WO2016161333A2 - Compositions comprising bioconjugates - Google Patents

Abstract

This disclosure provides extracellular matrix-binding bioconjugates comprised of one or more synthetic peptides conjugated to a glycan and methods of their use.

Description

COMPOSITIONS COMPRISING BIOCONJUGATES

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Serial No. 62/141,683 filed on April 1, 2015, the entire disclosure of which is hereby incorporated by reference.

GOVERNMENT INTERESTS STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

OR DEVELOPMENT This invention was made with government support under AR065398 awarded by the

National Institutes of Health. The government has certain rights in the invention.

FIELD

This disclosure provides extracellular matrix-binding bioconjugates 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). Lubricin 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, CP., K. Komvopoulos, and A.H. Reddi, Tissue Engineering, Part B: Reviews, 2008). In the absence of a strongly adsorbing, continuous, self -replenishing boundary lubricant layer, intermittent asperity-asperity interactions lead to rapid deterioration of the join surface by various mechanical wear processes, such as adhesion, abrasion, surface fatigue, and delamination. Lubricin tribo supplementation 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, 39(1): 164-72). Reducing friction at the articular cartilage interface will suppress cartilage wear and surface damage. Another extracellular matrix rich tissue is the vitreous humor, a complex 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 pathologies, 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 pressure 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 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.

U.S. Pub. No. 2014/0288002 discloses extracellular matrix -binding bioconjugates, the disclosure of which is incorporated herein by reference in its entirety, describing extracellular matrix -binding bioconjugates comprising a glycan, from about 1 to about 80 collagen-binding peptide(s) and from about 1 to about 80 hyaluronic acid-binding peptide(s). SUMMARY

It is desirable to have extracellular matrix -binding bioconjugates that reduce friction at the cartilage surface. It was unexpectedly found that the friction at the cartilage surface can be more effectively decreased by controlling the number of hyaluronic acid-binding peptide(s) in the bioconjugates and also by maintaining about 3: 1 or about 4: 1 ratio of the collagen-binding peptide(s) to hyaluronic acid-binding peptide(s). This disclosure provides bioconjugates that lower friction at the cartilage surface. Provided herein is a bioconjugate comprising: a) a glycan; b) from about 3 to about 30 collagen-binding peptides having at least one collagen-binding domain; and c) from about 1 to about 10 hyaluronic acid-binding peptide(s) having at least one hyaluronic acid-binding domain; wherein the ratio of collagen-binding peptides to hyaluronic acid-binding peptide(s) is about 3: 1 or about 4: 1, and wherein the peptides are covalently bonded to the glycan. The bioconjugate can comprise any glycan, including, but not limited to, alginate, dextran, dextran sulfate, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, heparan, heparan sulfate, heparin, keratin, keratan sulfate, or hyaluronic acid.

Also provided is a pharmaceutical composition comprising a bioconjugate

comprising: a) a glycan; b) from about 3 to about 30 collagen-binding peptides having at least one collagen-binding domain; and c) from about 1 to about 10 hyaluronic acid-binding peptide(s) having at least one hyaluronic acid-binding domain; wherein the ratio of collagen- binding peptides to hyaluronic acid-binding peptide(s) is about 3: 1 or about 4: 1, and wherein the peptides are covalently bonded to the glycan. Bioconjugates as described herein may be useful 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 degradation of a hyaluronic acid rich tissue in a patient comprising administering to a patient in need thereof the bioconjugate described herein and a viscosupplement. In addition, provided is a method of treating and/or preventing cartilage degeneration in a patient comprising administering to a patient in need thereof the bioconjugate described herein and a viscosupplement. Also provided is a method of treating and/or preventing vitreous humor degeneration in a patient comprising administering to a patient in need thereof the bioconjugate described herein. In addition, provided is a method of treating and/or preventing nucleus pulposus degeneration in a patient comprising administering to a patient in need thereof the bioconjugate described herein. Also, provided is a method of decreasing scar formation in a patient comprising administering to a patient in need thereof the bioconjugate described herein and a viscosupplement.

Also provided herein is a bioconjugate comprising: a) chondroitin sulfate; b) WYRGRL; and c) G AHWQFN ALT VRGG ; wherein the ratio of WYRGRL and GAHWQFNALTVRGG is about 3: 1, and

WYRGRL and GAHWQFNALTVRGG are covalently bonded to the chondroitin sulfate.

BRIEF DESCRIPTION OF THE DRAWINGS

Certain aspects are best understood from the following detailed description when read in conjunction with the accompanying drawings. Certain acronyms used in the description may be found in the Table of Abbreviations. Included in the drawings are the following figures:

FIG. 1 shows the results of a binding assay to assess mLublO binding with hyaluronic acid sodium salt.

FIG. 2 shows mLubl5 binding with hyaluronic acid sodium salt. Comparing with FIG. 1 shows that mLubl5 binds less HA than mLublO, which agrees with the decrease in the number of HA -binding peptides on the mLubl5 molecule.

FIG. 3 shows the results of a binding assay to assess mLublO binding with collagen II. This shows that the mLublO binds to collagen II.

FIG. 4 shows the results of a binding assay to assess mLubl5 binding with collagen II assay. This shows that the mLubl5 binds to collagen II.

FIG. 5 shows mLubl5 binding curves with collagen II and HA.

FIG. 6 shows typical normal force and torque graphs during macroscale coefficient of friction testing using a rheometer.

FIG. 7 shows total static and kinetic coefficient of friction (COF) data from rheometer. The rheometer data was of cartilage on glass. The static and kinetic COF values were calculated for each treatment group. In static COF, there is statistical difference between the trypsin treated plug and the WT, mLubl5 + Synvisc, and mLubl5 + HA treatments. In kinetic friction, there is statistical difference between the mLubl5 +Synvisc treatment and the trypsin, Synvisc, and mLublO + Synvisc treatments. Standard error bars are shown. ( p<0.05 for both static and kinetic COF differences). Each treatment group had a different n value (ranging from n=9 to 12). Thus, trypsin treated plugs that were treated with mLubl5 then Synvisc had lower static COF and significantly lower kinetic COF than the wild type, whereas trypsin treated plugs that were treated with mLublO then Synvisc had higher static as well as kinetic COF than the wild type. FIG. 8 shows fluorescent staining images of cryosections of cartilage with mLub probed with streptavidin and DAPI for nuclei. The left image represents a cartilage sample that did not go through the compression and shear movements on the rheometer while the right image was cryosectioned after the rheometer test. FIG. 8 shows that there is still mLub present after compression and shear movement. Arrows point to the mLub covered cartilage surface.

FIG. 9 shows 3D and 2D topography images of cartilage surfaces, WT, Trypsin treated, and Trypsin treated later followed by a mLub then Synvisc treatment. Images represent samples with roughness values near the values of those shown in FIG. 10. FIG. 10 shows friction coefficient, roughness and adhesion force on cartilage surface.

Friction coefficient and roughness values of selected areas of cartilage surfaces (n = 9) (p > 0.05) are shown. Standard error bars are shown. Friction was measured at selected areas within a 50 by 50 micron area where clear fibers are imaged and no AFM controller interference. This was done on 5 areas of each sample and averaged. COF was calculated by dividing the measured friction force by normal force. Adhesion values of a small subset of cartilage samples (n=2)(p < 0.05) are shown. Standard error bars are shown.

FIG. 11 shows fluorescently labeled cartilage cryosections with DAPI for cell nuclei and streptavidin for biotinylated mLubl5. FIG. 11 shows toluidine blue stained cartilage cryosections to show proteoglycan depletion with trypsin (scale bar for upper (top) panel = 50 μηι, scale bar for lower (bottom) panel = 200 μιη).

FIG. 12 shows fluorescent imaging of the articular cartilage of guinea pig joints injected with PBS (control) or mLub and harvested after at 6 hours, 1 week and 2 weeks after in vivo injection. PBS is on the top row and mLub 15 is on the bottom row (scale bar = 50 μιη). Fluorescent streptavidin is bound to the biotinylated mLub. The arrow points to mLub 15 present on the cartilage surface. The scale bar represents 50 mm The inset image in the bottom panel (on the left) shows native SZP staining alone after 6 h.

FIG. 13 shows an enlarged version of the inset image in the bottom panel (on the left) of FIG. 12.

FIG. 14 shows trypsin treated cartilage with mLub 15 treatment. Cell nuclei are stained with DAPI. Streptavidin that has bound to the biotinylated mLub showing that mLub is bound at the surface of cartilage. FIG. 15 shows 10X magnification of FIG. 14, trypsin treated cartilage with mLubl5 treatment.

FIG. 16 shows medial femur cartilage degeneration scores for three different zones of cartilage samples taken from guinea pigs treated with mLubl5 or a control.

FIG. 17 shows medial tibial cartilage degeneration scores for three different zones of cartilage samples taken from guinea pigs treated with mLubl5 or a control.

FIGS. 18-19 show % collagen degeneration measured in cartilage samples taken from guinea pigs treated with mLubl5 (or a control) across a population of guinea pigs having varying degrees of severity of osteoarthritis.

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 embodiments 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.

Table of Abbreviations

The following abbreviations used herein have the following meanings.

FPLC Fast protein liquid chromatography

HBSS Hank's Balanced Salt Solution

HEPES Hydroxyethyl piperazineethanesulfonic acid

hr Hour

kg Kilogram

M Molar

m Meter

MES 2-ethanesulfonic acid

mg Milligram

niL Milliliter

mm Millimeter

mM Millimolar

mV Millivolt

MOPS 3 -(N-morpholino)propanesulfonic acid

N Newton

nm Nanometer

nN Nanonewton

PBS Phosphate buffered saline

PIC Protease inhibitor cocktail

PIPES Piperazine-Ν,Ν '-bis(2-ethanesulfonic acid) rad/sec Radian per second

RMS Root mean square

RT Room temperature

Sec Seconds

3-[[l,3-dihydroxy-2-(hydroxymethyl)propan-2-

TAPS

yl]amino]propane-l-sulfonic acid

WT Wild type

wt% Weight percent

Lubricin mimic made with chondroitin-6-sulfate (CS) and 10 mol WYRGRL (collagen II binding) and 10 mol

mLublO GAHWQFNALTVRGG (HA-binding) wherein the peptides are covalently bonded to CS via a linker, N-[P-maleimidopropionic acid]hydrazide Lubricin mimic made with chondroitin-6-sulfate (CS) and 15

mol WYRGRL (collagen II binding) and 5 mol

mLubl5 GAHWQFNALTVRGG (HA-binding) wherein the peptides are

covalently bonded to CS via a linker, N-[P-maleimidopropionic acid]hydrazide

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 following 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 methods, 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 designation, e.g., temperature, time, amount, and concentration, including range, indicates approximations which may vary by (+) or (-) 10%, 5% or 1%.

