Heparan Sulfate Proteoglycan

(54) EXTRACELLULAR MATRIX-BINDING 7,732,427 B2 6/2010 Kiicket al. SYNTHETIC PEPTIDOGLYCANS 7,737,131 B2 6, 2010 Kiicket al. 7,803,905 B2 9/2010 Farach-Carson et al. 7,842,667 B2 11/2010 Seliktar et al. (71) Applicant: Symic Biomedical, Inc., San Francisco, 7,851,445 B2 12/2010 Stuppet al. CA (US) 7,855, 187 B1 12/2010 Prestwich et al. 7,862,831 B2 1/2011 Wang et al. (72) Inventors: Alyssa Panitch, West Lafayette, IN 7,897,165 B2 3/2011 Elisseeffet al. (US); John Eric Paderi, West Lafayette, 8. } }d R: $38H Sk st al IN (US); Shaili Sharma, West Lafayette, 8,114,834 B2 2, 2012 t a. IN (US); Katherine Allison Stuart, 8,88220 B2 5 2012 Ruosiahtietal. West Lafayette, IN (US); Nelda Marie 8,268,950 B2 9/2012 Elisseeff Vazquez-Portalatin, West Lafayette, IN 8,283,414 B2 10/2012 Yu et al. (US) 8,304,388 B2 11/2012 Chettibi et al. 8,314, 195 B2 11/2012 Elisseeff 8,329,673 B2 12/2012 Prestwich et al. (73) Assignee: systic IP, LLC, San Francisco, CA 85.65 55.5 K." 8,343,764 B2 1/2013 Abad et al. 8,343,942 B2 1, 2013 Oottamasathien et al. (*) Notice: Subject to any disclaimer, the term of this SE E: 5.38. t t al wapul et al. patent 1s st G adjusted under 35 8,415,325 B2 4/2013 Kiicket al. U.S.C. 154(b) by 0 days. 8,431,146 B2 4/2013 Harley et al. 8.450,271 B2 5, 2013 Shah et al. (21) Appl. No.: 14/214,220 8,470,780 B2 6/2013 Ruoslahti et al. 8,476,220 B2 7/2013 Barritault et al. 22) Filed: Mar 14, 2014 8,557,774 B2 10/2013 Vandroux et al. (22) 1. ar. 14, 8,703,740 B2 4/2014 Cho et al.

Page 3 (56) References Cited OTHER PUBLICATIONS Kirker, et al., (2002), "Glycosaminoglycan hydrogel films as bio interactive dressings for wound healing." Biomaterials, 23 (17)  ? w a r a n a t a u S s a e t r a e o p u o m r e a e + o ? t a n u u p p m a n n 8 s Note to .  Provisional Application Ser. No. 61/798,916, filed Mar. 15, 2013, which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 10, 2014, is named 39WG-197593 US SL.txt and is 22,178 bytes in size.

FIELD OF INVENTION
This disclosure provides extracellular matrix-binding syn thetic peptidoglycans comprised of one or more synthetic peptides conjugated to a glycan and methods of their use.

BACKGROUND
In most tissues, cells are surrounded by an extracellular matrix (ECM) containing proteins such as collagen, laminin, and fibronectin. As mammals age and in Some disease states, the extracellular matrix in certain areas of the body (e.g., in synovial joints, the vitreous humor, the spinal discs, the skin, etc.) can degrade, causing undesirable symptoms, such as various forms of arthritis, loss of vision, and the like.
Lubricin, also known as superficial Zone protein (SZP) or PRG4, is a mucinous glycoprotein secreted by tissues lining the interior Surfaces of animal joints (see Schumacher, B. L., et al., Arch Biochem Biophys, 1994, 311(1): 144-52). Lubri cin acts as a chondroprotective barrier against direct solid-to Solid contact in joints when the kinematic conditions are conducive to Surface sliding in the boundary lubrication regime, characterized by the formation of an adsorbed molecular layer conformal with the articular tissue surface topography (see Neu, C. P. K. Komvopoulos, and A. H. Reddi, Tissue Engineering, Part B: Reviews, 2008). In the absence of a strongly adsorbing, continuous, self-replenish ing boundary lubricant layer, intermittent asperity-asperity interactions lead to rapid deterioration of the join surface by various mechanical wear processes. Such as adhesion, abra Sion, Surface fatigue, and delamination. Lubricintribosupple mentation has been shown to reduce cartilage degeneration (see Jay, G. D., et al., Arthritis and rheumatism, 2012, 64(4): 1162-71, and Teeple, E., et al., The American Journal of Sports Medicine, 2011. Reducing friction at the articular cartilage interface will Suppress cartilage wear and Surface damage.
Another extracellular matrix is the vitreous humor, a com plex gel-like network which fills the posterior cavity of the eye, is composed of approximately 99 wt % water, 0.9 wt % salts, less than 0.1 wt % heterotypic collagen fibrils (type II, V/XI and IX), and a hyaluronan network. It serves several purposes (including developmental, optical, protective) and its degradation has been implicated in several ocular patholo gies, such as retinal tear, retinal detachment, retinal edema, choroidal detachment, vitreous hemorrhage, and glaucoma.
In addition, degeneration of the nucleus pulposus, a gel like Substance is the inner core of the spinal disc, results in reduced ability of the spinal disc to transmit loads evenly and efficiently between vertebral bodies, and leads to damage in the annular region of the disc, known as the annulus fibrosis. The nucleus pulposus functions to distribute hydraulic pres Sure in all directions within each disc under compressive loads and is comprised of chondrocyte-like cells, collagen fibrils, and proteoglycan aggrecans that aggregate through hyaluronic chains. Fissures or tears in the annulus can trans late into a disc that herniates orruptures, resulting in impinge ment of the nerves in the region of the disc and finally lower back or leg pain.
This disclosure provides extracellular matrix-binding syn thetic peptidoglycans for use in Supplementing and/or replac ing extracellular matrix fluids in the body, thus treating and/or preventing diseases or disorders resulting from the degrada tion thereof.

