking agent ~ce selected ~u that it eff°etively caus;.s the crocslinking of taoth the proteoglycan and collagen protein components thereof (as proteoglycan is the second most predominant protein in the arynulus fil~rasis). however, in some embadiments, the crosslinlcing agent is s selected so that it effectively causes the crosslinking of the proteoglycan protein component of the annulus f ibrosus. In other embodiments, the crosslinking agent is selected so that it effeczivety causes the crosslinking of essentially all the protein components present at the defect of the annulus fbrosus.
Chemical crosslinking of the selected molecular components can be accomplished to using a variety of methods. including bath direct and indirect crosslinking processes.
Typically, crosslinking of proteins is accomplished when ate aldehyde functional group reacts with an amino acid group to form a bond therebetween. In some embodiments of the present invention involving aldehyde and arnino acid functional groups, the aldehyde functional group may be either synthesized upon a naiive protein tsr pa~oviuau by a' iureign 15 agent, while the amino acid functional group is either present upon a native protein or provided by a foreign agent, provided that at least one of the functional groups is present on a native protein.
Far the purposes of the present invention, a crosslinking agent may "cause"
the crasslinking of proteins by methods including but not limited to:
zo a) directly chemically bridging two functional groups from two different protein molecules ("inter-protein direct crosslinking");
b) directly chemically bridging ruvo functional groups fmrn the same protein molecule ("intra-protein direct crosslinking");
c) reacting with a first protein molecule to farm a synthetic functional grnup on ~5 a first protein molecule {typically an aldehyde) which in turn reacts with a second functional group (typically an amino acid group) on a second protein molecule ("inter-protein indirect crosslinking"); or d) reacting with a protein molecule to form a synthesis functional group on the protein molecule (typically an aldehyde) which in turn reacts with a second 30 functional group (typically an atxxino acid group) on the same protein {intra protein indirece crosslinking).
s Accordingly, in some embodiments the in-situ crosslinking of the native proteins is accomplished by crosslinki:y a first sVnu°tic ~unctirnal group arcl a second fimctional group within a single native protein tnolecuie. In some embodiments, the single native protein molecule is a proteogiycan, while in others the single native protein molecule is catiagen_ In general, aldehyde groups do typically not exist on native proteins.
Accordingly, they must be provided as either a synthetic functional group (e.g., by using an oxidizing agent upon a protein) upon a native protein, or as a functional group of a foreign agent.
In other embodiments, the in-situ erosslinlcing of the native proteins is to accomplished by crosslinking a first f-uncrional group of a first protein molecule with a second functional group of a second protein molecule. In soave embodiments, the first protein molecule is the same type as the second protein molecule. In some cases thereof, the first and second protein molecules are Type I collagen, while in others the first and seconQ protein molecules are Type iI collagen, and in still others the first and second t 5 protein molecules are a eype of proteoglycan. In some embodiments, the first protein molecule is a different type than the second protein molecule. Ire some eraabodiments thereof , the first protein molecule is a type of collagen and the second protein molecule is a proteoglycan. In other etrrbodiments thereof, the first protein molecule is Type 1 collagen and the second protein molecule is Type II collagen. In other embodiments zo thereof, the first protein nnolecule is a glycosaminoglycan and the second protein molecule is Type II coltagen_ Direct erosslinkiuag can be used in accordance with the present invention, and typically involves using the crosslinking agent as a bridge to chemically connect one amino acid group on one protein molecule to another amino acid group on the same or 25 different protein molecule. These agents are called bifunctional agents. In some preferred emf~odirnents, the crosslinking agent comprises a pair of functional groups that react with amino acid groups on the native proteins to be linked. Preferably, these functional groups are aldehydes.
Direct crosslinking can be accomplished in either conventional one step or multi-3o step procedures. Conventional methods of direct cmsslinkiing are described by K.hor (Biomaterials 18:95-1~5, 1997), which discloses methods for crosslinking collagenous tissues to improve durability, particularly transplant tissues from xenogeneic or allogeneic aournes. In some embodiments of the present invenxion. di~dct crQsslinlcing is accomplished in a one-step reaction, preferably by using a bifunctional agent.
in some embodiments, the bifunctionai agent is selected froth the group consisting of dialdehydes, s , polyepoxy compounds and diisoryanates_ One more preferred bifuneti.onat agent used in accordance with the present invention is gluteraldehyde.
Conventional, muitistep. direct, erosslinking reactions are also described by Khar.
In some ernboditnents using muleistep reactions, the bifunetional crosslinking agent is selected from the group eonsistittg of aryl azide and carbodiirrtides.
to Pathak et al. (Soc for fdiomaterials 27'h Annual Meeting Transactions p.
130, 2001 ) describe an alternative to glutcraldehyde fixation of bovine pericardium using bis(sulfosuccinimidyl suberate) as the bifunetional agent. It is believed that bis(sulfosuccinimidyl suberate) may be less toxic than gluteraldehyde and is better resistant to calai~cation.
t s Crosslinking agents can also be made by attaching functional groups to synthetic polymers, which may improve biocompatibility. One particular example is fttrtctionally activating poty(ethylene glycol) with aldehyde groups to manufacture an agent that will directly erosslink tissues in a manner sinqilar to gluteraldehyde and forzna,ldehyde.
Preferably, the direct crosslinking agent of the present invention has a molecular Zo 'weight of no more than 1 million Daltorts, Above this preferred value, the crossiinking agent may be insoluble in water and may be very difficult: to inject and disperse into the disc tissues. More preferably, the crosslinking agent has a molecular weight of between about I d0 Daltons and about 10~,000 Daltons. When the molecular weight of the agent i5 below about 100 Daltons , the agent may too easily diffuse into bodily tissues outside 2S of the intervertebrai disc. When the molecular weight of the agent is above about 100,0n0 Daltons, the agent may not disperse adequately' within the disc tissues to cause uniform crosslinkiing of native molecules.
~ne preferred embodiment of this invention involves the direce, otle step, aQueous reaction of a water-soi>sble, bifunctional crosslinking agent with at least a portion of the 3o native proteins of the intervertebral disc. Preferred crosslinlcing agents for this method include gluteraldehyde, bis(sulfosuccinimidyl suberate), polyepoxy compounds and to bifunctionally activated synthetic polymers such as polyethylene glycol) dialdehyde.
Preferably. the agent is dilute.l in :.' pHf buffered physiological saline prio: so administration_ A buffered pl-i environment is desirable to n7aintain favorable reaction conditions during crosslinlcing.
Gluteraldehyde is particularly preferred as a crosslinlcing agent due to its medical history of use with biololgicat transplant tissues and injectable adhesives.
Preferably, the crosslinking agent (which is preferably glutcraldehyde) is delivered to the disc in a concentration of between approximately 0.1 and 20 volume percent (vol°l°), more preferably between 1 and Z 0 vo1% in saline buffered to a pH of approximately 6.5 to 8,d, l o more preferably between approximately 7.4 to 7.~.
Another method of causing the crosslinking of native intervertebral disc molecular components is to chemically react target portions of native proteins (such as nature collagen and/or native proteo~;lycan proteins) such that synthetic functional groups (such as aldehydes) are created directly an the native proteins.' i:rossnntcing then i5 preferably occurs between the synthetic functional groups (such as atdehyde groups) of the reacted native grotein and an unreacted functional group (such as an amino acid group) located on either the same native protein or another molecule.
Modification of the native component to form a functional group such as an aldehyde can be achieved using enzymatic oxidizing agents, and are preferably selected from the group consisting zo of lysine oxidase, xransgtutaminase, and mufti-copper oxidases.
Proteoglycans contain polysaccharide regions that may be modified to form aldehyde functional groups. This modifeation of proteoglycans may be accomplished by 'using either enzymatic or non-enzymatie agents. Examples of enzymatic polysaccharide oxidizing agents include catechol oxidase and tyrosinase. Examples of non-eru;yrnatac polysaccharide oxidizing 25 agents include periodate ions (periodic acid, sodium and potassiurty periodate), nitroprusside ions (sodium nitroprusside) and hydrogen peroxide, In a preferred embodiment of this invention, a non-enzymatic polysaccharide oxidizing agent is injected into the nucleus pulposus of a pathological intervertebral disc.
Because the dry weight corr~poncnt of the nucleus pulposus is rich in proteoglycans, there 3o are numerous sites chat can be oxidized to form functional aldehydes.
Subsequently, the ~t aldehydes can react with amino acid regions of both native collagens and native proteoglycans to form ;:rosslinks. . .
Chemical modification of proteoblycans, particularly the sulfated proteoglycans, in the nucleus pulposus can elicit the added benefit of reducing the swelling pressure in the nucleus, thereby reducing both the potential for exuding out of the disc and the tendency for the nucleus pulposus to cause bulging of the annulus. This is one proposed mechanism for the effectiveness of chernonucleolysis in reducing pain in properly selected patients (Ifato er u!., _Spin's l7; 534-939. 199}. Furthermore, this mechanism is the basis far clinical experiments conducted with aprotini:n, a protease Inhibitor that to forms strong complexes with sulfated glycosaminoglycans (Kraemer er al..
Spine 7.~73-74. d 982).
Preferably, the indirect crosstinking agent of the present invention has a relatively low molecular weight, is subseantially completely reacted in the crosslinking process, and iorans icy-produces (such as gases and water) which can relatively easily exit the reaevion site.
As is frequently the case with pathological intervertebral discs, concomitant with the presence of pain is a narrowing of the disc space and/or exuding of disc material (herniation) into the neural foramen. Lil~ewise, the quantity of native tissue present in the disc may be lower than desired in order for crosslinking to restore the stability, biomechanics and disc height desired. Therefore, it may be desirable to add a supplemental protein into the disc as an adjunct to the crossiinking treatment. Preferably, the supplemental protein can be crosslinked by the same crosslinking agent used to crosslink the native proteins of the disc. Examples of supplemental proteins that are useful with the crosslinking strategies described above include but are not limited to collagen in various types and farms, hyaluronan, chondroitin sulfate, keratin sulfate, albumin, elastin, fibrin, fabronectin and casein.
Although it is within the scope of the invention for the suppleznentai protein to be added before or after the crosslinking step, a preferred embodiment comprises the steps of first adding the supplemental material, then allowing or effecting the dispersion of the 3o material into the disc space, then adding the crosslinking agent.
Ultimately, this helps provide a tx~ore homogeneous crosslinked structure.
t~