As used herein, the terms "bioconjugate", " synthetic peptidoglycan",

"peptidoglycan", "proteoglycan", and "synthetic proteoglycan" are used interchangeably and refer to a synthetic conjugate that comprises a glycan and one or more synthetic peptides covalently bonded thereto. The glycan portion can be made synthetically or derived from animal sources. In certain embodiments, the peptides are covalently bound to the glycan. The synthetic peptides can be covalently bonded directly to the glycan or via a linker. For methods of conjugating 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" and "bonded" are used interchangeably and refer 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 having a large number of monosaccharides linked glycosidically. In certain embodiments, the glycan is a

glycosaminoglycans, which comprise 2-aminosugars linked in an alternating fashion with uronic acids, and include polymers such as heparin, heparan sulfate, chondroitin, keratin, and dermatan. Accordingly, non-limiting examples of glycans which can be used in the embodiments described herein include alginate, agarose, dextran, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, heparan, heparin, keratin, keratan sulfate, and hyaluronic acid, including derivatives thereof. In another embodiment the molecular weight of the glycan is varied to tailor the effects of the bioconjugate (see e.g., Radek, K. A., et al., Wound Repair Regen., 2009, 17: 118-126; and Taylor, K. R., et al., J. Biol. Chem., 2005, 280:5300- 5306). In one embodiment, the glycan is degraded by oxidation and alkaline elimination (see e.g., Fransson, L. A., et al., Eur. J. Biochem., 1980, 106 :59-69) to afford degraded glycan having a lower molecular weight (e.g., from about 10 kDa to about 50 kDa.

Hyaluronic Acid-Binding Peptides

As used herein, "hyaluronic acid-binding peptide" refers to a synthetic peptide comprising a hyaluronic acid-binding unit (or sequence). As used herein, "hyaluronic acid- binding unit" is intended to refer to an amino acid sequence within a peptide which binds to hyaluronic acid. "Hyaluronic acid-binding" indicates an interaction with hyaluronic acid that could include hydrophobic, ionic (charge), and/or Van der Waals interactions, such that the compound binds or interacts favorably with hyaluronic acid. This binding (or interaction) is intended to be differentiated from covalent bonds and nonspecific interactions with common functional groups, such that the hyaluronic acid-binding peptide would interact with any species containing that functional group to which the peptide binds on the hyaluronic acid. See, e.g., Becerra, S.P., et al. J. Biol. Chem., 2008, 283: 33310-33320. In one embodiment, the peptide, or the hyaluronic acid-binding unit, binds to hyaluronic acid with a dissociation constant (¾) of less than about 1 mM, or less than about 900 μΜ, or less than about 800 μΜ, or less than about 700 μΜ, or less than about 600 μΜ, or less than about 500 μΜ, or less than about 400 μΜ, or less than about 300 μΜ, or less than about 200 μΜ, or less than about 100 μΜ. In certain embodiments, the hyaluronic acid-binding unit of the synthetic bioconjugate can comprise an amino acid sequence derived from hyaluronan-mediated motility receptor (RHAMM) (exemplary sequences include, but are not limited to,

NP_001136028, NP_001136029, NP_036616, and NP_036617). In the various embodiments described herein, the peptide component of the bioconjugate can comprise an amino acid sequence selected from the group consisting of: GAHWQFNALTVR (SEQ ID NO: ), GDRRRRRMWHRQ(SEQ ID NO: ),

GKHLGGKHRRSR (SEQ ID NO: ), RGTHHAQKRRS (SEQ ID NO: ), RRHKSGHIQGSK (SEQ ID NO: ), SRMHGRVRGRHE (SEQ ID NO: ), RRRAGLTAGRPR (SEQ ID NO: ), RYGGHRTS RKW V (SEQ ID NO: ), RSARYGHRRGVG (SEQ ID NO: ),

GLRGNRRVFARP(SEQ ID NO: ), SRGQRGRLGKTR (SEQ ID NO: ),

DRRGRS S LPKLAGP VEFPDRKIKGRR (SEQ ID NO: ), RMRRKGRVKHWG (SEQ ID NO: ), RGGARGRHKTGR (SEQ ID NO: ), TGARQRGLQGGWGPRHLRGKDQPPGR (SEQ ID NO: ), RQRRRDLTRVEG (SEQ ID NO: ),

STKDHNRGRRNVGPVSRSTLRDPIRR (SEQ ID NO: ), RRIGHQVGGRRN (SEQ ID NO: ), RLES R A AGQRR A (SEQ ID NO: ), GGPRRHLGRRGH (SEQ ID NO: ),

VS KRGHRRT AHE (SEQ ID NO: ), RGTRSGSTR (SEQ ID NO: ), RRRKKIQGRSKR (SEQ ID NO: ), RKSYGKYQGR (SEQ ID NO: ), KNGRYSISR (SEQ ID NO: ),

RRRCGQKKK (SEQ ID NO: ), KQKIKHVVKLK (SEQ ID NO: ), KLKSQLVKRK (SEQ ID NO: ), RYPISRPRKR (SEQ ID NO: ), KVGKSPPVR (SEQ ID NO: ), KTFGKMKPR

(SEQ ID NO: ), RIKWSRVSK (SEQ ID NO: ) KRTMRPTRR (SEQ ID NO: ), or a sequence having at least about 80% sequence identity, or at least about 83% 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 thereto, provided the sequence is capable of binding to hyaluronic acid. Additional peptides that can be included as the peptide component of the hyaluronic acid-binding bioconjugates include peptides which have an Arg-Arg (R-R) motif, such as one or more peptides selected from the group consisting of RRASRSRGQVGL (SEQ ID NO: ), GRGTHHAQKRRS (SEQ ID NO: ), QPVRRLGTPVVG (SEQ ID NO: ), ARRAEGKTRMLQ (SEQ ID NO: ),

PKVRGRRHQ AS G (SEQ ID NO: ), SDRHRRRREADG (SEQ ID NO: ),

NQRVRRVKHPPG (SEQ ID NO: ), RERRERHAVARHGPGLERDARNLARR (SEQ ID NO: ), TVRPGGKRGGQVGPPAGVLHGRRARS (SEQ ID NO: ), N VRS RRGHRMNS (SEQ ID NO: ), DRRRGRTRNIGN (SEQ ID NO: ), KTAGHGRRWSRN (SEQ ID NO: ), AKRGEGRREWPR (SEQ ID NO: ), GGDRRKAHKLQA (SEQ ID NO: ),

RRGGRKWGSFEG (SEQ ID NO: ) and RQRRRDLTRVEG (SEQ ID NO: ) (see, e.g., Amemiya et al, Biochem. Biophys. Acta, 2005, 1724, 94-99, incorporated herein by reference). In another embodiment, the peptide is selected from the group consisting of RDGTRYVQKGEYR (SEQ ID NO: ), HREARSGKYK (SEQ ID NO: ), PDKKHKLYGV (SEQ ID NO: ), and WDKERSRYDV (SEQ ID NO: ) (see, e.g., Yang et al, EMBO Journal, 1994, 13, 286-296, and Goetinck et al, J. Cell. Biol, 1987, 105, 2403-2408, both of which are incorporated herein by reference).

In other embodiments described herein, the hyaluronic acid-binding peptide component of the synthetic bioconjugate can comprise an amino acid sequence selected from GAHWQFNALTVR (SEQ ID NO: ), S TMMS RS HKTRS HH V (SEQ ID NO: ),

TMTRPHFHKRQLVLS (SEQ ID NO: ), STMMSRSHKTRSCHH (SEQ ID NO: ),

S TMMS RS HKTRS HH (SEQ ID NO: ), GDRRRRRMWHRQ (SEQ ID NO: ),

GKHLGGKHRRSR (SEQ ID NO: ), RGTHHAQKRRS (SEQ ID NO: ), RRHKSGHIQGSK (SEQ ID NO: ), SRMHGRVRGRHE (SEQ ID NO: ), RRRAGLTAGRPR (SEQ ID NO: ), RYGGHRTS RKW V (SEQ ID NO: ), RS ARYGHRRG VG (SEQ ID NO: ),

GLRGNRRVFARP (SEQ ID NO: ), SRGQRGRLGKTR (SEQ ID NO: ),

DRRGRS S LPKLAGP VEFPDRKIKGRR (SEQ ID NO: ), RMRRKGRVKHWG (SEQ ID NO: ), RGGARGRHKTGR (SEQ ID NO: ), TGARQRGLQGGWGPRHLRGKDQPPGR (SEQ ID NO: ), RQRRRDLTRVEG (SEQ ID NO: ),

S TKDHNRGRRN VGP VS RS TLRDPIRR (SEQ ID NO: ), RRIGHQVGGRRN (SEQ ID NO: ), RLES R A AGQRR A (SEQ ID NO: ), GGPRRHLGRRGH (SEQ ID NO: ),

VS KRGHRRT AHE (SEQ ID NO: ), RGTRSGSTR (SEQ ID NO: ), RRRKKIQGRSKR (SEQ ID NO: ), RKSYGKYQGR (SEQ ID NO: ), KNGRYSISR (SEQ ID NO: ),

RRRCGQKKK (SEQ ID NO: ), KQKIKHVVKLK (SEQ ID NO: ), KLKSQLVKRK (SEQ ID NO: ), RYPISRPRKR (SEQ ID NO: ), KVGKSPPVR (SEQ ID NO: ), KTFGKMKPR (SEQ ID NO: ), RIKWSRVSK (SEQ ID NO: ) and KRTMRPTRR (SEQ ID NO: ), or a sequence having at least about 80% sequence identity, or at least about 83% 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 thereto, provided the sequence is capable of binding to hyaluronic acid.

Additional peptides that can be included as the peptide component of the hyaluronic acid-binding synthetic bioconjugate include peptides which have an Arg-Arg (R-R) motif, such as one or more peptides selected from RRASRSRGQVGL (SEQ ID NO: ), GRGTHHAQKRRS (SEQ ID NO: ), QPVRRLGTPVVG (SEQ ID NO: ),

ARRAEGKTRMLQ (SEQ ID NO: ), PKVRGRRHQ AS G (SEQ ID NO: ),

SDRHRRRREADG (SEQ ID NO: ), NQRVRRVKHPPG (SEQ ID NO: ),

RERRERHAVARHGPGLERDARNLARR (SEQ ID NO: ),

TVRPGGKRGGQVGPPAGVLHGRRARS (SEQ ID NO: ), N VRS RRGHRMNS (SEQ ID NO.), DRRRGRTRNIGN (SEQ ID NO: ), KTAGHGRRWSRN (SEQ ID NO: ),

AKRGEGRREWPR (SEQ ID NO: ), GGDRRKAHKLQA (SEQ ID NO: ),

RRGGRKWGSFEG (SEQ ID NO: ), and RQRRRDLTRVEG (SEQ ID NO: ) (see, e.g., Amemiya et al., Biochem. Biophys. Acta, 2005, 1724, 94-99, incorporated herein by reference); or a sequence having at least about 80% sequence identity, or at least about 83% 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 thereto, provided the sequence is capable of binding to hyaluronic acid. In another embodiment, the peptide is selected from RDGTRYVQKGEYR (SEQ ID NO: ),

HREARSGKYK (SEQ ID NO: ), PDKKHKLYGV (SEQ ID NO: ), and WDKERSRYDV (SEQ ID NO: ) (see, e.g., Yang, B., et al, EMBO Journal, 1994, 13, 286-296, and Goetinck, P.F. et al, J. Cell. Biol, 1987, 105, 2403-2408, both of which are incorporated herein by reference); or a sequence having at least about 80% sequence identity, or at least about 83% 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 thereto, provided the sequence is capable of binding to hyaluronic acid.