SUMMARY
Provided herein is an extracellular matrix-binding syn thetic peptidoglycan, wherein the extracellular matrix-bind ing synthetic peptidoglycan comprises a glycan; from about 1 to 60 collagen binding peptide(s); and from about 1 to 60 hyaluronic acid binding peptide(s). In the extracellular matrix-binding synthetic peptidoglycan, the collagenbinding peptide(s) and the hyaluronic acid binding peptide(s) are covalently bonded to the glycan. The  Also provided is a pharmaceutical composition comprising an extracellular matrix-binding synthetic peptidoglycan comprising a glycan, from about 1 to 80 collagen binding peptide(s); and from about 1 to 80 hyaluronic acid binding peptide(s), wherein the collagen binding peptide(s) and the hyaluronic acid binding peptide(s) are covalently bonded to the glycan.
Synthetic peptidoglycans as described herein may be use ful in Supplementing and/or protecting tissues that have both collagen and hyaluronic acid, such as cartilage, the nucleus pulposus, and the vitreous humor of the eye. Accordingly, provided is a method of treating and/or preventing cartilage degeneration in a patient comprising administering to a patient in need thereof a pharmaceutical composition com prising the extracellular matrix-binding synthetic peptidogly can described herein. In addition, provided is a method of treating and/or preventing vitreous humor degeneration in a patient comprising administering to a patient in need thereof the pharmaceutical composition comprising the extracellular matrix-binding synthetic peptidoglycan described herein. Also provided is a method of treating and/or preventing nucleus pulposus degeneration in a patient comprising administering to a patient in need thereof the pharmaceutical composition comprising the extracellular matrix-binding synthetic peptidoglycan described herein.
It is further contemplated that the extracellular matrix binding synthetic peptidoglycans and pharmaceutical com positions thereof can be used in vascular intervention proce dures including, for example, to prevent any one or a combination of platelet binding to exposed collagen of the denuded endothelium, platelet activation, thrombosis, inflammation resulting from denuding the endothelium, inti mal hyperplasia, and vasospasm. The extracellular matrix binding synthetic peptidoglycans described herein can also 3 be used to stimulate endothelial cell proliferation and can bind to collagen in a denuded vessel.

BRIEF DESCRIPTION OF THE DRAWINGS
Certain aspects are best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various fea tures are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures: FIG. 1 shows the frictional force in the case of undamaged cartilage (with no aggrecan depletion) and shows that with a synthetic peptidoglycan according to according to the disclo Sure, the friction increases between the cartilage Surfaces.
FIG . 2 shows the frictional force in the case of damaged cartilage (with aggrecan depletion to simulate osteoarthritis) and shows that when the synthetic peptidoglycan according to Example 1 is added to the cartilage, friction is lowered between the cartilage surfaces.
FIG . 3 shows the rheological measurements on bovine vitreous with no treatment (control), PBS buffer and trypsin (FIG. 3A), the rheological measurements on bovine vitreous treated with trypsin, and trypsin in combination with three different peptidoglycans (FIG.3B), and the rheological mea Surements on bovine vitreous with no treatment (control), PBS buffer, and three different peptidoglycans without trypsin (FIG. 3C).

DETAILED DESCRIPTION
It is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodi ments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular forms "a", "an', and "the include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide' includes a plurality of peptides.