Therefore, in accordance with the present iw~~:~,tion, ther~:.xa provided 3 method of treating in a living being an intervertebral disc having native molecular proteins, comprising the steps of:
a) injecting a supglemental protein into the intervertebral disc, and b) injecting a crosslinking agent into the intervertebral disc.
In preferred embodiments, the method comprises the sequential steps of a) injecting a supplemental protein into the intervertebral disc, b) optionally dispersing the supplemental protein throughout at least the nucleus t0 pulposus portion of the disc. and c) injecting a crossiinking agent into the intervertebral disc.
Preferably, the erosslinlcing agent is injected in an amount suf~eient tc~
erossiink at least a portion of the native proteins. More preferably, the crosslinking agent is injected in an amount suf#icient to crosslituc at least a portion of the supplemental protein. Irt some ~ 5 ernboditnents, the supplemental protein is selected from the group consisting of collagen, hyaiuronan, chondroitin sulfate, keratan sulfate, albumin, elastin, fibrin, ~bronectin arid casein. Preferably, when direct crosslinking is performed, the supplemental protein is collagen, and gluteraldehyde is preferably selected as the crosslinking agent.
Preferably, when indirect cross linking is performed, the supplemental protein is a polysaccharide 20 (more preferably, hyaluronan) and sodium periodate is preferably selected as the crosslinking agent (which functions as a polysaccharide oxidizing agent). In some embodiments, the supplemental protein is injected substantially into the nucleus pulposus. More preferably, the crossiinking agent is also injected substantially into the nucleus pulposus. Because the supplemental protein can react with the crosslinking agent, is in preferred embodiments, the crosslinking agent as provided in first sterile container and the supplemental protein is provided in a second sterile container.
Although the agents used in the disclosed invention are preferably non-toxic at the concentrations employed for tz~eatrxtent, it may desirable to substantially inactivate any residual erosslinking agent with an inactivating agent once adequate crosslinking has 3o been achieved in order to reduce any toxic potential the crosslinking agent may still posse5s_ For example, a diluted solution of glyeine will inactivate unreacted gluteraldehyde as described by Aksan (PCTIWO 0170151). Because the inactivating _7~~Lnr can react with the erosslin!,~ng agent, in preferred embodiments, the crr~sslin:king agent is provided in first sterile container and the inactivating agent is provided is a second sterile container.
s In certain crosslinking procedures, tt~e reaction is reversible, particularly if there is a dramatic change in pH. For chose reversible reactions, it rnay be preferable to add a stabilizing agent once the crosslinking has been achieved. l~or example, the reaction of aldehyde funceionat groups with proteins generally forms what is know as a Schiff base, which may be a seversible reaction (with the exception of gluteraldehyde, which may to undergo an irreversible reaction with proteins). In these cases where reverse reactions are possible, it rnay be preferable to include a later step of adding a stabilizing agent that substantially prevents the reversal of the crosslinking reaction. Preferred stabilizing agents comprise boron compounds. More preferred stabilizing agents are selected froraa the group consisting of sodium borohydride, sodium cyanoooranyciricle or pyridine t 5 borane_ Pyridine borane is a rraorc preferred agent for this invention due to its relatively higher biocompatibility. Because the stabilizing agent can react with the crosslinking agent, in preferred embodiments, the orosslinking agent is provided in a first sterile container and the stabilizing agent is provided in a second sterile container.
in some embodiments, one or mare of the cor~tpounds selected from the group zo consisting of the crosslinking agent, the supplemental protein, the inactivating agent and the stabilizing agent is delivered (preferably, separately) to the disc in a buffered saline solution capable of controlling pH during the crosslinlsixig reaction.
Preferably, the solution is buffered to a pFi of approximately 6.5 to 8.0, more preferably between approximately 7.0 to 7.~.
z5 In some embodiments, one or more of the compounds selected from the group consisting of the crosslinking agent, the supplemental protein, the inactivating agent and the stabilizing agent is delivered (,preferably, separately) to the disc in a composition further comprising a radio-opaque contrast medium present in an amount sufficient to monitor the flow of the composition through fluoroscopy. In some embodiments, the 3o radio-opaque contrast medium comprises iodine. In some embodiments, the radio-opaque contrast medium is an iodine-containing dye. In some embodiments, the radio-opaque t.~