Peptides may also be selected by phage display, utilizing positive selection for binding to hyaluronic acid. A hyaluronic acid-binding peptide may be determined by its interaction with hyaluronic acid, and measured by any of the techniques used to evaluate molecular interactions (such as surface plasmon resonance, ELISA, competitive binding assays, isothermal titration calorimetry, affinity chromatography, rheology and/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 residues in the peptide. The interaction may be measured by 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 chromophore. The interaction may also be measured by mechanical influence on hyaluronic acid-containing fluids, gels, or tissues that have been incubated with the hyaluronic acid-binding peptide or with a synthetic bioconjugate containing an hyaluronic acid-binding peptide or peptides. For identifying a peptide, a peptide selected from phage display, or one that is identified from a hyaluronic acid-binding 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.

Other various methods for screening peptide sequences for hyaluronic acid-binding affinity (or a hyaluronic acid-binding domain/unit), which are routine in the art, may be used to select collagen-binding peptides suitable for use in bioconjugates described herein.

Collagen-Binding Peptides As used herein, the term "collagen-binding peptide" refers to a synthetic peptide comprising a collagen -binding unit (or sequence). As used herein, the term "collagen- binding unit" is intended to refer to an amino acid sequence within a peptide which binds to collagen. "Collagen-binding" indicates an interaction with collagen that could include hydrophobic, ionic (charge), and/or Van der Waals interactions, such that the compound binds or interacts favorably with collagen. This binding (or interaction) is intended to be differentiated from covalent bonds and nonspecific interactions with common functional groups, such that the peptide would interact with any species containing that functional group to which the peptide binds on the collagen. Peptides can be tested and assessed for binding to collagen using any method known in the art. See, e.g., Li, Y., et al., Current Opinion in Chemical Biology, 2013, 17: 968-975, Helmes, B.A., et al., J. Am. Chem. Soc. 2009, 131, 11683-11685, and Petsalaki, E., et al., PLoS Comput Biol, 2009, 5(3): el000335. In one embodiment, the peptide, or the collagen-binding unit of the peptide, binds to collagen with a dissociation constant (¾) of less than about 1 mM, or less than about 900 μΜ, or less than about 800 μΜ, or less than about 700 μΜ, or less than about 600 μΜ, or less than about 500 μΜ, or less than about 400 μΜ, or less than about 300 μΜ, or less than about 200 μΜ, or less than about 100 μΜ. 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 embodiment, the collagen-binding 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 fibrillo genesis.

In various embodiments, the collagen-binding peptide comprises an amino acid sequence selected from the group consisting of WYRGRL (SEQ ID NO: ),

RRANAALKAGELYKSILY (SEQ ID NO: ), RLDGNEIKR (SEQ ID NO: ),

AHEEISTTNEGVM (SEQ ID NO: ), GELYKSILY (SEQ ID NO: ),

NGVFKYRPRYFLYKHAYFYPPLKRFPVQ (SEQ ID NO: ), CQDSETRTFY (SEQ ID NO: ), TKKTLRT (SEQ ID NO: ), GLRS KS KKFRRPDIQYPD ATDEDITSHM (SEQ ID NO: ), SQNPVQP (SEQ ID NO: ), SYIRIADTNIT (SEQ ID NO: ), KELNLVYT (SEQ ID NO: ), GSITTIDVPWNVGC (SEQ ID NO: ), GSITTIDVPWNV (SEQ ID NO: ) or a sequence having at least about 80% sequence identity, or at least about 83% 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 thereto, provided the sequence is capable of binding to collagen.

In other embodiments, the peptide comprises an amino acid sequence selected from RRANAALKAGELYKSILY (SEQ ID NO: ), GELYKSILY (SEQ ID NO: ),

RRANAALKAGELYKCILY (SEQ ID NO: ), GELYKCILY (SEQ ID NO: ), RLDGNEIKR (SEQ ID NO: ), AHEEISTTNEGVM (SEQ ID NO: ),

NGVFKYRPRYFLYKHAYFYPPLKRFPVQ (SEQ ID NO: ), CQDSETRTFY (SEQ ID NO: ), TKKTLRT (SEQ ID NO: ), GLRS KS KKFRRPDIQYPD ATDEDITSHM (SEQ ID NO: ), SQNPVQP (SEQ ID NO: ), SYIRIADTNIT (SEQ ID NO: ), KELNLVYT (SEQ ID NO: ), GSIT (SEQ ID NO: ), GSITTIDVPWNV (SEQ ID NO: ), GQLYKSILY (SEQ ID NO: ), RR AN A ALKAGQLYKS ILY (SEQ ID NO: ), or a sequence having at least about 80% sequence identity, or at least about 83% 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 thereto, provided the sequence is capable of binding to collagen.

In certain embodiments, the collagen-binding peptide comprises an amino acid sequence that has about 80%, about 85%, about 90%, about 95%, about 98%, or about 100% sequence identity 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 (1997), Romijn, et al., J. Biol. Chem. 278: 15035-15039 (2003), and Chiang, et al., Cardio. & Haemato. Disorders-Drug Targets 7: 71- 75 (2007), each incorporated herein by reference. A non-limiting example is WREPSFCALS (SEQ ID NO: ), derived from vWF.

Various methods for screening peptide sequences for collagen-binding affinity (or a collagen-binding domain/unit) are routine in the art. Other peptide sequences shown to have collagen-binding affinity (or a collagen-binding unit) which can be used in the bioconjugates and methods disclosed herein include but are not limited to, pAWHCTTKFPHHYCLYBip (SEQ ID NO: ), pAHKCPWHLYTTHYCFTBip (SEQ ID NO: ), pAHKCPWHLYTHYCFT (SEQ ID NO: ), etc., where Bip is biphenylalanine and βΑ is beta-alanine (see, Abd-Elgaliel, W.R., et al., Biopolymers, 2013, 100(2), 167-173), GROGER (SEQ ID NO: ), GMOGER (SEQ ID NO: ), GLOGEN (SEQ ID NO: ), GLOGER (SEQ ID NO: ), GLKGEN (SEQ ID NO: ), GFOGERGVEGPOGPA (SEQ ID NO: ), etc., where O is 4-hydroxyproline (see, Raynal, N., et al., J. Biol. Chem., 2006, 281(7), 3821-3831), HVWMQAPGGGK (SEQ ID NO: ) (see, Helms, B.A., et al., J. Am. Chem. Soc. 2009, 131, 11683-11685),

WREPSFCALS (SEQ ID NO: ) (see, Takagi, J., et al., Biochemistry, 1992, 31, 8530-8534), WYRGRL (SEQ ID NO: ), etc. (see, Rothenfluh D.A., et al., Nat Mater. 2008, 7(3), 248-54), WTCS GDE YTWHC (SEQ ID NO: ), WTCVGDHKTWKC (SEQ ID NO: ),

QWHCTTRFPHHYCLYG (SEQ ID NO: ), etc. (see, U.S. 2007/0293656),

STWTWNGSAWTWNEGGK (SEQ ID NO: ), STWTWNGTNWTRNDGGK (SEQ ID NO: ), etc. (see, WO/2014/059530), CVWLWEQC (SEQ ID NO: ) cyclic CVWLWENC (SEQ ID NO: ), cyclic CVWLWEQC (SEQ ID NO: ), (see, Depraetere H., et al., Blood. 1998, 92, 4207-4211, and Duncan R., Nat Rev Drug Discov, 2003, 2(5), 347-360), CMTSPWRC (SEQ ID NO: ), etc. (see, Vanhoorelbeke, K., et al., J. Biol. Chem., 2003, 278, 37815-37821),

CPGRVMHGLHLGDDEGPC (SEQ ID NO: ) (see, Muzzard, J., et al., PLoS one. 4 (e 5585) I- 10), KLWLLPK (SEQ ID NO: ) (see, Chan, J. M., et al., Proc Natl Acad Sci U.S.A., 2010, 107, 2213- 2218), and CQDSETRTFY (SEQ ID NO: ), etc. (see, U.S. 2013/0243700), H-V- F/W-Q/ M-Q-P/A-P/K (Helms, B.A., et al., J. Am. Chem. Soc, 2009, 131(33), 11683- 11685), wherein each is hereby incorporated by reference in its entirety.

Additional peptide sequences shown to have collagen-binding affinity (or a collagen- binding unit) which can be used in the bioconjugates and methods disclosed herein include but are not limited to, LSELRLHEN (SEQ ID NO: ), LTELHLDNN (SEQ ID NO: ), LSELRLHNN (SEQ ID NO: ), LSELRLHAN (SEQ ID NO: ), and LRELHLNNN (SEQ ID NO: ) (see, Fredrico, S., Angew. Chem. Int. Ed. 2015, 37, 10980-10984).

In certain embodiments, the peptides include one or more sequences selected from the group consisting of RVMHGLHLGDDE (SEQ ID NO: ), D-amino acid EDDGLHLGHMVR (SEQ ID NO: ), RVMHGLHLGNNQ (SEQ ID NO: ), D-amino acid QNNGLHLGHMVR (SEQ ID NO: ), RVMHGLHLGNNQ (SEQ ID NO: ), GQLYKSILYGSG-4K₂K (SEQ ID NO: ) (a 4-branch peptide), GSGQLYKSILY (SEQ ID NO: ), GSGGQLYKSILY (SEQ ID NO: ), KQLNLVYT (SEQ ID NO: ), CVWLWQQC (SEQ ID NO: ), WREPSFSALS (SEQ ID NO: ), GHRPLDKKREEAPSLRPAPPPISGGGYR (SEQ ID NO: ), and

GHRPLNKKRQQ APS LRP APPPIS GGG YR (SEQ ID NO: ).

Additionally, peptides may be identified from a phage display library selected for collagen 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 (e.g., SILY_bi_otin) 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, the peptides comprise one or more collagen-binding units which binds any one or more of collagen type I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV. In one embodiment, the peptide binds to type I collagen with a dissociation constant (¾) of less than about 1 mM, or less than about 900 μΜ, or less than about 800 μΜ, or less than about 700 μΜ, or less than about 600 μΜ, or less than about 500 μΜ, or less than about 400 μΜ, or less than about 300 μΜ, or less than about 200 μΜ, or less than about 100 μΜ. In one embodiment, the peptide binds to type II collagen with a dissociation constant (¾) of less than about 1 mM, or less than about 900 μΜ, or less than about 800 μΜ, or less than about 700 μΜ, or less than about 600 μΜ, or less than about 500 μΜ, or less than about 400 μΜ, or less than about 300 μΜ, or less than about 200 μΜ, or less than about 100 μΜ. In one embodiment, the peptide binds to type III collagen with a dissociation constant (K^) of less than about 1 mM, or less than about 900 μΜ, or less than about 800 μΜ, or less than about 700 μΜ, or less than about 600 μΜ, or less than about 500 μΜ, or less than about 400 μΜ, or less than about 300 μΜ, or less than about 200 μΜ, or less than about 100 μΜ. In one embodiment, the peptide binds to type IV collagen with a dissociation constant (¾) of less than about 1 mM, or less than about 900 μΜ, or less than about 800 μΜ, or less than about 700 μΜ, or less than about 600 μΜ, or less than about 500 μΜ, or less than about 400 μΜ, or less than about 300 μΜ, or less than about 200 μΜ, or less than about 100 μΜ.

A non-limiting example of a collagen-binding unit which binds type IV collagen is TLTYTWS (SEQ ID NO: _) which binds specifically to MMP 2 and 9-degraded basement membrane type IV collagen. Likewise, TLTYTWS GSG (SEQ ID NO: _) which further includes a GSG linker can also bind to cleaved or degraded type IV collagen specifically.

Another example is KLWVLPK (SEQ ID NO: ) which selectively binds to intact type IV collagen.