DEFINITIONS
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein the following terms have the follow ing meanings.
As used herein, the term "comprising or "comprises" is intended to mean that the compositions and methods include the recited elements, but not excluding others. "Consisting essentially of when used to define compositions and meth ods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) claimed. "Consisting of shall mean excluding more than trace elements of other ingredients and Substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure. The term "about when used before a numerical designa tion, e.g., temperature, time, amount, and concentration, including range, indicates approximations which may vary by (+) or (-) 10%, 5% or 1%.
As used herein, the term "synovial cavity" refers to the space between the bones of a synovial joint that is filled with synovial fluid.
As used herein, the term "treating and/or preventing refers to preventing, curing, reversing, attenuating, alleviating, minimizing, Suppressing or halting the deleterious effects of a disease state, disease progression or other abnormal condi tion.
As used herein, the term "patient" refers to a subject (i.e., human) at risk for or Suffering from a disease state, disease progression or other abnormal or deleterious condition.
As used herein, the term "synthetic peptidoglycan refers to a synthetic conjugate that comprises a glycan and one or more synthetic peptides covalently bonded thereto. The gly can portion can be made synthetically or derived from animal sources. The synthetic peptides can be covalently bonded directly to the glycan or via a linker. For methods of conju gating hyaluronic acid binding peptides to glycans, see, e.g., WO 2012/162534. For methods of conjugating collagen binding peptides to glycans, see, e.g., US 2013/0190246, US 2012/0100106, and US 2011/0020298, the disclosures of which are incorporated herein by reference in their entirety.
As used herein, the term "covalently bonded refers to a bond in which one or more pairs of electrons are shared by tWO atOmS.
As used herein, the term 'glycan refers a compounds consisting of a large number of monosaccharides linked gly cosidically. In certain embodiments, the glycan is a gly cosaminoglycans, which comprise 2-amino Sugars linked in an alternating fashion with uronic acids, and include poly mers such as heparin, heparan Sulfate, chondroitin, keratin, and dermatan. Accordingly, non-limiting examples of gly cans which can be used in the embodiments described herein include alginate, agarose, dextran, chondroitin, chondroitin Sulfate, dermatan, dermatan Sulfate, heparan, heparin, kera tin, keratan Sulfate, and hyaluronic acid.
As used herein, "hyaluronic acid binding peptide' refers to a synthetic peptide comprising a hyaluronic acid binding sequence. In the various embodiments described herein, the peptide component of the synthetic peptidoglycan can com prise an amino acid sequence selected from the group con  EMBO Journal, 1994, 13, 286-296, and Goetinck et al., J. Cell. Biol, 1987, 105, 2403-2408 which are incorporated herein by reference).
As used herein, the term "collagen binding peptide' refers to a synthetic peptide comprising a collagen binding sequence. The "collagen binding peptide' can have amino acid homology with a portion of a protein normally or not normally involved in collagen fibrillogenesis. In one embodi ment, the collagenbinding peptide comprises from about 5 to about 40 amino acids, or from about 5 to about 20 amino acids, or from about 5 to about 10 amino acids. In some embodiments, these peptides have homology or sequence identity to the amino acid sequence of a small leucine-rich proteoglycan, a platelet receptor sequence, or a protein that regulates collagen fibrillogenesis. In various embodiments, the collagen binding peptide comprises an amino acid sequence selected from the group consisting of RRANAALKAGELYKSILY ( In any of the embodiments described herein, any one or more of the synthetic peptides (e.g., the hyaluronic acid bind ing peptide and/or the collagen binding peptide) may have a glycine-cysteine (GC) attached to the C-terminus of the pep As used herein, the term "amino acid refers to either a natural and/or unnatural or synthetic amino acid, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics. Single letter and three letter abbreviations of the naturally occurringamino acids are listed below in Table 1. A peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein.
In any of the embodiments described herein, a synthetic peptide (e.g., a hyaluronic acid binding peptide and/or a col lagen binding peptide) comprises any amino acid sequence described in the preceding paragraph or an amino acid sequence with 80%, 85%, 90%. 95%, 98%, or 100% homol ogy to any of these amino acid sequences. In various embodi ments, the peptide components of the synthetic peptidogly can described herein can be modified by the inclusion of one or more conservative amino acid Substitutions. As is well known to those skilled in the art, altering any non-critical amino acid of a peptide by conservative Substitution should not significantly alter the activity of that peptide because the side-chain of the replacement amino acid should be able to form similar bonds and contacts to the side chain of the amino acid which has been replaced. Non-conservative substitutions are possible provided that these do not excessively affect the hyaluronic acid binding activity of the peptide.
As used herein, the term "homology" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is 7 occupied by the same base oramino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A peptide (or a polypeptide or peptide region) has a certain percentage (for example, 60%. 65%, 70%, 75%, 80%, 85%, 90%. 95%, 98% or 99%) of "homology" or "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using Software programs known in the art (e.g., BLAST), and for example, those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology.
As is well-known in theart, a "conservative substitution' of an amino acid or a "conservative substitution variant' of a peptide refers to an amino acid Substitution which maintains: 1) the secondary structure of the peptide; 2) the charge or hydrophobicity of the amino acid; and 3) the bulkiness of the side chain or any one or more of these characteristics. Illus tratively, the well-known terminologies "hydrophilic resi dues' relate to serine or threonine. "Hydrophobic residues' refer to leucine, isoleucine, phenylalanine, Valine or alanine, or the like. "Positively charged residues' relate to lysine, arginine, ornithine, or histidine. "Negatively charged resi dues' refer to aspartic acid or glutamic acid. Residues having "bulky side chains" refer to phenylalanine, tryptophan or tyrosine, or the like. A list of illustrative conservative amino acid Substitutions is given in Table 2.  In one embodiment, the synthetic peptidoglycans of the disclosure bind, either directly or indirectly to collagen and/or hyaluronic acid. The terms "binding or "bind as used herein are meant to include interactions between molecules that may be detected using, for example, a hybridization assay, Surface plasmon resonance, ELISA, competitive binding assays, iso thermal titration calorimetry, phage display, affinity chroma tography, rheology or immunohistochemistry. The terms are also meant to include "binding interactions between mol ecules. Binding may be "direct" or "indirect". "Direct" bind ing comprises direct physical contact between molecules. "Indirect binding between molecules comprises the mol ecules having direct physical contact with one or more mol ecules simultaneously. For example, it is contemplated that synthetic peptidoglycans of the disclosure directly bind and interact with both hyaluronic acid and type II collagen, and can be used to restore the low friction properties of articular cartilage, thus protect the Surface from mechanical wear. This binding can result in the formation of a "complex' compris ing the interacting molecules. A "complex' refers to the bind ing of two or more molecules held together by covalent or non-covalent bonds, interactions or forces.