contrast medium coFnprises a compound selected from the group consisting of barium-containing compounds (such as t~arurrt sr.Ifate), zircon:a and tantalum, is preferably a barium-containing compound, and more preferably is barium sulfate.
Therefore, in accordance with the present invention, there is provided a composition present within a naturat intervertebral disc having a nucleus pulposus and an annulus fibroses, comprising a crosslinked stnacture comprising:
a) proteins which are native to the intervertebrai disc, and b)a radio-opaque contrast medium present in an effective amount to be detected by fluoroscopy.
io Also in accordance with the present invention, there is provided a composition for trea.eing a natural intervertebral disc having native proteins, the eonnposition comprising:
a) a erossiinking agent present in an effective amount for crosslinking both at least a portion of the native proteins, and is b) a radio-opaque contrast medium present in an effective amount to be detected by fluroscopy.
In preferred embodiments, the compound is delivered. in a buffered saline solution containing the radio-opaque contrast medium.. In more preferred embodiments, each compound used in the procedure is delivered in a buffered saline solution containing the 20 radio-opaque contrast medium.
When certain embodiments of the present invention are practiced, there is produced a composition present within gun intervertebraa disc having a nucleus pulposus, comprising a cFOSSlullCed structure comprising:
a) proteins which are native to the intervertebs~'! disc, and 25 b) supplemental proteins.
In some embodiments, the crosslinked structure further comprises:
c) a direct crosstinking agent, preferably, gluteraldehyde.
In some embodiments, the native proteins are substantially derived from the nucleus pulposus_ 3o When certain embodiments of the present invention are practiced, there is produced a composition formed within an intervertebrat disc having a level of naturally crosslinked proteins, the composition comprising a crosslinked structure comprising proteins which .are n.a:ive to the natural intervertebral disc, u-lmrein the cross!inkad structure has a degree of crosslinkin? which is higher than the unmodified level of naturally erosslinked proteins. Preferably, the naturally crosslinked proteins are unmodified.
In some embodiments, the crosslinked structure further comprises a direct cross linking agent. preferably glutcratdehyde. In others, the crvsslinked structure has been crosslinked using an indirect crosslinking ageztt which is not incorporated into the crosslinked structure. Optionally, the crosslinked structure further comprises supplemental proteins. In preferred embodiments, the disc further comprises an annulus ~o fibrosus having an inner wail, and wherein the crosslinked structure includes the inner wall of the annulus fibrosus. In others, the crosslinked st~eture is present essentially within the nucleus puiposus. in others, the crosslinked structure is present essentially throughout the entire disc, In a preferred embodiment of the invention, the agents and materials to be delivered to the intervertebral disc are injected percutaneously under fluoroscopic control using a syringe and appropriate gauge needle, for example 25G. Such a minimally invasive procedure is highly desirable for minixmi~ing damage to musculoskeleta!
structures and can be perforn~ed using an outpatient proeedure_ Although this infection can be delivered from any approach to the disc, it is preferable to use a posterior or 2o posterolateral approach such that the needle is required to pass through a shortest distance before reaching the disc without penetrating major va.acular or neural structures_ Preferably, the agents and materials of this invention are injected into the center of the nucleus pulposus of the disc such that the material can then disperse radially into the remainder of the nucleus pulposus and/or zemainder of the disc.
25 The volume of diluted crosslinking solution injected into the disc is preferably between approximately O.I and 10 ml, more preferably between approximately 1 and 5 ml. This ensures that adequate crosslinking agent is delivered to the disc yet does not produce high pressures in the disc, potentially causing failure of the disc and extruding of crosslinking agent and disc material. The volume of supplemental protein needed is 3D preferably determined based on the amount of volume restoration needed to restore the i~