Other various methods for screening peptide sequences for collagen-binding affinity (or a collagen-binding domain/unit), which are routine in the art, may be used to select collagen-binding peptides suitable for use in bioconjugates described herein.

In any of the embodiments described herein, any one or more of the synthetic peptides (e.g., the hyaluronic acid-binding peptide and/or the collagen-binding peptide) may have a glycine-cysteine (GC) attached to the C-terminus of the peptide, and/or a glycine-cysteine- glycine (GCG) attached to the N-terminus of the peptide. For example, the collagen-binding peptide may comprise an amino acid sequence selected from the group consisting of

WYRGRLGC, RRANAALKAGELYKSILYGC, RLDGNEIKRGC, AHEEISTTNEGVMGC , GCGGELYKSILY, NGVFKYRPRYFLYKHAYFYPPLKRFPVQGC, CQDSETRTFYGC, TKKTLRTGC, GLRSKSKKFRRPDIQYPDATDEDITSHMGC, SQNPVQPGC,

SYIRIADTNITGC, KELNLV YTGC , GSITTIDVPWNVGC, GCGGELYKS ILYGC and GELYKS ILYGC .

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 occurring amino 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.

Table 1

1-Letter 3-Letter Amino Acid

F Phe L-phenylalanine

M Met L-methionine

A Ala L- alanine

S Ser L- serine

I He L-isoleucine

L Leu L-leucine

T Thr L-threonine

V Val L-valine

P Pro L-proline

K Lys L-lysine

H His L-histidine

Q Gin L-glutamine

E Glu L-glutamic acid

W Trp L-tryptohan

R Arg L-arginine

D Asp L-aspartic acid

N Asn L-asparagine

C Cys L-cysteine

In any of the embodiments described herein, a synthetic peptide (e.g., a hyaluronic acid-binding peptide and/or a collagen-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% homology to any of these amino acid sequences. In various embodiments, the peptide components of the bioconjugate 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 occupied by the same base or amino 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 the art, 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. Illustratively, the well-known terminologies "hydrophilic residues" 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 residues" 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.

Table 2

For Amino Acid Replace With

Phenylalanine D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-Trp

Proline D-Pro

Serine D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-Cys

Threonine D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Val, D-Val

Tyrosine D-Tyr, Phe, D-Phe, His, D-His, Trp, D-Trp

Valine D-Val, Leu, D-Leu, He, D-Ile, Met, D-Met

In one embodiment, the bioconjugates 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, isothermal titration calorimetry, phage display, affinity chromatography, rheology or immunohistochemistry. The terms are also meant to include "binding" interactions between molecules. Binding may be "direct" or "indirect". "Direct" binding comprises direct physical contact between molecules. "Indirect" binding between molecules comprises the molecules having direct physical contact with one or more molecules simultaneously. For example, it is contemplated that bioconjugates of the disclosure directly bind and interact with both hyaluronic acid and collagen (e.g., 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" comprising the interacting molecules. A "complex" refers to the binding of two or more molecules held together by covalent or non-covalent bonds, interactions or forces.

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 "extracellular matrix" refers to the extracellular part of tissue that provides structural and biochemical support to the surrounding cells.

As used herein, the term "treating and/or preventing" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting one or more clinical symptoms of a disease or disorder prior to, during, and/or after the deleterious effects of a disease state, disease progression or other abnormal condition related to the degradation of a collagen- and/or hyaluronic acid-rich tissue (e.g., 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). 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 "viscosupplement" refers to any substance that is used to restore and/or increase the cushioning and lubrication of arthritic synovial fluid.

Viscosupplements include hylan, hyaluronic acid and other hyaluronan (sodium hyaluronate) compounds, which are natural complex sugars of the glycosaminoglycan family.

Hyaluronan, in particular, is a long-chain polymer containing repeating disaccharide units of Na-glucoronate-N-acetylglucosamine. By way of example, commercially available hyaluronan viscosupplements include Synvisc, Hyalgan, Supartz, and Orthovisc.

Viscosupplements may also contain additional active or inactive components including, for example, the non-steroidal anti-inflammatory drugs (NSAIDs), e.g., Ibuprofen, Diclofenac, and; anesthetics, e.g., Lidocaine and Bupivacaine; opioid analgesics, e.g., codeine and morphine; corticosteroids, e.g., dexamethasone and prednisone; antineoplastic agents such a Methotrexate, 5-fluorouracil and Paclitaxel; and anti-viral agents, e.g., Acyclovir and

Vidarabine. Viscosupplements may also contain components such as cells (e.g., chondrocytes or mesenchymal stem cells), proteins, DNA, vitamins or other desirable biologically active material. Viscosupplementation may also refer to the maintenance of the cartilage surface properties through the inhibition of synovial cell overgrowth (see Rhee DK, et al., The secreted glycoprotein lubricin protects cartilage surfaces and inhibits synovial cell overgrowth, J. Clin. Invest. 2005 Mar; 115(3):622-31). Viscosupplements may be used individually, or in combination, for improved efficacy, e.g. lubricin and hyaluronan (see Schmidt TA, et al., Boundary lubrication of articular cartilage: role of synovial fluid constituents, Arthritis Rheum. 2007 Mar; 56(3):882-91).

As used herein, the term "composition" refers to a preparation suitable for

administration to an intended patient for therapeutic purposes that contains at least one pharmaceutically active ingredient, including any solid form thereof. The composition may include at least one pharmaceutically acceptable component to provide an improved formulation of the compound, such as a suitable carrier. In certain embodiments, the composition is formulated as a film, gel, patch, or liquid solution. As used herein, the term "topically" refers to administering a composition non-systemically to the surface of a tissue and/or organ (internal or, in some cases, external) to be treated, for local effect.

As used herein, the term "pharmaceutically acceptable" indicates that the indicated material does not have properties that would cause a reasonably prudent medical practitioner to avoid administration to a patient, taking into consideration the amount used and/or the disease or conditions to be treated and the respective route of administration. Typical pharmaceutically acceptable materials are essentially sterile.

As used herein, the term "pharmaceutically acceptable carrier" refers to

pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any supplement or composition, or component thereof, from one organ, or portion of the body, to another organ, or portion of the body, or to deliver an agent to the internal surface of a vein.

As used herein, the term "formulated" or "formulation" refers to the process in which different chemical substances, including one or more pharmaceutically active ingredients, are combined to produce a dosage form. In certain embodiments, two or more pharmaceutically active ingredients can be coformulated into a single dosage form or combined dosage unit, or formulated separately and subsequently combined into a combined dosage unit. A sustained release formulation is a formulation which is designed to slowly release a therapeutic agent in the body over an extended period of time, whereas an immediate release formulation is a formulation which is designed to quickly release a therapeutic agent in the body over a shortened period of time .

As used herein, the term "delivery" refers to routes, approaches, formulations, technologies, and systems for transporting a pharmaceutical composition in the body as needed to safely achieve its desired therapeutic effect. The route of delivery can be any suitable route, including but not limited to, intravascular, intravenous, intraarterial, intramuscular, cutaneous, subcutaneous, percutaneous, intradermal, and intraepidermal routes.

As used herein, the term "concurrently" refers to simultaneous (i.e., in conjunction) administration. In one embodiment, the administration is co-administration such that two or more pharmaceutically active ingredients, including any solid form thereof, are delivered together at one time.

As used herein, the term "sequentially" refers to separate (i.e., at different times) administration. In one embodiment, the administration is staggered such that two or more pharmaceutically active ingredients, including any solid form thereof, are delivered separately at different times.

As used herein, the term "solution" refers to solutions, suspensions, emulsions, drops, ointments, liquid wash, sprays, and liposomes, which are well known in the art. In some embodiments, the liquid solution contains an aqueous pH buffering agent which resists changes in pH when small quantities of acid or base are added. In certain embodiments, the liquid solution contains a lubricity enhancing agent.

As used herein, the term "polymer," "polymer matrix" or "polymeric agent" refers to a biocompatible polymeric material. The polymeric material described herein may comprise, for example, sugars (such as mannitol), peptides, protein, laminin, collagen, hyaluronic acid, ionic and non-ionic water soluble polymers; acrylic acid polymers; hydrophilic polymers such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers, and

polyvinylalcohol; cellulosic polymers and cellulosic polymer derivatives such as

hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, methyl cellulose, carboxymethyl cellulose, and etherified cellulose; poly(lactic acid), poly(glycolic acid), copolymers of lactic and glycolic acids, or other polymeric agents, both natural and synthetic.

In certain embodiments, the polymeric matrix is absorbable, such as, for example collagen, polyglycolic acid, polylactic acid, polydioxanone, and caprolactone. As used herein, the term "absorbable" refers to the ability of a material to be absorbed into the body. In other embodiments, the polymer is non-absorbable, such as, for example polypropylene, polyester or nylon.

As used herein, the term "pH buffering agent" refers to an aqueous buffer solution which resists changes in pH when small quantities of acid or base are added to it. pH

Buffering solutions typically comprise a mixture of weak acid and its conjugate base, or vice versa. For example, pH buffering solutions may comprise phosphates such as sodium phosphate, sodium dihydrogen phosphate, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate, disodium hydrogen phosphate dodecahydrate, potassium phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate; boric acid and borates such as, sodium borate and potassium borate; citric acid and citrates such as sodium citrate and disodium citrate; acetates such as sodium acetate and potassium acetate; carbonates such as sodium carbonate and sodium hydrogen carbonate, etc. pH Adjusting agents can include, for example, acids such as hydrochloric acid, lactic acid, citric acid, phosphoric acid and acetic acid, and alkaline bases such as sodium hydroxide, potassium hydroxide, sodium carbonate and sodium hydrogen carbonate, etc. In some embodiments, the pH buffering agent is a phosphate buffered saline (PBS) solution (i.e., containing sodium phosphate, sodium chloride and in some formulations, potassium chloride and potassium phosphate).

Bioconjugates

Some embodiments of the present disclosure are directed to localizing bioconjugates to the articular surface, so as to provide bioconjugates that bring down the COF of a trypsin treated plug that were treated with the bioconjugate, to either match the COF or provide lower COF than that of the WT cartilage. As shown in FIG. 7, trypsin treated plugs that were treated with mLublO and Synvisc did not show the desired results. Trypsin treated plugs that were treated with mLubl5 and Synvisc decreased the static and kinetic COF to below that of the WT cartilage, whereas similar treatment with mLublO and then Synvisc resulted in higher COF to that of the WT cartilage. Thus, surprisingly and unexpectedly, mLubl5 decreased the COF of a trypsin treated plug to below that of the WT cartilage.

Specifically, trypsin treated plugs that were treated with mLubl5 and Synvisc had lower static COF and significantly lower kinetic COF than the wild type, whereas trypsin treated plugs that were treated with mLublO and Synvisc had higher static as well as kinetic COF than the wild type. Accordingly, provided herein is a bioconjugate comprising: a) a glycan, b) from about

3 to about 30 collagen-binding peptides having at least one collagen-binding domain; and c) from about 1 to about 10 hyaluronic acid-binding peptide(s) having at least one hyaluronic acid-binding domain, wherein the ratio of collagen-binding peptides to hyaluronic acid- binding peptide(s) is about 3: 1 or about 4: 1, and wherein the peptides are covalently bonded to the glycan.