Synthetic Peptidoglycans
The present disclosure provides an extracellular matrix binding synthetic peptidoglycan comprising: a) a glycan; b) from about 1 to about 80 collagen binding peptide(s); and c) from about 1 to about 80 hyaluronic acid binding peptide(s): wherein the peptides ofb) and c) are covalently bonded to the glycan.
In the synthetic peptidoglycan disclosed herein, the glycan can be any glycan, or polysaccharide, including but not lim ited to, dextran, chondroitin, chondroitin Sulfate, dermatan, dermatan Sulfate, heparan, heparin, keratin, keratan Sulfate, and hyaluronic acid. In some embodiments, the glycan is chondroitin Sulfate. In some embodiments, the glycan is der matan Sulfate. In some embodiments, the glycan is hyaluronic acid.
The peptides can be bonded to the glycan directly or via a linker. In certain embodiments, the peptides are covalently bonded to the glycan via a linker. The linker can be any Suitable bifunctional linker, e.g., 3-(2-pyridyldithio)propio nyl hydrazide (PDPH), N-B-maleimidopropionic acid hy drazide (BMPH), and the like. In any of the various embodi ments described herein, the sequence of the peptide may be modified to include a glycine-cysteine segment to provide an attachment point for a glycan. In certain embodiments, the linker is N-B-maleimidopropionic acid hydrazide (BMPH).
Depending on the desired properties of the synthetic pep tidoglycan, the total number of peptides bound to the glycan certain embodiments, the total number of peptides present in the synthetic peptidoglycan is less than about 50. In certain embodiments, the total number of peptides present in the synthetic peptidoglycan is from about 10 to about 40. In certain embodiments, the total number of peptides present in the synthetic peptidoglycan is about 22. In certain embodi ments, the total number of collagen binding peptides is from about 5 to about 20, or about 10, or about 11. In certain embodiments, the total number of hyaluronic acid binding peptides is from about 5 to about 20, or about 10, or about 11.
In one aspect, the collagen binding peptides present in the extracellular matrix-binding synthetic peptidoglycans described herein comprise have binding affinity to one or more of collagen types I, II, III, or IV. One or more collagen binding peptide having a specified binding affinity can be used in the extracellular matrix-binding synthetic peptidogly cans described herein. For example, the extracellular matrix binding synthetic peptidoglycans can comprise at least one collagen binding peptide which has binding affinity to type I collagen and at least one collagen binding peptide which has binding affinity to type II collagen. In another aspect, the collagen binding peptides have binding affinity to type I col lagen. In certain aspects, the collagen binding peptides have binding affinity to type II collagen.
Suitable collagen binding peptides are known in the art (see, e.g., US 2013/0190246, US 2012/0100106, and US 2011/0020298, the disclosures of which are incorporated herein by reference in their entirety). In one embodiment, the collagen binding peptide comprises from about 5 to about 40 amino acids. In some embodiments, these peptides have homology to the amino acid sequence of a small leucine-rich proteoglycan, a platelet receptor sequence, or a protein that regulates collagen fibrillogenesis.
In various embodiments, the collagen binding peptide comprises an amino acid sequence selected from the group consisting of WYRGRLGC ( , or any peptide sequence comprising a sequence with at least about 80% sequence identity, or at least about 80% sequence identity, or at least about 85% sequence identity, or at least about 90% sequence identity, or at least about 95% sequence identity, or at least about 98% sequence identity. In certain embodiments, the collagen binding peptide is WYRGRLGC (SEQID NO: 81), or any peptide that has at least about 80% sequence identity, or at least about 85% sequence identity, or at least about 90% sequence identity, or at least about 95% sequence identity, or at least about 98% sequence identity. In certain embodiments, the collagen binding peptide is about 80%, about 85%, about 90%, about 95%, about 98%, or about 100% homologous with the collagen binding domain(s) of the von Willebrand factor or a platelet collagen receptor as described in Chiang, et al. J. Biol. Chem. 277: 34896-34901 (2002), Huizing a, et al., Structure 5: 1147-1156, Romijn, et al., J. Biol. Chem. 278: 15035-15039 (2003) with at least about 80% sequence identity, or at least about 80% sequence identity, or at least about 85% sequence iden tity, or at least about 90% sequence identity, or at least about 95% sequence identity, or at least about 98% sequence iden tity.
Additional peptides that can be included as the , or any peptide sequence comprising a sequence with at least about 80% sequence identity, or at least about 80% sequence iden tity, or at least about 85% sequence identity, or at least about 90% sequence identity, or at least about 95% sequence iden tity, or at least about 98% sequence identity (see, e.g., Amemiya et al. Biochem. Biophys. Acta, 2005, 1724, 94-99, incorporated herein by reference). In certain embodiments, the collagen binding peptide is GAHWOFNALTVRGG (SEQ ID NO: 2), or any peptide sequence comprising a sequence with at least about 80% sequence identity, or at least about 80% sequence identity, or at least about 85% sequence identity, or at least about 90% sequence identity, or at least about 95% sequence identity, or at least about 98% sequence identity. In another embodiment, the peptide is selected from the group consisting of RDGTRYVQKGEYR (SEQID NO: 51), HREARSGKYK (SEQ ID NO. 52), PDKKHKLYGV (SEQ ID NO. 53), and WDKERSRYDV (SEQ ID NO. 54) (see, e.g., Yang et al., EMBO Journal, 1994, 13, 286-296, and Goetinck et al., J. Cell. Biol., 1987, 105, 2403-2408 which are incorporated herein by reference).
In another embodiment, the hyaluronic acid binding pep tide may be selected from a group consisting of the B-X7-B homology, in which B is either lysine or arginine and X is any non-acidic amino acid residue (i.e., any amino acid other than aspartic acid or glutamic acid), where at least one of the 7x residues is a basic residue (i.e., arginine, lysine, or histidine). Peptides may also be selected by phage display, utilizing positive selection for binding to hyaluronic acid. An hyalu ronic acid binding peptide may be determined by its interac tion with hyaluronic acid, and measured by any of the tech niques used to evaluate molecular interactions. For example, Surface plasmon resonance, ELISA, competitive binding assays, isothermal titration calorimetry, affinity chromatog raphy, rheology or immunohistochemistry. Peptides that are considered "hyaluronic acid binding may interact with hyaluronic acid or hyaluronic acid-containing tissues such that the interaction is not attributed to known chemically reactive groups. The interaction may be due to hydrophobic and charge interactions resulting from the amino acid resi dues in the peptide. The interaction may be measured by 11 immobilizing hyaluronic acid on a microplate and incubating with hyaluronic acid binding peptides followed by detection techniques such as biotin-avidin with the use of a chro mophore. The interaction may also be measured by mechani cal influence on hyaluronic acid-containing fluids, gels, or tissues that have been incubated with the hyaluronic acid binding peptide, or with a peptidoglycan containing anhyalu ronic acid binding peptide or peptides.
For identifying a peptide, a peptide selected from phage display, or one that is identified from a hyaluronic acid bind ing motif in a protein, can be synthesized and evaluated for its interaction with hyaluronic acid. For example, a B-X7-B sequence could be synthesized with a biotin modification at the N-terminus and incubated on a hyaluronic acid coated microplate. A dose response binding curve can be generated to determine the ability of the peptide to bind to hyaluronic acid.
Similarly for a collagen binding peptide, a synthetic pep tide derived from a phage display library selected for collagen binding can be generated. The peptide can be synthesized and evaluated for binding to collagen by any of the techniques such as SPR, ELISA, ITC, affinity chromatography, or others known in the art. An example could be a biotin modified peptide sequence that is incubated on a microplate containing immobilized collagen. A dose response binding curve can be generated using a streptavidin-chromophore to determine the ability of the peptide to bind to collagen.
In one embodiment, provided herein is an extracellular matrix-binding synthetic peptidoglycan comprising: wherein the peptides ofb) and c) are covalently bonded to the glycan via N-IB-maleimidopropionic acid hydrazide (BMPH); and further wherein the collagenbinding peptide(s) are WYRGRLGC (SEQID NO: 81) and the hyaluronic acid binding peptide(s) are GAHWOFNALTVRGGGC (SEQ ID NO: 82).
In various embodiments described herein, the peptides described herein can be modified by the inclusion of one or more conservative amino acid Substitutions. As is well known to those skilled in the art, altering any non-critical amino acid of a peptide by conservative Substitution should not signifi cantly alter the activity of that peptide because the side-chain of the replacement amino acid should be able to form similar bonds and contacts to the side chain of the amino acid which has been replaced. Non-conservative substitutions may too be possible, provided that they do not substantially affect the binding activity of the peptide (i.e., hyaluronic acid or col lagen binding affinity). In various embodiments described herein, the synthetic peptidoglycan is resistant to aggrecanase. An aggrecanase is characterized in the art as any enzyme known to cleave aggre can. In one embodiment, the synthetic peptidoglycan does not contain a polymerizable group. Such as methacrylates, ethacrylates, itaconates, acrylamides, thiols, peptides and aldehydes.