disc to its natural size. This can be determined using a eornbin.ation of x-rays and IVtRI
scans.
In another preferred embodiment, light exercising is used as an adjunct to the procedure in order to aid in the dispersing of agents and materials within the disc and to s ensure complete reaction of the functional materials injected andlor generated.
Examples of light exercise include limited left and right lateral bending, flexion and extension, and torso twists (axial rotation). Preferably, the light exercising is performed after each step of the treatment to disperse each agent or material prior to delivering a subsequent material.
to As noted above, it is desirable to provide the different compounds used in the present invention in separate sterile containers in order to avoid undesirable reactions therebetween prior to their injection into the disc.
Therefore, in accordance with the present invention, there is provided a lcit for injecting therapeutic solutions into an intervertebrai disc rravtng native proteins, 15 comprising:
a) a first container having a sterile inner surface and containing a first compound, and b) a second container having a sterile iruner surface and containing a second different compound, 2o wherein the first and second compounds are selected from the group consisting of:
i) a crosslinking agent present in an effective amount for cross linking at least a portion of the native proteins, ii) a supplemental protein, iii) an inactivating agent, and z5 iv) a stabilizing agent.
In some eanbodiments, there is provided a third co~.~tainer having a sterile inner surface containing a third different compound selected from the above group of compounds. In other embodiments, there is provided a fourth container having a sterile 3o inner surface containing a fourth different compound selected from the above group of compounds.
1~