In the bioconjugate disclosed herein, the glycan can be any glycan,

glycosaminoglycan, or polysaccharide, including but not limited to, alginate, dextran, dextran sulfate, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, heparan, heparan sulfate, heparin, keratin, keratan sulfate, and hyaluronic acid. In some embodiments, the glycan is dermatan sulfate, chondroitin sulfate, or heparin. In some embodiments, the glycan is dermatan sulfate. In some embodiments, the glycan is hyaluronic acid. In some embodiments, the glycan is heparin. In some embodiments, the glycan is chondroitin sulfate.

The peptides can be bonded to the glycan directly or via a linker or suitable functional group. 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)propionyl hydrazide (PDPH), N-[P-maleimidopropionic acid]hydrazide (BMPH), and the like. In any of the various embodiments 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 other embodiments, the In certain embodiments, the linker is N-[P-maleimidopropionic

acid]hydrazide (BMPH).

Depending on the desired properties of the bioconjugate, the total number of peptides bound to the glycan can be varied. In certain embodiments, the total number of peptides present in the bioconjugate is from about 4 to about 40, or from about 8 to about 40, or from about 12 to about 40, or from about 16 to about 40, or from about 20 to about 40, or from about 24 to about 40, or from about 28 to about 40, or from about 32 to about 40, or from about 36 to about 40, or from about 4 to about 36, or from about 4 to about 32, or from about 4 to about 28, or from about 4 to about 24, or from about 4 to about 20, or from about 4 to about 16, or from about 4 to about 12, or from about 4 to about 8, or from about 8 to about 36, or from about 8 to about 32, or from about 8 to about 28, or from about 8 to about 24, or from about 8 to about 20, or from about 8 to about 16, or from about 8 to about 12, or from about 12 to about 36, or from about 12 to about 32, or from about 12 to about 28, or from about 12 to about 24, or from about 12 to about 20, or from about 8 to about 16, or from about 16 to about 36, or from about 16 to about 32, or from about 16 to about 28, or from about 16 to about 24, or from about 16 to about 20, or from about 20 to about 36, or from about 20 to about 32, or from about 20 to about 28, or from about 20 to about 24, or from about 24 to about 36, or from about 24 to about 32, or from about 24 to about 28. In certain embodiments, the total number of peptides present in the bioconjugate is less than about 33. In certain embodiments, the total number of peptides present in the bioconjugate is from about 4 to about 36. In certain embodiments, the total number of peptides present in the bioconjugate is about 28. In certain embodiments, the total number of collagen -binding peptides is from about 6 to about 30, or about 9, or about 15, or about 16. In certain embodiments, the total number of hyaluronic acid-binding peptides is from about 2 to about 10, or about 3, or about 4, or about 5.

In one aspect, the collagen-binding peptides present in the extracellular matrix- binding bioconjugates described herein 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 bioconjugates described herein. For example, the extracellular matrix -binding bioconjugates 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 at least one collagen-binding domain which binds to type I collagen and the at least one collagen-binding domain that binds to type II collagen are present on a single collagen-binding peptide. In another aspect, the collagen-binding peptides have binding affinity to type I collagen. 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 WYRGRL (SEQ ID NO: ),

RRANAALKAGELYKSILY (SEQ ID NO: ), RRANAALKAGELYKCILY (SEQ ID NO: ), RLDGNEIKR (SEQ ID NO: ), AHEEISTTNEGVMGC (SEQ ID NO: ),

NGVFKYRPRYFLYKHAYFYPPLKRFPVQ (SEQ ID NO: ), CQDSETRTFY (SEQ ID NO: ), TKKTLRT (SEQ ID NO: ), GLRS KS KKFRRPDIQYPD ATDEDITSHM (SEQ ID NO: ), SQNPVQP (SEQ ID NO: ), SYIRIADTNIT (SEQ ID NO: ), KELNLVYT (SEQ ID NO: ), GELYKSILY (SEQ ID NO: ), GELYKCILY (SEQ ID NO: ) and GSITTIDVPWNV (SEQ ID NO: ), 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 some embodiments, the collagen-binding peptide comprises an amino acid sequence selected from the group consisting of WYRGRL (SEQ ID NO: ), RRANAALKAGELYKSILY (SEQ ID NO: ), RRANAALKAGELYKCILY (SEQ ID NO: ), RLDGNEIKR (SEQ ID NO: ), AHEEISTTNEGVMGC (SEQ ID NO: ),

NGVFKYRPRYFLYKHAYFYPPLKRFPVQ (SEQ ID NO: ), CQDSETRTFY (SEQ ID NO: ), TKKTLRT (SEQ ID NO: ), GLRS KS KKFRRPDIQYPD ATDEDITSHM (SEQ ID NO: ), SQNPVQP (SEQ ID NO: ), SYIRIADTNIT (SEQ ID NO: ), KELNLVYT (SEQ ID NO: ), GELYKSILY (SEQ ID NO: ), GELYKCILY (SEQ ID NO: ) or GSITTIDVPWNV (SEQ ID NO: ). In certain embodiments, the collagen-binding peptide is WYRGRL (SEQ ID NO: ), 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 (1997), Romijn, et al., J. Biol. Chem. 278: 15035-15039 (2003), and Chiang, et al., Cardio. & Haemato. Disorders-Drug Targets 7: 71-75 (2007), each incorporated herein by reference.

Suitable hyaluronic acid-binding peptides are known in the art (see, e.g., WO

2012/162534). In the various embodiments described herein, the peptide component of the bioconjugate can comprise an amino acid sequence selected from the group consisting of GAHWQFNALTVR (SEQ ID NO: ), GDRRRRRMWHRQ (SEQ ID NO: ),

GKHLGGKHRRSR (SEQ ID NO: ), RGTHHAQKRRS (SEQ ID NO: ), RRHKS GHIQGS K (SEQ ID NO: ), SRMHGRVRGRHE (SEQ ID NO: ), RRRAGLTAGRPR (SEQ ID NO: ), RYGGHRTS RKW V (SEQ ID NO: ), RSARYGHRRGVG (SEQ ID NO: ),

GLRGNRRVFARP (SEQ ID NO: ), SRGQRGRLGKTR (SEQ ID NO: ),

DRRGRS S LPKLAGP VEFPDRKIKGRR (SEQ ID NO: ), RMRRKGRVKHWG (SEQ ID NO: ), RGGARGRHKTGR (SEQ ID NO: ), TGARQRGLQGGWGPRHLRGKDQPPGR (SEQ ID NO: ), RQRRRDLTRVEG (SEQ ID NO: ),

S TKDHNRGRRN VGP VS RS TLRDPIRR (SEQ ID NO: ), RRIGHQVGGRRN (SEQ ID NO: ), RLES R A AGQRR A (SEQ ID NO: ), GGPRRHLGRRGH (SEQ ID NO: ),

VS KRGHRRT AHE (SEQ ID NO: ), RGTRSGSTR (SEQ ID NO: ), RRRKKIQGRSKR (SEQ ID NO: ), RKSYGKYQGR (SEQ ID NO: ), KNGRYSISR (SEQ ID NO: ),

RRRCGQKKK (SEQ ID NO: ), KQKIKHVVKLK (SEQ ID NO: ), KLKSQLVKRK (SEQ ID NO: ), RYPISRPRKR (SEQ ID NO: ), KVGKSPPVR (SEQ ID NO: ), KTFGKMKPR (SEQ ID NO: ), RIKWSRVSK (SEQ ID NO: ) and KRTMRPTRR (SEQ ID NO: ), or any, 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 some embodiments, the hyaluronic acid-binding peptide comprises an amino acid sequence selected from the group consisting of GAHWQFNALTVR (SEQ ID NO: ), GDRRRRRMWHRQ (SEQ ID NO: ), GKHLGGKHRRSR (SEQ ID NO: ),

RGTHHAQKRRS (SEQ ID NO: ), RRHKSGHIQGSK (SEQ ID NO: ), SRMHGRVRGRHE (SEQ ID NO: ), RRRAGLTAGRPR (SEQ ID NO: ), RYGGHRTS RKW V (SEQ ID NO: ), RSARYGHRRGVG (SEQ ID NO: ), GLRGNRRVFARP (SEQ ID NO: ),

SRGQRGRLGKTR (SEQ ID NO: ), DRRGRS SLPKLAGPVEFPDRKIKGRR (SEQ ID NO: ), RMRRKGRVKHWG (SEQ ID NO: ), RGGARGRHKTGR (SEQ ID NO: ),

TG ARQRGLQGGWGPRHLRGKD QPPGR (SEQ ID NO: ), RQRRRDLTRVEG (SEQ ID NO: ), S TKDHNRGRRN VGP VS RS TLRDPIRR (SEQ ID NO: ), RRIGHQVGGRRN (SEQ ID NO: ), RLES R A AGQRRA (SEQ ID NO: ), GGPRRHLGRRGH (SEQ ID NO: ),

VS KRGHRRT AHE (SEQ ID NO: ), RGTRSGSTR (SEQ ID NO: ), RRRKKIQGRSKR (SEQ ID NO: ), RKSYGKYQGR (SEQ ID NO: ), KNGRYSISR (SEQ ID NO: ),

RRRCGQKKK (SEQ ID NO: ), KQKIKHVVKLK (SEQ ID NO: ), KLKSQLVKRK (SEQ ID NO: ), RYPISRPRKR (SEQ ID NO: ), KVGKSPPVR (SEQ ID NO: ), KTFGKMKPR (SEQ ID NO: ), RIKWSRVSK (SEQ ID NO: )and KRTMRPTRR (SEQ ID NO: ).

Additional peptides that can be included as the peptide component of the hyaluronic acid-binding bioconjugates include peptides which have an Arg-Arg (R-R) motif, such as one or more peptides selected from the group consisting of RRASRSRGQVGL (SEQ ID NO: ), GRGTHHAQKRRS (SEQ ID NO: ), QPVRRLGTPVVG (SEQ ID NO: ),

ARRAEGKTRMLQ (SEQ ID NO: ), PKVRGRRHQ AS G (SEQ ID NO: ),

SDRHRRRREADG (SEQ ID NO: ), NQRVRRVKHPPG (SEQ ID NO: ),

RERRERHAVARHGPGLERDARNLARR (SEQ ID NO: ),

TRPGGKRGGQVGPPAGVLHGRRARS (SEQ ID NO: ), N VRS RRGHRMNS (SEQ ID NO: ), DRRRGRTRNIGN (SEQ ID NO: ), KTAGHGRRWSRN (SEQ ID NO: ),

AKRGEGRREWPR (SEQ ID NO: ), GGDRRKAHKLQA (SEQ ID NO: ),

RRGGRKWGSFEG (SEQ ID NO: ) and RQRRRDLTRVEG (SEQ ID NO: ), 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 (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

GAHWQFNALTVR (SEQ ID NO: ), 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 (SEQ ID NO: ), HREARSGKYK (SEQ ID NO: ), PDKKHKLYGV (SEQ ID NO: ), and WDKERSRYDV (SEQ ID NO: ) (see, e.g., Yang et al, EMBO Journal, 1994, 13, 286-296, and Goetinck et al, J. Cell. Biol., 1987, 105, 2403-2408, both of which are incorporated herein by reference).