Synthesis of Peptidoglycans
The peptides used in the synthetic peptidoglycans described herein (i.e., the hyaluronic acid binding peptide and the collagen peptide) may be purchased from a commercial Source or partially or fully synthesized using methods well known in the art (e.g., chemical and/or biotechnological methods). In certain embodiments, the peptides are synthe sized according to Solid phase peptide synthesis protocols that are well known in the art. In another embodiment, the peptide is synthesized on a solid Support according to the well-known Fmoc protocol, cleaved from the support with trifluoroacetic acid and purified by chromatography accord ing to methods known to persons skilled in the art. In other embodiments, the peptide is synthesized utilizing the meth ods of biotechnology that are well known to persons skilled in the art. In one embodiment, a DNA sequence that encodes the amino acid sequence information for the desired peptide is ligated by recombinant DNA techniques known to persons skilled in the art into an expression plasmid (for example, a plasmid that incorporates an affinity tag for affinity purifica tion of the peptide), the plasmid is transfected into a host organism for expression, and the peptide is then isolated from the host organism or the growth medium, e.g., by affinity purification. Recombinant  In certain embodiments, the peptides are covalently bonded to the glycan directly (i.e., without a linker). In Such embodiments, the synthetic peptidoglycan may be formed by covalently bonding the peptides to the glycan through the formation of one or more amide, ester or imino bonds between an acid, aldehyde, hydroxy, amino, or hydrazo group on the glycan. All of these methods are known in the art or are further described in the Examples section of this application or in Hermanson G. T., Bioconjugate Techniques, Academic Press, pp. 169-186 (1996), incorporated herein by reference. As shown in Scheme 1, the glycan (e.g., "CS") can be oxi dized using a periodate reagent, such as sodium periodate, to provide aldehyde functional groups on the glycan (e.g., "ox CS) for covalently bonding the peptides to the glycan. In such embodiments, the peptides may be covalently bonded to a glycan by reacting a free amino group of the peptide with an aldehyde functional groups of the oxidized glycan, e.g., in the presence of a reducing agent, utilizing methods known in the art.
In embodiments where the peptides are covalently bonded to the glycan via a linker, the oxidized glycan (e.g., "ox-CS") can be reacted with a linker (e.g., any suitable bifunctional liker, such as 3-(2-pyridyldithio)propionyl hydrazide (PDPH) or N-B-maleimidopropionic acid hydrazide (BMPH)) prior to contacting with the peptides. The linker typically comprises about 1 to about 30 carbon atoms, or about 2 to about 20 carbon atoms. Lower molecular weight linkers (i.e., those having an approximate molecular weight of about 20 to about 500) are typically employed. In addition, structural modifications of the linker are contemplated. For example, amino acids may be included in the linker, including but not limited to, naturally occurring amino acids as well as 13 those available from conventional synthetic methods, such as beta, gamma, and longer chain amino acids.
As shown in Scheme 1, in one embodiment, the peptides are covalently bonded to the glycan (e.g., "CS") by reacting an aldehyde function of the oxidized glycan (e.g., "ox-CS) with 3-(2-pyridyldithio)propionyl hydrazide (PDPH) or N-B-maleimidopropionic acid hydrazide (BMPH) to form an glycan intermediate (e.g., "BMPH-CS) and further react ing the glycan intermediate with peptides containing at least one free thiol group (i.e., -SH group) to yield the synthetic peptidoglycan. In yet another embodiment, the sequence of the peptides may be modified to include anamino acid residue or residues that act as a spacer between the HA-or Collagen binding peptide sequence and a terminating cysteine (C). For example a glycine-cysteine (GC) or a glycine-glycine-gly cine-cysteine (GGGC) (SEQ ID NO: 83) or glycine-serine glycine-cysteine (GSGC) (SEQID NO: 84) segment may be added to provide an attachment point for the glycan interme diate. Accordingly, in one embodiment, the synthetic peptidogly cans described herein are provided by a) oxidizing at least one vicinal diol group of a glycan to provide a glycan having at least two aldehyde functional groups; b) optionally reacting the glycan with a linker; and reacting the glycan with from about 1 to about 80 collagen binding peptide(s); and from about 1 to about 80 hyaluronic acid binding peptide(s), such that the peptides are covalently bonded to the glycan.
The synthetic peptidoglycan can be synthesized by sequen tially adding the peptides having different binding affinities to the glycan (i.e., oxidized glycan or glycan intermediate), or alternatively, adding all peptides simultaneously. The Syn thetic peptidoglycans can be isolated and/or purified using known methods, such as size exclusion chromatography, at any point in the synthesis.