In preferred embodiments, at least one container containing a compound further contains 4 baffered salin ~ solution, and preferably øurthPr contains a radio-opaque contrast medium. In more preferred embodiments, each provided container containing a eompoand further contains a buffered saline solutifln, and preferably further contains a radio-opaque contrast medium.
In preferred embodiments, the first container contains the crosslinking agent present in an effective amount for crosslinlsing at least a portion of the native proteins, and the second container contains the supplemental protein, In some preferred kit embodiments, the crosslinking agent is a direct crosslinking to agent (such as gluteraldehyde) and the supplemental protein as collagen. In others, the crosslinking agent is an indirect cross linking agent (such as sodium periodate) and the supplemental protein is hyaluronan.
In some embodiments, the kit further comprises first and second sterile syringes for separately injecting the compounds. In other embodiments, third and preferably iourtn t5 syringes are also provided. In some embodiments, the contaitxers of the kit are provided in a case. Likewise the syringes o~f the kit are provided in the case.
E~~.4MPL1~ 1 zo $iomeelhanical Testing of Cadaveric Functional Spine Units after Crosslinking Treatment In a prophetic experiment, four human eadaverie lumbar spine segments (L1 -LS) are tested biomechanicatly to determine range-of ~nraotion (IZ.OM) in flexion-25 extension, lateral bending, axial torsion and pure compression to establish a baseline.
Three crosslinking solutions plus one control solution are prepared: 1 ) 10%
w/v gluteraldehyde in 0.1M phosphate buffered saline (PBS) with pH 7.4; 2) 10% wlv bis(sulfosuccinimidyl suberate) in PBS; 3) 10% sodium periodate in PBS; 4) )?BS alone_ For a given treatment, 2 rnl of solution is injected into the center of one of the four intervertebral discs of the cadaveric lumbar spine using a 5 ml syringe and 25G needle.
For each spine segment, all treatments are applied, one to each level.