In another embodiment, provided herein is a bioconjugate comprising: a) a glycan, b) from about 3 to about 30 collagen-binding peptides having at least one collagen-binding domain; and c) from about 1 to about 10 hyaluronic acid-binding peptide(s) having at least one hyaluronic acid-binding domain, wherein the ratio of collagen -binding peptides to hyaluronic acid-binding peptide(s) is about 3: 1 or about 4: 1, and wherein the peptides are covalently bonded to the glycan, wherein the collagen-binding domain binds to collagen with a dissociation constant (¾) of less than about 1 mM. In another embodiment, the collagen- binding domain binds to type I collagen, type II collagen or type III collagen with a dissociation constant (¾) of less than about 1 mM. In another embodiment, the hyaluronic acid-binding domain binds to hyaluronic acid with a dissociation constant (¾) of less than about 1 mM. In another embodiment, the collagen-binding peptides comprise up to 40 amino acids. In another embodiment, the collagen-binding peptides comprise up to 25 amino acids. In another embodiment, the bioconjugate comprises about 15 collagen-binding peptides. In another embodiment, the bioconjugate comprises about 5 hyaluronic acid-binding peptides.

In another embodiment, the hyaluronic acid-binding peptide 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 7 X 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 hyaluronic acid-binding peptide may be determined by its interaction with hyaluronic acid, and measured by any of the techniques used to evaluate molecular interactions. For example, surface plasmon resonance, ELISA, competitive binding assays, isothermal titration calorimetry, affinity chromatography, 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 residues in the peptide. The interaction may be measured by 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 chromophore. The interaction may also be measured by mechanical influence on hyaluronic acid-containing fluids, gels, or tissues that have been incubated with the hyaluronic acid-binding peptide, or with a peptidoglycan containing an hyaluronic acid- binding peptide or peptides.

For identifying a peptide, a peptide selected from phage display, or one that is identified from a hyaluronic acid-binding 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 peptide 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 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 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 may too be possible, provided that they do not substantially affect the binding activity of the peptide (i.e., hyaluronic acid or collagen-binding affinity). In various embodiments described herein, the bioconjugate is resistant to aggrecanase. An aggrecanase is characterized in the art as any enzyme known to cleave aggrecan. In one embodiment, the bioconjugate does not contain a polymerizable group, such as

methacrylates, ethacrylates, itaconates, acrylamides, thiols, peptides and aldehydes. In another embodiment, provided herein is a bioconjugate comprising: a) chondroitin sulfate (CS); b) from about 3 to about 30 collagen-binding peptide(s); and c) from about 1 to about 10 hyaluronic acid-binding peptide(s); wherein the ratio of collagen-binding peptides to hyaluronic acid-binding peptide(s) is about 3: 1 or about 4: 1 and the peptides of b) and c) are covalently bonded to the chondroitin sulfate; and further wherein the collagen-binding peptide(s) are WYRGRL (SEQ ID NO: ) and the hyaluronic acid-binding peptide(s) are GAHWQFNALTVRGG (SEQ ID NO: ). In certain embodiments, the peptides are covalently bonded to the CS via a linker, such as Ν-[β- maleimidopropionic acid]hydrazide (BMPH). In another embodiment, provided herein is a bioconjugate comprising: a) chondroitin sulfate; b) WYRGRL (SEQ ID NO: ); and c) GAHWQFNALTVRGG (SEQ ID NO: ); wherein the ratio of WYRGRL (SEQ ID NO: ) and GAHWQFNALTVRGG (SEQ ID NO: ) is about 4: 1, and WYRGRL (SEQ ID NO: ) and GAHWQFNALTVRGG (SEQ ID NO: ) are covalently bonded to the chondroitin sulfate via N-[P-maleimidopropionic acid]hydrazide (BMPH).

In another embodiment, provided herein is a bioconjugate comprising: a) chondroitin sulfate; b) WYRGRL (SEQ ID NO: ); and c) GAHWQFNALTVRGG (SEQ ID NO: ); wherein the ratio of WYRGRL (SEQ ID NO: ) and GAHWQFNALTVRGG (SEQ ID NO: ) is about 3: 1, and WYRGRL (SEQ ID NO: ) and GAHWQFNALTVRGG (SEQ ID NO: ) are covalently bonded to the chondroitin sulfate via N-[P-maleimidopropionic acid]hydrazide (BMPH).

Synthesis of Peptidoglycans

The peptides used in the bioconjugates 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 bio technological methods). In certain embodiments, the peptides are synthesized 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 according to methods known to persons skilled in the art. In other embodiments, the peptide is synthesized utilizing the methods 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 purification 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 DNA technology methods are described in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd Edition, Cold Spring Harbor Laboratory Press, (2001), incorporated herein by reference, and are well- known to the skilled artisan.

In certain embodiments, the peptides are covalently bonded to the glycan directly (i.e., without a linker). In such embodiments, the bioconjugate 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 oxidized 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 Ν-[β- 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 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-[P-maleimidopropionic

acid] hydrazide (BMPH) to form an glycan intermediate (e.g., "BMPH-CS") and further reacting the glycan intermediate with peptides containing at least one free thiol group (i.e., - SH group) to yield the bioconjugate. In yet another embodiment, the sequence of the peptides may be modified to include an amino 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-glycine-cysteine (GGGC) or glycine- serine-glycine-cysteine (GSGC) segment may be added to provide an attachment point for the glycan intermediate.

Scheme 1

Accordingly, in one embodiment, the bioconjugates 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 3 to about 30 collagen-binding peptide(s); and from about 1 to about 10 hyaluronic acid-binding peptide(s), such that the peptides are covalently bonded to the glycan.

The bioconjugate can be synthesized by sequentially adding the peptides having different binding affinities to the glycan (i.e., oxidized glycan or glycan intermediate), or alternatively, adding all peptides simultaneously. The bioconjugates can be isolated and/or purified using known methods, such as size exclusion chromatography, at any point in the synthesis.

Methods of Use

The bioconjugates described herein may be useful in replacing, rejuvenating, or supplementing tissues that have both collagen and hyaluronic acid, such as cartilage, synovial fluid, and the vitreous humor. In particular, peptidoglycans having both collagen-binding and hyaluronic acid-binding peptides may be especially useful as a lubricin mimetic and in the methods and uses described below.

Cartilage degeneration

A well-lubricated surface on articular cartilage leads to optimal functionality of synovial joints. As occurs in osteoarthritis, 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 the examples, the bioconjugates provided 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 bioconjugates of the disclosure thus may be used to enhance lubrication at an articular cartilage surface, thereby reducing wear-induced erosion of the cartilage.

Specifically, the bioconjugates of the disclosure can be used to protect articular cartilage in vivo. For example, the bioconjugate mLubl5 showed in vivo efficacy in the protection of articular cartilage and attenuation of osteoarthritis in a natural disease model.

Accordingly, provided is a method of treating and/or preventing cartilage

degeneration in a patient comprising administering to the patient in need thereof a

pharmaceutical composition comprising the bioconjugate described herein and a

viscosupplement.

In one embodiment, the bioconjugate used in the methods described above comprises about 5 hyaluronic acid-binding peptides and about 15 collagen-binding peptides conjugated to a glycan. In one embodiment, the glycan is chondroitin-6-sulfate (CS) glycan, the hyaluronic acid-binding peptides comprise the sequence GAHWQFNALTVRGG (SEQ ID NO: ), and the collagen-binding peptides the comprise the sequence WYRGRL (SEQ ID NO: ),

In one embodiment, the patient is treated by injecting the pharmaceutical composition comprising the bioconjugate and a viscosupplement into a synovial cavity.

Degradation of a hyaluronic acid rich tissue The bioconjugate 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 preventing degradation of a hyaluronic acid rich tissue in a patient comprising administering to a patient in need thereof a pharmaceutical composition comprising the bioconjugate described herein and a

viscosupplement. In one embodiment, the hyaluronic acid rich tissue is the skin. Also, provided is a method of decreasing scar formation in a patient comprising administering to a patient in need thereof a pharmaceutical composition comprising the bioconjugate described herein and a viscosupplement.

It is also contemplated that the bioconjugates can be used to treat and/or prevent articular cartilage disease by protecting the articular cartilage matrix from traumatic and cytokine-induced enzymatic degradation.

In one embodiment, the bioconjugate used in the methods described above comprises about 5 hyaluronic acid-binding peptides and about 15 collagen-binding peptides conjugated to a glycan. In one embodiment, the glycan is chondroitin-6-sulfate (CS) glycan, the hyaluronic acid-binding peptides comprise the sequence GAHWQFNALTVRGG (SEQ ID NO: ), and the collagen-binding peptides the comprise the sequence WYRGRL (SEQ ID NO: ),

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 temporary or permanent vitreous replacement during retinal surgery. 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 preventing vitreous humor degeneration in a patient comprising administering to a patient in need thereof a

pharmaceutical composition comprising the bioconjugate described herein. In another embodiment, the method further comprises a viscosupplement.

In one embodiment, the bioconjugate used in the methods described above comprises about 5 hyaluronic acid-binding peptides and about 15 collagen-binding peptides conjugated to a glycan. In one embodiment, the glycan is chondroitin-6-sulfate (CS) glycan, the hyaluronic acid-binding peptides comprise the sequence GAHWQFNALTVRGG (SEQ ID NO: ), and the collagen-binding peptides the comprise the sequence WYRGRL (SEQ ID NO:

),

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 proteoglycan aggrecans that aggregate through hyaluronic chains. Degeneration of the nucleus pulposus results in reduced ability of the 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. 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 prevented and the mechanical function is restored to the vertebral segment.

It is contemplated that the bioconjugates described herein will bind to and protect the annulus fibrosis. Accordingly, provided is a method of treating and/or preventing annulus fibrosis degeneration in a patient comprising administering to a patient in need thereof a pharmaceutical composition comprising the bioconjugate described herein and a

viscosupplement.

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 comprising the extracellular matrix-binding bioconjugate described herein. In another embodiment, the method further comprises a viscosupplement.

In one embodiment, the bioconjugate used in the methods described above comprises about 5 hyaluronic acid-binding peptides and about 15 collagen-binding peptides conjugated to a glycan. In one embodiment, the glycan is chondroitin-6-sulfate (CS) glycan, the hyaluronic acid-binding peptides comprise the sequence GAHWQFNALTVRGG (SEQ ID NO: ), and the collagen-binding peptides the comprise the sequence WYRGRL (SEQ ID NO: )· Compositions

In one embodiment, the bioconjugate is administered in a composition. The present disclosure provides compositions comprising a bioconjugate and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are known to one having ordinary skill in the art may be used, including water or saline.

Disclosed herein is a pharmaceutical composition comprising a bioconjugate comprising: a) a glycan, b) from about 3 to about 30 collagen-binding peptides having at least one collagen-binding domain; and c) from about 1 to about 10 hyaluronic acid-binding peptide(s) having at least one hyaluronic acid-binding domain, wherein the ratio of collagen- binding peptides to hyaluronic acid-binding peptide(s) is about 3: 1 or about 4: 1, and wherein the peptides are 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 compositions may be for oral, topical or parenteral delivery, including intra- articular, intervertebral or intraocular delivery. In any of the embodiments described herein, the bioconjugate can be administered alone or in combination with suitable pharmaceutical carriers or diluents. Diluent or carriers used in the compositions can be selected so that they do not diminish the desired effects of the bioconjugate. The composition may be in any suitable form. Examples of suitable

compositions include aqueous solutions, for example, a solution in isotonic saline, 5% glucose, and/or other well-known pharmaceutically acceptable liquid carriers such as alcohols, glycols, esters and amides. In certain embodiments, the composition further comprises one or more excipients, such as, but not limited to pH buffering agents, surfactants, solubility enhancing agents, sugars such as mannitol or sorbitol, ionic strength modifying agents, stabilizing polymer, preservatives, co-solvents, and/or viscosity modulating (lubricity enhancing) agents.