Pharmaceutical Compositions and Administration
Disclosed herein is a pharmaceutical composition com prising the extracellular matrix-binding synthetic peptidogly can, wherein the synthetic peptidoglycan comprises a glycan having from about 1 to about 80 collagen binding peptide(s) and from about 1 to about 80 hyaluronic acid binding peptide(s) covalently bonded to the glycan.
As is known in the art, the components as well as their relative amounts are determined by the intended use and method of delivery. The pharmaceutical compositions may be "BMPH-CS for oral, topical or parenteral delivery, including intra-articu lar, intervertebral or intraocular delivery. In any of the embodiments described herein, the synthetic peptidoglycan can be administered alone or in combination with suitable pharmaceutical carriers or diluents. Diluent or carriers used in the pharmaceutical compositions can be selected so that they 15 do not diminish the desired effects of the synthetic pepti doglycan. The pharmaceutical composition may be in any suitable form. Examples of suitable dosage forms include aqueous Solutions, for example, a solution in isotonic saline, 5% glucose or other well-known pharmaceutically accept able liquid carriers such as alcohols, glycols, esters and amides. In certain embodiments, the pharmaceutical compo sition further comprises one or more pH buffering agent, one or more ionic strength modifying agent, and/or one or more Viscosity modulating agent.
In certain embodiments, the pharmaceutical composition further comprises a collagen binding peptidoglycan. Suitable "collagen binding peptidoglycans' are described in the art (see, e.g., US 2013/0190246, US 2012/0100106, and US 2011/0020298). In certain embodiments, the collagen bind ing peptidoglycan comprises chondroitin Sulfate conjugated to from about 1 to about 20 collagen binding peptide(s) as described herein. In certain embodiments, the collagen bind ing peptide(s) of the collagenbinding peptidoglycan is WYR GRLGC (SEQID NO: 81).
In certain embodiments, the pharmaceutical composition further comprises a hyaluronic acid binding peptidoglycan. Suitable "hyaluronic acid binding peptidoglycans' are described in the art (see, e.g., WO 2012/162534). In certain embodiments, the hyaluronic acid binding peptidoglycan comprises chondroitin Sulfate conjugated to from about 1 to about 20 hyaluronic acid binding peptide(s) as described herein. In certain embodiments, the hyaluronic acid binding peptide(s) of the hyaluronic acid binding peptidoglycan is GAHWOFNALTVRGGGC (SEQID NO: 82).
Suitable viscosity modulating agents include but are not limited to, ionic and non-ionic water soluble polymers; crosslinked acrylic acid polymers such as the "carbomer' family of polymers, e.g., carboxypolyalkylenes that may be obtained commercially under the CarbopolTM trademark: hydrophilic polymers such as polyethylene oxides, polyoxy ethylene-polyoxypropylene copolymers, and polyvinylalco hol; cellulosic polymers and cellulosic polymer derivatives Such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellu lose phthalate, methyl cellulose, carboxymethyl cellulose, and etherified cellulose; gums such as tragacanth and Xanthan gum, sodium alginate; gelatin, hyaluronic acid and salts thereof, chitosans, gellans or any combination thereof. Typi cally, non-acidic viscosity enhancing agents, such as a neutral or basic agent are employed in order to facilitate achieving the desired pH of the formulation.
In some embodiments, the synthetic peptidoglycans can be combined with minerals, amino acids, Sugars, peptides, pro teins, vitamins (such as ascorbic acid), or laminin, collagen, fibronectin, hyaluronic acid, fibrin, elastin, or aggrecan, or growth factors such as epidermal growth factor, platelet-de rived growth factor, transforming growth factor beta, or fibro blast growth factor, and glucocorticoids such as dexametha Sone or viscoelastic altering agents, such as ionic and non ionic water Soluble polymers; acrylic acid polymers; hydrophilic polymers such as polyethylene oxides, polyoxy ethylene-polyoxypropylene copolymers, and polyvinylalco hol; cellulosic polymers and cellulosic polymer derivatives Such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellu lose phthalate, methyl cellulose, carboxymethyl cellulose, and etherified cellulose; poly(lactic acid), poly(glycolic acid), copolymers of lactic and glycolic acids, or other poly meric agents both natural and synthetic.
Parenteral formulations may be suitably formulated as a sterile non-aqueous Solution or as a dried form to be used in conjunction with a suitable vehicle Such as sterile, pyrogen free water. The preparation of parenteral formulations under sterile conditions, for example, by lyophilization, may readily be accomplished using standard pharmaceutical techniques well known in the art.
In certain embodiments, the solubility of the synthetic peptidoglycan may need to be enhanced. In Such cases, the solubility may be increased by the use of appropriate formu lation techniques, such as the incorporation of solubility enhancing compositions such as mannitol, ethanol, glycerin, polyethylene glycols, propylene glycol, poloXomers, and oth ers known in the art.
The synthetic peptidoglycan may be sterilized to remove unwanted contaminants including, but not limited to, endot oxins and infectious agents. Sterilization techniques which do not adversely affect the structure and biotropic properties of the synthetic peptidoglycan can be used. In certain embodi ments, the synthetic peptidoglycan can be disinfected and/or sterilized using conventional sterilization techniques includ ing propylene oxide or ethylene oxide treatment, sterile fil tration, gas plasma sterilization, gamma radiation, electron beam, and/or sterilization with a peracid, Such as peracetic acid. In one embodiment, the synthetic peptidoglycan can be Subjected to one or more sterilization processes. Alterna tively, the synthetic peptidoglycan may be wrapped in any type of container including a plastic wrap or a foil wrap, and may be further sterilized.
In various embodiments, the synthetic peptidoglycan can be administered intravenously or into muscle, for example. Suitable routes for parenteral administration include intravas cular, intravenous, intraarterial, intramuscular, cutaneous, Subcutaneous, percutaneous, intradermal, and intraepidermal delivery. Suitable means for parenteral administration include needle (including microneedle) injectors, infusion techniques, and catheter-based delivery.
In various embodiments described herein, formulations for parenteral administration may be formulated to be for imme diate and/or modified release. Modified release formulations include delayed, Sustained, pulsed, controlled, targeted and programmed release formulations. Thus, a synthetic pepti doglycan may be formulated as a solid, semi-solid, or thixo tropic liquid for administration as an implanted depot provid ing modified release of the active compound. Illustrative examples of Such formulations include drug-coated Stents and copolymeric (dl-lactic, glycolic)acid (PGLA) micro spheres. In another embodiment, the synthetic peptidoglycan or composition comprising the synthetic peptidoglycan may be continuously administered, where appropriate.
In any of the embodiments described herein, the synthetic peptidoglycan or composition comprising the synthetic pep tidoglycan can be administered intravascularly into the patient (e.g., into an artery or vein) in any suitable way. In various embodiments described herein, the synthetic pepti doglycan or composition comprising the synthetic pepti doglycan can be administered into a vessel of a patient prior to, during, or after vascular intervention. In various embodi ments, vascular interventions, such as percutaneous coronary intervention (PCI), can include, for example, stenting, atherectomy, grafting, and angioplasty. Such as balloon angioplasty. Illustratively, the vascular intervention can be one which involves temporarily occluding an artery, such as a coronary artery or a vein (e.g., balloon angioplasty), or it can be one which does not involve temporarily occluding an artery or a vein (e.g., non-balloon angioplasty procedures, stenting procedures that do not involve balloon angioplasty, etc.). Illustrative modes of delivery can include a catheter, parenteral administration, a coating on a balloon, through a porous balloon, a coated Stent, and any combinations thereof or any other known methods of delivery of drugs during a vascular intervention procedure. In one illustrative embodi ment, the target vessel can include a coronary artery, e.g., any blood vessel which supplies blood to the heart tissue of a patient, including native coronary arteries as well as those which have been grafted into the patient, for example, in an earlier coronary artery bypass procedure.
Exemplary pharmaceutical compositions for use with the synthetic peptidoglycans for parenteral administration or catheter-based delivery may comprise: a) a synthetic pepti doglycan as described herein; b) a pharmaceutically accept able pH buffering agent to provide a pH in the range of about pH 4.5 to about pH 9; c) an ionic strength modifying agent in the concentration range of about 0 to about 300 millimolar; and d) water Soluble viscosity modifying agent in the concen tration range of about 0.25% to about 10% total formula weight or any individual componenta), b), c), or d) or any combinations of a), b), c) and d).
Suitable dosages of the synthetic peptidoglycan can be determined by standard methods, for example by establishing dose-response curves in laboratory animal models or in clini cal trials and can vary significantly depending on the patient condition, the disease state being treated, the route of admin istration and tissue distribution, and the possibility of co usage of other therapeutic treatments. The effective amount to be administered to a patient is based on body Surface area, patient weight or mass, and physician assessment of patient condition. In various exemplary embodiments, an effective dose ranges from about 1 ng/kg to about 10 mg/kg, 100 ng/kg to about 1 mg/kg, from about 1 g/kg to about 500 g/kg, or from about 100 g/kg to about 400 g/kg. In each of these embodiments, dose/kg refers to the dose per kilogram of patient mass or body weight. In other illustrative aspects, effective doses ranges from about 0.01 ug to about 1000 mg per dose, 1 g to about 100 mg per dose, or from about 100 ug to about 50 mg per dose, or from about 500 ug to about 10 mg per dose or from about 1 mg to 10 mg per dose, or from about 1 to about 100 mg perdose, or from about 1 mg to 5000 mg per dose, or from about 1 mg to 3000 mg per dose, or from about 100 mg to 3000 mg per dose, or from about 1000 mg to 3000 mg per dose. In any of the various embodiments described ug, 450 ug, 500 ug, 550 ug, 575 ug, 600 ug. 625 ug, 650 jug, 675 ug. 700 ug. 800 ug, 900 ug, 1.0 mg, 1.5 mg, 2.0 mg, 10 mg, 100 mg, or 100 mg to 30 grams.
Any effective regimen for administering the synthetic pep tidoglycan can be used. For example, the synthetic pepti doglycan can be administered as a single dose, or as a mul tiple-dose daily regimen. Further, a staggered regimen, for example, one to five days per week can be used as an alter native to daily treatment.
In various embodiments described herein, the patient is treated with multiple injections of the synthetic peptidogly can. In one embodiment, the patientis injected multiple times (e.g., about 2 up to about 50 times) with the synthetic pepti doglycan, for example, at 12-72 hour intervals or at 48-72 hour intervals. Additional injections of the synthetic pepti doglycan can be administered to the patient at an interval of days or months after the initial injections(s).