Following the injections, the spine segments are subjected to simulated light exercise, i.e. flexion-extension. lateral bending and ~:=ial rotation. Tlhese exercises are repeated at various times during the course of the experiment. After 2 hours and 24 hours, the biomechanical range-of rrtotion (ROM) testing is repeated to measure the effect of crossiinking on each motion segfnent.
The range of motion testing should indicate that the stiffness of the segrrmnts significantly increased over that of a normal untreated disc.

to Outpatient Procedure for In Sita Crosslinkiag Treatment of the Intervertebrai Disc After complaining of low back and leg pain, a patient is referred to a spine speeialist_ Using x-rays and MRI, the physician determines that the pain is being caused by a bulging intervertebral disc with loss of disc height_ The recommended treatment is restoration of disc heiga~t with an mjeetion of soluble Type 1 atelc~collagen followed by crosslinking treatment with 10°/m gluteraldehyde.
In the first outpatient procedure, 2, ml of atelopsptide Type I collagen solution is injected percutaneously into the center of the intervertebrai disc. The patient then follows a prescribed light exercise regimen of limited right and left lateral bending, flexion and extension, and torsional twisting and told to refrain from lifting heavy objects and engaging in high impact exercises.
The second outpatient procedure is performed on the following day. In this procedure , 2 rnl of 10% gluteraldehyde solution in O.1M phosphate buffered saline (pfI
7.4) is injected percutaneously into the center of the intemertebral disc.
Again, the patient then follows a prescribed light exercise regimen of lirniced right and left lateral ?s bending, flexion and extension, and tortional twisting and told to refrain from lifting heavy objects and engaging in high impact exercises for at least two days.
!9

Claims (88)

1. A method of treating in a living being an intervertebral disc having a nucleus pulposus, comprising the step of a) injecting a crosslinking agent into the nucleus pulposus.

2. The method of claim 1 wherein the crosslinking agent is injected in an effective amount to cause in-situ crosslinking of native proteins present in the nucleus pulposus.

3. The method of claim 2 wherein the crosslinking agent is a bifunctional agent having at least one aldehyde functional group.

4. The method of claim 3 wherein the crosslinking agent is a bifunctional agent having at least two aldehyde functional groups.

5. The method of claim 3 wherein the bifunctional agent is selected from a group consisting of gluteraldehyde, formaldehyde, a polyepoxy compound, and a diisocyanate.

6. The method of claim 3 wherein the bifunctional agent is gluteraldehyde.

7. The method of claim 3 wherein the bifunctional agent is selected froth a group consisting of acyl azide and carbodiimde.

8. The method of claim 3 wherein the bifunctional agent is bis(sulfosuccicimidyl suberate).

9. The method of claim 3 wherein the bifunctional agent is a synthetic polymer modified with aldehyde groups.

10. The method of claim 2 wherein the crosslinking agent is an indirect agent capable of synthesizing at least one aldehyde group upon a first native protein.

11. The method of claim 10 where the first nature protein is a proteoglycan.

12. The method of claim 11 wherein the indirect crosslinking agent is further capable of synthesizing at least one aldehyde group upon a collagen molecule.

13. The method of claim 10 wherein the crosslinking agent is an enzymatic oxidizing agent.

14. The method of claim 13 wherein the enzymatic oxidizing agent is selected from the group consisting of lysine oxidase, transglutaminase, and a multi-copper oxidase.

15. The method of claim 13 wherein the enzymatic oxidizing agent is a polysaccharide oxidizing agent.

16. The method of claim 15 wherein the enzymatic polysaccharide oxidising agent is selected from the group consisting of catechol oxidase and tyrosinase.

17. The method of claim 10 wherein the crosslinking agent is a non-enzymatic crosslinking agent.

18. The method of claim 17 wherein the non-enzymatic crosslinking agent is selected from the group consisting of a periodate ion, a nitroprusside son, and hydrogen peroxide.

19. The method of claim 17 wherein the non-enzymatic crosslinking agent is sodium periodate.

20. The method of claim 2 wherein the in-situ crosslinking of the native proteins is accomplished by crosslinking first and second functional groups within a single protein molecule.