In certain embodiments, the 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-binding peptidoglycan comprises chondroitin sulfate conjugated to from about 1 to about 20 collagen-binding peptide(s) as described herein. In certain embodiments, the collagen-binding peptide(s) of the collagen-binding peptidoglycan comprises WYRGRL (SEQ ID NO: ). In certain embodiments, the 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 comprises GAHWQFNALTVR (SEQ ID NO: ).

Suitable pH buffering agents for use in the compositions herein described include, for example, acetate, borate, carbonate, citrate, and phosphate buffers, as well as hydrochloric acid, sodium hydroxide, magnesium oxide, monopotassium phosphate, bicarbonate, ammonia, carbonic acid, hydrochloric acid, sodium citrate, citric acid, acetic acid, disodium hydrogen phosphate, borax, boric acid, sodium hydroxide, diethyl barbituric acid, and proteins, as well as various biological buffers, for example, TAPS, Bicine, Tris, Tricine, HEPES, TES, MOPS, PIPES, cacodylate, or MES. In certain embodiments, an appropriate buffer system (e.g., sodium phosphate, sodium acetate, sodium citrate, sodium borate or boric acid) is added to the composition to prevent pH drift under storage conditions. In some embodiments, the buffer is a phosphate buffered saline (PBS) solution (i.e., containing sodium phosphate, sodium chloride and in some formulations, potassium chloride and potassium phosphate). The particular concentration will vary, depending on the agent employed. In certain embodiments, the pH buffer system (e.g., sodium phosphate, sodium acetate, sodium citrate, sodium borate or boric acid) is added to maintain a pH within the range of from about pH 4 to about pH 8, or about pH 5 to about pH 8, or about pH 6 to about pH 8, or about pH 7 to about pH 8. In some embodiments, the buffer is chosen to maintain a pH within the range of from about pH 4 to about pH 8. In some embodiments, the pH is from about pH 5 to about pH 8. In some embodiments, the buffer is a saline buffer. In certain embodiments, the pH is from about pH 4 and about pH 8, or from about pH 3 to about pH 8, or from about pH 4 to about pH 7. In some embodiments, the composition is in the form of a film, gel, patch, or liquid solution which comprises a polymeric matrix, pH buffering agent, a lubricity enhancing agent and a bioconjugate wherein the composition optionally contains a preservative; and wherein the pH of said composition is within the range of about pH 4 to about pH 8.

Surfactants are employed in the composition to deliver higher concentrations of bioconjugate. The surfactants function to solubilize the inhibitor and stabilize colloid dispersion, such as micellar solution, microemulsion, emulsion and suspension. Suitable surfactants comprise c polysorbate, poloxamer, polyosyl 40 stearate, polyoxyl castor oil, tyloxapol, triton, and sorbitan monolaurate. In one embodiment, the surfactants have hydrophile/lipophile/balance (HLB) in the range of 12.4 to 13.2 and are acceptable for ophthalmic use, such as TritonXl 14 and tyloxapol.

In certain embodiments, stabilizing polymers, i.e., demulcents, are added to the composition. The stabilizing polymer should be an ionic/charged example, more specifically a polymer that carries negative charge on its surface that can exhibit a zeta-potential of (-)IO- 50 mV for physical stability and capable of making a dispersion in water (i.e. water soluble). In one embodiment, the stabilizing polymer comprises a poly electrolyte or polyectrolytes if more than one, from the family of cross-linked polyacrylates, such as carbomers and

Pemulen®, specifically Carbomer 974p (polyacrylic acid), at a range of about 0.1% to about 0.5% w/w.

In one embodiment, the composition comprises an agent which increases the permeability of the bioconjugate to the extracellular matrix of blood vessels. Preferably the agent which increases the permeability is selected from benzalkonium chloride, saponins, fatty acids, polyoxyethylene fatty ethers, alkyl esters of fatty acids, pyrrolidones,

polyvinylpyrrolidone, pyruvic acids, pyroglutamic acids or mixtures thereof.

Suitable ionic strength modifying agents include, for example, glycerin, propylene glycol, mannitol, glucose, dextrose, sorbitol, sodium chloride, potassium chloride, and other electrolytes.

In certain embodiments, the composition contains a lubricity enhancing agent. As used herein, lubricity enhancing agents refer to one or more pharmaceutically acceptable polymeric materials capable of modifying (modulating) the viscosity of the pharmaceutically acceptable carrier. Suitable lubricity enhancing 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 Carbopol™ trademark; hydrophilic polymers such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers, and polyvinylalcohol; hyaluronic acid and its salts, chondroitin sulfate and its salts, dextrans, gelatin, chitosans, gellans, other bioconjugates or polysaccharides, or any combination thereof; cellulosic polymers and cellulosic polymer derivatives such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, methyl cellulose, carboxymethyl cellulose, and etherified cellulose; collagen and modified collagens;

galactomannans, such as guar gum, locust bean gum and tara gum, as well as polysaccharides derived from the foregoing natural gums and similar natural or synthetic gums containing mannose and/or galactose moieties as the main structural components (e.g., hydroxypropyl guar); gums such as tragacanth and xanthan gum; sodium alginate; gelatin, hyaluronic acid and salts thereof, chitosans, gellans or any combination thereof. Typically, 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 one embodiment, a lubricity enhancing agent is selected from the group consisting of hyaluronic acid, dermatan, chondroitin, heparin, heparan, keratin, dextran, chitosan, alginate, agarose, gelatin, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, methyl cellulose, carboxymethyl cellulose, and etherified cellulose, polyvinyl alcohol, polyvinylpyrrolidinone, povidone, carbomer 941, carbomer 940, carbomer 97 IP, carbomer 974P, or a pharmaceutically acceptable salt thereof. In one embodiment, a lubricity enhancing agent is applied concurrently with the bioconjugate. Alternatively, in one embodiment, a lubricity enhancing agent is applied sequentially to the bioconjugate. In one embodiment, the lubricity enhancing agent is chondroitin sulfate. In one embodiment, the lubricity enhancing agent is hyaluronic acid. The lubricity enhancing agent can change the viscosity of the composition.

For further details pertaining to the structures, chemical properties and physical properties of the above lubricity enhancing agents, see e.g., U.S. 5,409,904, U.S. 4,861,760 (gellan gums), U.S. 4,255,415, U.S. 4,271,143 (carboxyvinyl polymers), WO 94/10976 (polyvinyl alcohol), WO 99/51273 (xanthan gum), and WO 99/06023

(galactomannans).

In some embodiments, the bioconjugates can be combined with minerals, amino acids, sugars, peptides, proteins, vitamins (such as ascorbic acid), or laminin, collagen, fibronectin, hyaluronic acid, fibrin, elastin, or aggrecan, or growth factors such as epidermal growth factor, platelet-derived growth factor, transforming growth factor beta, or fibroblast growth factor, and glucocorticoids such as dexamethasone or viscoelastic altering agents, such as ionic and non-ionic water soluble polymers; acrylic acid polymers; hydrophilic polymers such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers, and polyvinylalcohol; cellulosic polymers and cellulosic polymer derivatives such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, methyl cellulose, carboxymethyl cellulose, and etherified cellulose; poly(lactic acid), poly(glycolic acid), copolymers of lactic and glycolic acids, or other polymeric 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 bioconjugate may need to be enhanced. In such cases, the solubility may be increased by the use of appropriate formulation techniques, such as the incorporation of solubility-enhancing compositions such as mannitol, ethanol, glycerin, polyethylene glycols, propylene glycol, poloxomers, and others known in the art.

The bioconjugate may be sterilized to remove unwanted contaminants including, but not limited to, endotoxins and infectious agents. Sterilization techniques which do not adversely affect the structure and biotropic properties of the bioconjugate can be used. In certain embodiments, the bioconjugate can be disinfected and/or sterilized using conventional sterilization techniques including propylene oxide or ethylene oxide treatment, sterile filtration, gas plasma sterilization, gamma radiation, electron beam, and/or sterilization with a peracid, such as peracetic acid. In one embodiment, the bioconjugate can be subjected to one or more sterilization processes. Alternatively, the bioconjugate may be wrapped in any type of container including a plastic wrap or a foil wrap, and may be further sterilized. In some embodiments, preservatives are added to the composition to prevent microbial contamination during use. Suitable preservatives added to the compositions comprise benzalkonium chloride, benzoic acid, alkyl parabens, alkyl benzoates,

chlorobutanol, chlorocresol, cetyl alcohols, fatty alcohols such as hexadecyl alcohol, organometallic compounds of mercury such as acetate, phenylmercury nitrate or borate, diazolidinyl urea, diisopropyl adipate, dimethyl polysiloxane, salts of EDTA, vitamin E and its mixtures. In certain embodiments, the preservative is selected from benzalkonium chloride, chlorobutanol, benzododecinium bromide, methyl paraben, propyl paraben, phenylethyl alcohol, edentate disodium, sorbic acid, or polyquarternium-1. In certain embodiments, the compositions comprise a preservative. In some embodiments, the preservatives are employed at a level of from about 0.001% to about 1.0% w/v. In certain embodiments, the compositions do not contain a preservative and are referred to as

"unpreserved". In some embodiments, the unit dose compositions are sterile, but

unpreserved.

Exemplary compositions contemplated by the present disclosure may also be for administration by injection include aqueous or oil suspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles. Aqueous solutions in saline are also conventionally used for injection, but less preferred in the context of the present disclosure. Ethanol, glycerol, propylene glycol, liquid polyethylene glycol, and the like (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils may also be employed. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

Sterile injectable solutions are prepared by incorporating the component in the required amount in the appropriate solvent with various other ingredients as enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

In making pharmaceutical compositions that include bioconjugates described herein, the active ingredient is usually diluted by an excipient or carrier and/or enclosed within such a carrier that can be in the form of a capsule, sachet, paper or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material (as above), which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of films, gels, patches, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compounds, soft and hard gelatin films, gels, patches, sterile injectable solutions, and sterile packaged powders. Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents.

Films used for drug delivery are well known in the art and comprise non-toxic, non- irritant polymers devoid of leachable impurities, such as polysaccharides (e.g., cellulose, maltodextrin, etc.). In some embodiments, the polymers are hydrophilic. In other embodiments, the polymers are hydrophobic. The film adheres to tissues to which it is applied, and is slowly absorbed into the body over a period of about a week. Polymers used in the thin-film dosage forms described herein are absorbable and exhibit sufficient peel, shear and tensile strengths as is well known in the art. In some embodiments, the film is injectable. In certain embodiments, the film is administered to the patient prior to, during or after surgical intervention.