Methods
The synthetic peptidoglycans described herein may be use ful in replacing, rejuvenating, or Supplementing tissues that have both collagen and hyaluronic acid, such as cartilage, synovial fluid, and the vitreous humor.

Cartilage Degeneration
A well-lubricated surface on articular cartilage leads to optimal functionality of synovial joints. As occurs in osteoar thritis, however, a reduced lubrication results in cartilage degradation and fibrillation; which in turn contribute to joint dysfunction and pain. Reduced lubrication also leads to joint dysfunction and pain in other forms of arthritis, including rheumatoid arthritis.
As shown in Example 2, the synthetic peptidoglycans pro vided herein can be used to mimic some of the functions of lubricin, a mucinous glycoprotein secreted by tissues lining the interior Surfaces of animal joints. The synthetic pepti doglycan thus has the potential to enhance lubrication at an articular cartilage surface, thereby reducing wear-induced erosion of the cartilage. The synthetic peptidoglycan also has the potential to protect macromolecules, like hyaluronic acid and type II collagen, from enzyme-induced degradation.
Accordingly, provided is a method of treating and/or pre venting cartilage degeneration in a patient comprising admin istering to a patient in need thereof a pharmaceutical compo sition comprising the extracellular matrix-binding synthetic peptidoglycan described herein. In one embodiment, the patient is treated by injecting the pharmaceutical composition comprising the extracellular matrix-binding synthetic pepti doglycan into a synovial cavity.
It is also contemplated that the synthetic peptidoglycans can be used to treat and/or prevent articular cartilage disease by protecting the articular cartilage matrix from traumatic and cytokine-induced enzymatic degradation.

Vitreous Humor Degeneration
The vitreous humor is a viscoelastic, gel-like Substance that fills the posterior cavity of the eye. Vitreous replacements have been used to replace a dysfunctional vitreous humor, for example in cases where opacification or the physical collapse and liquefaction of the vitreous has occurred, and as a tem porary or permanent vitreous replacement during retinal Sur gery. A suitable vitreous replacement should be transparent, biocompatible, and they should have a density and refractive index close to the natural vitreous.
Accordingly, provided is a method of treating and/or pre venting vitreous humor degeneration in a patient comprising administering to a patient in need thereof a pharmaceutical composition comprising the extracellular matrix-binding synthetic peptidoglycan described herein. Nucleus Pulposus Degeneration The nucleus pulposus is a gel-like Substance present in spinal discs, and functions to distribute hydraulic pressure in all directions within each disc under compressive loads and is comprised of chondrocyte-like cells, collagen fibrils, and pro teoglycan aggrecans that aggregate through hyaluronic chains. Degeneration of the nucleus pulposus results in reduced ability of the disc to transmit loads evenly and effi ciently between vertebral bodies, and leads to damage in the annular region of the disc, known as the annulus fibrosis.
Fissures or tears in the annulus can translate into a disc that herniates or ruptures, resulting in impingement of the nerves in the region of the disc and finally lower back or leg pain.
Attempts have also been made to replace only the nucleus pulposus. Replacement of the nucleus pulposus is expected to arrest the initial dehydration of the degenerated nucleus and return the disc to a fully hydrated state so that the degenerative process, including the associated pain, is postponed or pre vented and the mechanical function is restored to the vertebral Segment.
It is contemplated that the synthetic peptidoglycans described herein will bind to and protect the annulus fibrosis. Accordingly, provided is a method of treating and/or prevent ing annulus fibrosis degeneration in a patient comprising administering to a patient in need thereof a pharmaceutical composition comprising the extracellular matrix-binding synthetic peptidoglycan described herein. Also provided is a method of treating and/or preventing nucleus pulposus degeneration in a patient comprising administering to a patient in need thereof a pharmaceutical composition com prising the extracellular matrix-binding synthetic peptidogly can described herein. propan2-yl)aminopropane-1-sulfonic acid