21. The method of claim 20 wherein the first functional group is an aldehyde group and the second functional group is an amino acid group.

22. The method of claim 20 wherein the single protein molecule is selected from the group consisting of a proteoglycan and collagen.

23. The method of claim 20 wherein each of the first and second functional groups are amino acid groups.

24. The method of claim 2 wherein the in situ crosslinking of the native proteins is accomplished by crosslinking a first functional group of a first protein molecule with a second functional group of a second protein molecule.

25. The method of claim 24 wherein the first protein molecule and the second protein molecule are of the same type.

26. The method of claim 25 wherein the type of protein molecule is selected from the group consisting of a collagen and a proteoglycan.

27. The method of claim 24 wherein the first protein molecule and the second protein molecule are different types of proteins.

28. The method of claim 24 wherein the first functional group is an aldehyde group and the second functional group is an amino acid group.

29. The method of claim 24 wherein the first protein molecule is collagen and the second protein molecule is a proteoglycan.

30. The method of claim 24 wherein each of the first and second functional groups are amino acid groups.

31. The method of claim 2 further comprising the subsequent step of:
b) injecting an inactivating agent into the intervertebral disc in an amount sufficient to substantially inactivate the crosslinking agent.

32. The method of claim 37 wherein the inactivating agent is a solution comprising glycine.

33. The method of claim 2 further comprising the subsequent step of:
b) injecting a stabilizing agent into the intervertebral disc in an amount sufficient to stabilize the crosslinked proteins and inhibit reversal of the crosslinking reaction.

34. The method of claim 33 wherein the stabilizing agent comprises boron.

35. The method of claim 2 wherein the native proteins are untreated.

36. A method of treating in a living being an intervertebral disc having native molecular proteins, comprising the steps of:
a) injecting a supplemental protein into the intervertebral disc, and b) injecting a crosslinking agent into the intervertebral disc.

37. The method of claim 36 wherein the crosslinking agent is injected in an amount sufficient to crosslink at least a portion of the native proteins.

38. The method of claim 37 wherein the crosslinking agent is injected in an amount sufficient to crosslink the supplemental protein.

39. The method of claim 38 wherein the supplemental protein is selected from the group consisting of collagen hyaluronan, chondroitin sulfate, keratan sulfate, albumin, elastin, fibrin, fibronectin and casein.

40. The method of claim 36 wherein the supplemental protein is injected into the nucleus pulposus.

41. The method of claim 36 wherein the crosslinking agent is injected in an amount sufficient to crosslink the supplemental protein with the native proteins.

42. The method of claim 36 wherein the crosslinking agent is injected into the nucleus pulposus.

43. The method of claim 36 wherein step a) is performed before step b).

44. The method of claim 43 further comprising the step of c) dispersing the supplemental protein throughout at least the nucleus pulposus portion of the disc, wherein dispersing step c) is performed before step b).

45. A kit for injecting therapeutic solutions into an intervertebral disc having native proteins, comprising.

a) a first container having a sterile inner surface and containing a first compound, and b) a second container having a sterile inner surface and containing a second different compound, wherein the first and second compounds are selected from the group consisting of i) a crosslinking agent present in an effective amount for crosslinking at least a portion of the native protein, ii) a supplemental protein, iii) an inactivating agent, and iv) a stabilizing agent.

46. The kit of claim 45 further comprising a third container having a sterile inner surface containing a third different compound selected from the group.

47. The kit of claim 4b further comprising a fourth container having a sterile inner surface containing a fourth different compound selected from the group.

48. The kit of claim 45 wherein at least one container containing a compound further contains a buffered saline solution and a radio-opaque contrast medium.

49. The kit of claim 45 wherein each container of the kit further contains a buffered saline solution, and preferably further contains a radio-opaque contrast medium.

50. The kit of claim 45 wherein the first container contains the crosslinking agent present in an effective amount for crosslinking at least a portion of the native proteins, and the second container contains the inactivating agent.

51. The kit of claim 45 wherein the first container contains the crosslinking agent o present in an effective amount for crosslinking at least a portion of the native proteins, and the second container contains the supplemental protein.

52. The kit of claim 51 wherein each container contain further contains a buffered saline solution and a radio-opaque contrast medium.

53. The kit of claim 45 wherein the crosslinking agent it present in the first container in a concentration of between 0.1 and 20 volume percent.

54. The kit of claim 45 wherein the cross linking agent is present in the first container in a concentration of between t and 10 volume percent.

55. The kit of claim 45 further comprising first and second sterile syringes.

56. The kit of claim 45 further comprising a case, wherein the containers are provided in the case.

57. A method of treating in a diving being an intervertebral disc having a nucleus pulposus, an annulus fibrosus. and native proteins, comprising the step of a) injecting an effective amount of a crosslinking agent into the intervertebral disc to cause in situ crosslinking of at least a portion of the native proteins present in the disc.

58. The method of claim 57 wherein the crosslinking agent is injected into at least the nucleus pulposus.

59. The method of claim 57 wherein the native proteins are unmodified native proteins.

60. The method of claim 57 wherein the injection produces a therapeutic effect of relieving pressure on a neural element.

61. The method of claim 60 wherein the neural element is a spinal cord.

62. The method of claim 60 wherein the neural element is a nerve root.

63. The method of claim 57 wherein the injection substantially prevents prolapse of a portion of the nucleus pulposus.

64. The method of claim 63 wherein the substantial prevention of prolapse of the portion of the nucleus pulposus sustains disc height.

65. The method of claim 63 wherein the substantial prevention of prolapse prevents leakage of material of the nucleus pulposus from the disc into a neural foramen.

66. The method of claim 57 wherein the cross linking agent is injected only into the nucleus pulposus.

67. The method of claim 57 wherein both the nucleus pulposus and the annulus fibrosus are treated with the same injection of crosslinking agent.

68. The method of claim 57 wherein the injection causes crosslinking of substantially the entire nucleus pulposus.

69. The method of claim 68 wherein the nucleus pulposus has a periphery and the annulus fibrosus has an inner wall, and wherein the injection further causes crosslinking of the periphery of the nucleus pulposus to the inner wall of the annulus fibrosus.

70. The method of claim 57 wherein the crosslinking agent is injected into a defect in the annulus fibrosus.

71. A composition present within an intervertebral disc having a nucleus pulposus and an annulus fibrosus, comprising a crosslinked structure comprising:
a) proteins which are native to the intervertebral disc, and b) supplemental proteins.

72. The composition of claim 71 wherein the crosslinked structure further comprises a direct crosslinking agent.

73. The composition of claim 71 wherein the direct crosslinking agent is gluteraldehyde.

74. The composition of claim 71 wherein the cross linked structure is present in the annulus fibrosus.

75. The composition of claim 71 wherein the crosslinked structure is present substantially in the nucleus pulposus.

76. A composition formed within a natural intervertebral disc having a level of naturally crosslinked proteins, the composition comprising a crosslinked structure comprising proteins which are native to the natural intervertebral disc, wherein the crosslinked structure has a degree of crosslinking which is higher than the level of naturally crosslinked proteins in the disc.

77. The disc of claim 76 wherein the cross linked structure further comprises a direct cross linking agent.

78. The disc of claim 76 wherein the naturally crosslinked proteins in the disc are unmodmea.

79. The disc of claim 76 wherein the cross linked structure further comprises an indirect cross linking agent.

80. The disc of claim 76 wherein the cross linked structure further comprises supplemental proteins.

81. The disc of claim 76 wherein the disc further comprises are annulus fibrosus having an inner wall, and wherein the crosslinked structure includes the inner wall of the annulus fibrosus.

82. The disc of claim 76 wherein the cross linked structure is present essentially within the nucleus pulposus.

83. A composition present within a natural intervertebral disc having a nucleus pulposus and an annulus fibrosus, comprising a crosslinked structure comprising:
b) proteins which are native to the intervertebral disc, and c) a radio-opaque contrast medium present in an effective amount to be detected by fluoroscopy.

84. The composition of claim 83 wherein the radio-opaque contrast medium comprises iodine.

85. The composition of claim 83 wherein the radio-opaque contrast medium is an iodine-containing dye.

86. The composition of claim 83 wherein the radio-opaque contrast medium comprises a compound selected from the group consisting of barium-containing compounds, zirconia and tantalum,

87. The composition or claim t53 whereas me medium is barium sulfate.

88. A composition for treating a natural intervertebral disc having native proteins, the composition comprising:
b) a crosslinking agent present in an effective amount for crosslinking both at least a portion of the native proteins, and c) a radio-opaque contrast medium present in an effective amount to be detected by fluroscopy.