Methods
The synthesis of collagen type I and hyaluronic acid bind ing peptidoglycans has been previously described (see, e.g., Bernhard J. C., Panitch A. Acta Biomater 2012:8: 1543-1550, and Paderi J. E., Panitch A. Biomacromolecules 20089: 2562-2566). The present synthetic peptidoglycan was pre pared according to the methods described therein and modi fied as follows.
Chondroitin sulfate (10 mg/mL) was oxidized with sodium meta-periodate in 0.1 M sodium acetate buffer pH 5.5. The degree of oxidation was controlled by the concentration of sodium meta-periodate. Heterobifunctional crosslinker BMPH was conjugated to functionalized chondroitin sulfate by reacting about 40-fold molar excess BMPH to chondroitin sulfate in 1xRBS pH 7.2 buffer at room temperature for 2 hours. The degree of functionalization was determined by measuring the consumption of BMPH during purification of CS-BMPH from excess BMPH. In the final step of conjuga tion, peptides WYRGRLGC (SEQ ID NO: 81) and GAH WQFNALTVRGGGC (SEQ ID NO: 82) were dissolved in water and each was added at one-half molar equivalent to the number of BMPH reactive groups on chondroitin. For example, chondroitin Sulfate with an average of about 22 BMPH functional groups was conjugated with about 11 WYRGRLGC (SEQID NO: 81) peptides and about 11 GAH WQFNALTVRGGGC (SEQID NO: 82) peptides. The mix ture containing both peptides was reacted for 2 hours at room temperature followed by purification in ultrapure water and lyophilization.
All intermediates were purified by size-exclusion chroma tography using an AKTA Purifier FPLC (GE Healthcare) and a column packed with polyacrylamide beads (Bio-Rad Labs). The final product was similarly purified using a column packed with Sephadex G-25 beads. After synthesis, the syn thetic peptidoglycan can be lyophilized and stored for extended periods of time at -80° C. Example 2

Lubricin Mimetic
The ability of the lubrican mimetic peptidoglycan to reduce the frictional force between cartilage surfaces was tested as follows. A cartilage plug was glued to the plate of a rheometer, and a second cartilage plug was glued to the rotat ing fixture of the rheometer. The two cartilage surfaces were put together with a 5N force, and then a shear was applied. The torque was measured and used to calculate the frictional force between the surfaces. Rheometer  2 shows the frictional force in the case of damaged cartilage (with aggrecan depletion to simulate osteoarthritis) and shows that when the lubricin mimetic peptidoglycan accord ing to the disclosure is added to the cartilage, friction is lowered between the cartilage Surfaces. Accordingly, when the lubrican mimetic synthetic peptidoglycan according to the disclosure was applied between the two cartilage Surfaces, the frictional force was decreased at higher oscillatory shear, Suggesting that it can protect the cartilage during rapid move mentS.

Example 3
Vitreous Humor Mimetic

Methods
The synthetic peptidoglycans according to the present dis closure can be tested as follows. Viscoelastic properties can be measured using a rheometer, for example the TAInstru ments ARG2 rheometer. A cone and plate geometry or a parallel plate geometry can be used. Vitreous humor (e.g., bovine) can be placed within the geometry. The temperature can be controlled and frequency Sweeps can be performed from ranges of about 0.1 Hz to about 100 Hz, (e.g., 1 Hz) and storage, loss, and complex modulus can be measured at an oscillatory shear stress between about 0.1 Pa and about 10 Pa (e.g., 10 Pa). Temperature: 37° C. Data FIG. 3A shows the rheological measurements on bovine vitreous with no treatment (control), PBS buffer and trypsin, where the trypsin treated bovine vitreous exhibited a lower Viscosity, thus mimicking the disease state.
In FIG. 3B and FIG. 3C, GAH is a synthetic peptidoglycan comprising chondroitin Sulfate with hyaluronic acid binding peptide (i.e., GAHWOFNALTVRGGGC (SEQID NO: 82)) covalently bonded thereto (via BMPH), WYR is a synthetic peptidoglycan comprising chondroitin Sulfate with collagen II binding peptide (i.e., WYRGRLGC (SEQ ID NO: 81)) covalently bonded thereto (via BMPH), and WYRGAH is a mixture of the two synthetic peptidoglycans WYR and GAH just described. Based on the data shown in FIGS. 3A, 3B and 3C, it is contemplated that the present synthetic peptidoglycans can be used to treat diseases and disorders affecting the vitreous humor.

Synthetic -Continued
Gly Ala His Trp Glin Phe Asn Ala Lieu. Thr Val Arg 24. A method of treating and/or preventing degradation of a hyaluronic acid rich tissue in a patient comprising admin istering to a patient in need thereof the pharmaceutical com position of claim 23.
25. The method of claim 24, wherein the hyaluronic acid rich tissue is the skin.
26. A method of treating and/or preventing cartilage degen eration in a patient comprising administering to a patient in need thereof the pharmaceutical composition of claim 23. 27. A method of treating and/or preventing cartilage degen eration in a patient comprising injecting the pharmaceutical composition of claim 23 into a synovial cavity of a patient in need thereof. 28. A method of treating and/or preventing vitreous humor degeneration in a patient comprising administering to a patient in need thereof the pharmaceutical composition of claim 23. 29. A method of treating and/or preventing nucleus pulpo SuS degeneration in a patient comprising administering to a patient in need thereof the pharmaceutical composition of claim 23.