Gels are used herein refer to a solid, jelly-like material that can have properties ranging from soft and weak to hard and tough. As is well known in the art, a gel is a non- fluid colloidal network or polymer network that is expanded throughout its whole volume by a fluid. A hydrogel is a type of gel which comprises a network of polymer chains that are hydrophilic, sometimes found as a colloidal gel in which water is the dispersion medium. Hydrogels are highly absorbent and can contain a high degree of water, such as, for example greater than 90% water. In some embodiments, the gel described herein comprises a natural or synthetic polymeric network. In some embodiments, the gel comprises a hydrophilic polymer matrix. In other embodiments, the gel comprises a hydrophobic polymer matrix. In some embodiments, the gel possesses a degree of flexibility very similar to natural tissue. In certain embodiments, the gel is biocompatible and absorbable. In certain embodiments, the gel is administered to the patient prior to, during or after surgical intervention. Liquid solution as used herein refers to solutions, suspensions, emulsions, drops, ointments, liquid wash, sprays, liposomes which are well known in the art. In some embodiments, the liquid solution contains an aqueous pH buffer agent which resists changes in pH when small quantities of acid or base are added. In certain embodiments, the liquid solution is administered to the patient prior to, during or after surgical intervention.

Exemplary formulations may comprise: a) one or more bioconjugate as described herein; b) pharmaceutically acceptable carrier; and c) hydrophilic polymer as matrix network, wherein said compositions are formulated as viscous liquids, i.e., viscosities from several hundred to several thousand cps, gels or ointments. In these embodiments, the bioconjugate is dispersed or dissolved in an appropriate pharmaceutically acceptable carrier.

In certain embodiments, the bioconjugate, or a composition comprising the same, is lyophilized prior to, during, or after, formulation. Accordingly, also provided herein is a lyophilized composition comprising a bioconjugate or composition comprising the same as described herein. In various embodiments, the bioconjugate can be administered intravenously or into muscle, for example. Suitable routes for parenteral administration include intravascular, 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, the bioconjugate can be administered topically, such as by film, gel, patch, or liquid solution. In some of the embodiments, the compositions provided are in a buffered, sterile aqueous solution. In certain embodiments, the solutions have a viscosity of from about 1 to about 100 centipoises (cps), or from about 1 to about 200 cps, or from about 1 to about 300 cps, or from about 1 to about 400 cps. In some embodiments, the solutions have a viscosity of from about 1 to about 100 cps. In certain embodiments, the solutions have a viscosity of from about 1 to about 200 cps. In certain embodiments, the solutions have a viscosity of from about 1 to about 300 cps. In certain embodiments, the solutions have a viscosity of from about 1 to about 400 cps. In certain embodiments, the solution is in the form of an injectable liquid solution. In other embodiments, the

compositions are formulated as viscous liquids, i.e., viscosities from several hundred to several thousand cps, gels or ointments. In these embodiments, the bioconjugate is dispersed or dissolved in an appropriate pharmaceutically acceptable carrier. In various embodiments described herein, formulations for parenteral administration may be formulated to be for immediate and/or modified release. Modified release

formulations include delayed, sustained, pulsed, controlled, targeted and programmed release formulations. Thus, a bioconjugate may be formulated as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot providing modified release of the active compound. Illustrative examples of such formulations include drug-coated stents and copolymeric (dl-lactic, glycolic)acid (PGLA) microspheres. In another embodiment, the bioconjugate or composition comprising the bioconjugate may be continuously administered, where appropriate. In any of the embodiments described herein, the bioconjugate or composition comprising the bioconjugate can be administered intravascularly into the patient (e.g., into an artery or vein) in any suitable way. In various embodiments described herein, the

bioconjugate or composition comprising the bioconjugate can be administered into a vessel of a patient prior to, during, or after vascular intervention. In various embodiments, 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 embodiment, 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 compositions for use with the bioconjugates for parenteral administration or catheter-based delivery may comprise: a) a bioconjugate as described herein; b) a pharmaceutically acceptable 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 concentration range of about 0.25% to about 10% total formula weight or any individual component a), b), c), or d) or any combinations of a), b), c) and d). Dosing

Suitable dosages of the bioconjugate can be determined by standard methods, for example by establishing dose-response curves in laboratory animal models or in clinical trials and can vary significantly depending on the patient condition, the disease state being treated, the route of administration 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 μg/kg to about 500 μg/kg, or from about 100 μg/kg to about 400 μg/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 μg to about 1000 mg per dose, 1 μg to about 100 mg per dose, or from about 100 μg to about 50 mg per dose, or from about 500 μg to about 10 mg per dose or from about 1 mg to 10 mg per dose, or from about 1 to about 100 mg per dose, 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 herein, effective doses ranges from about 0.01 μg to about 1000 mg per dose, 1 μg to about 100 mg per dose, about 100 μg to about 1.0 mg, about 50 μg to about 600 μg, about 50 μg to about 700 μg, about 100 μg to about 200 μg, about 100 μg to about 600 μg, about 100 μg to about 500 μg, about 200 μg to about 600 μg, or from about 100 μg to about 50 mg per dose, or from about 500 μg to about 10 mg per dose or from about 1 mg to 10 mg per dose. In other illustrative embodiments, effective doses can be 1 μg, 10 μg, 25 μg, 50 μg, 75 μg, 100 μg, 125 μg, 150 μg, 200 μg, 250 μg, 275 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 550 μg, 575 μg, 600 μg, 625 μg, 650 μg, 675 μg, 700 μg, 800 μg, 900 μg, 1.0 mg, 1.5 mg, 2.0 mg, 10 mg, 100 mg, or 100 mg to 30 grams.

In some embodiments, separate or sequential administration of the bioconjugate and other agent is necessary to facilitate delivery of the composition. In certain embodiments, the bioconjugate and the other agent can be administered at different dosing frequencies or intervals. For example, the bioconjugate can be administered daily, while the other agent can be administered less frequently. Additionally, as will be apparent to those skilled in the art, the bioconjugate and the other agent can be administered using the same route of

administration or different routes of administration. Any effective regimen for administering the bioconjugate can be used. For example, the bioconjugate can be administered as a single dose, or as a multiple-dose daily regimen. Further, a staggered regimen, for example, one to five days per week can be used as an alternative to daily treatment. In various embodiments described herein, the patient is treated with multiple injections of the bioconjugate. In one embodiment, the patient is injected multiple times (e.g., about 2 up to about 50 times) with the bioconjugate, for example, at 12-72 hour intervals or at 48-72 hour intervals. Additional injections of the bioconjugate can be administered to the patient at an interval of days or months after the initial injections(s). In various embodiments described herein, the non-limiting examples of administration sites include articular joints of body, e.g., knee, shoulder, temporo-mandibular and carpometacarpal joints, elbow, hip, wrist, ankle, and lumbar zygapophysial (facet) joints in the spine, and lining regions, e.g. endothelium or vitreous humor of the eye. Examples of administration to connective and other tissues include articular cartilage, tendon, meniscus, ligament, synovium, etc.

In various embodiments described herein, the viscosupplement is also administered using the similar means and modes of administration as described herein for the

bioconjugates. In some embodiments, this disclosure further provides a

viscosupplementation device comprising a pre-filled, single-use syringe having a single unit dosage of the viscosupplement. In another embodiment, the viscosupplement comprises sodium hyaluronate.

Examples

Example 1: Synthesis of Pep tidogl cans Reagents

The peptides GAHWQFNALTVRGG (SEQ ID NO: ) (GAH) and WYRGRL (SEQ

ID NO: ) were purchased from Genscript (Piscataway, NJ). N-[B-maleimidopropionic acid] hydrazide, trifluoroacetic acid salt (BMPH) was purchased from Pierce (Rockford, IL).

Methods

The synthesis of collagen type I and hyaluronic acid-binding 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 2008; 9: 2562-2566). The present bioconjugate was prepared according to the methods described therein and modified as described in Example 2.

All intermediates were purified by size-exclusion chromatography 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 bioconjugate can be lyophilized and stored for extended periods of time at -80 °C.

Example 2: Lubricin Mimic Fabrication:

Chondroitin-6 sulfate (CS) backbone was oxidized to create about 20 aldehydes for crosslinker BMPH to bind. Purification through size exclusion chromatography rids of excess BMPH. BMPH links to HA-binding peptides (GAH (SEQ ID NO: )) and collagen II binding peptide (WYRGRL (SEQ ID NO: )). Initially 10 mols GAH (SEQ ID NO: )and 10 mols WYRGRL (SEQ ID NO: ) were added per 1 mol of CS. Later the ratios changed to 5 mol GAH (SEQ ID NO: ) and 15 mol WYRGRL (SEQ ID NO: ) for more collagen II binding. Also, 1 mol of biotinylated GAH was added to each CS for detection purposes.

Using this protocol, mLublO and mLubl5 were synthesized. The compositions of mLublO and mLubl5 are as follows: mLublO = lubricin mimic with 10 mol WYRGRL (SEQ ID NO: ) (collagen II binding) and 10 mol GAH (SEQ ID NO: ) (HA) mLubl5 = lubricin mimic with 15 mol WYRGRL (SEQ ID NO: ) (collagen II) and 5 mol GAH (SEQ ID NO: ) (HA)

Example 3: Binding Assay to Assess mLub Binding to Hyaluronic Acid

The following protocol is based on the method described by Peach et al.,

Identification of hyaluronic acid-binding sites in the extracellular domain of CD44, JCB, vol. 122, no. 1, 257-265, 1993.

96-well plate were coated with 50 μΐ of 50 μg/ml HA (hyaluronic acid sodium salt from strep, equi) in 50 mM sodium carbonate and incubated overnight. Wells were rinsed with PBS + Tween in between each step. Wells were blocked with 1% BSA in PBS solution. 10: 1 serial dilutions of a 10 mM solution of the lubricin mimic (e.g., mLublO or mLubl5) was were made. Solutions of the lubricant mimetic was added to each well and incubated at 37°C. The wells were rinsed and a streptavidin-HRP solution was added to each well and incubated in the dark. The wells were rinsed and a color solution was added to each well and incubated in the dark. 2N sulfuric acid was added to each well. The absorbance was measured on a plate reader at 450 and 540 nm.

FIG. lshows mLublO binding with hyaluronic acid sodium salt in assay. FIG. 2 shows mLubl5 binding with hyaluronic acid sodium salt assay. Comparing with FIG. 1, mLubl5 binds less than the mLublO, which agrees with the decrease in HA-binding peptides on the molecule.

Example 4: Binding Assay to Assess mLub Binding to Collagen II

Collagen II was dissolved in 10 mM HC1 to make a 0.5 mg/ml solution. The solution was added to a 96-well plate and incubated overnight at 4 °C. Wells were rinsed with PBS + Tween in between each step. The wells were blocked with 1% BSA in PBS for 1 hour in RT. 10: 1 serial dilutions of a 10 mM solution of the lubricin mimic (e.g., mLublO or mLubl5) was were made. A solution of the lubricant mimetic was and added to each well and incubated at 37 °C. The wells were rinsed and a streptavidin-HRP solution was added to each well and incubated in the dark. The wells were rinsed and a color solution was added to each well and incubated in the dark. 2N sulfuric acid was added to each well. The absorbance was measured on a plate reader at 450 and 540 nm.

FIG. 3shows mLublO binding with collagen II the binding assay. This shows that the mLublO binds to collagen II. FIG. 4 shows mLubl5 binding with collagen II binding assay. This shows that the mLub 15 binds to collagen II. FIG. 5 shows combined collagen II and HA data with mLub 15.

Example 5: Friction

Cartilage extraction

Seven millimeter diameter cartilage plugs were harvested from bovine knees joints at the load bearing regions on the femoral condyles. The plugs were stored in Hank's Balanced Salt Solution (HBSS) plus a protease inhibitor cocktail (PIC) at 4C. Right after harvest, some of the plugs are treated with 0.5% trypsin solution for 3 hours to deplete the cartilage of some peptidoglycans to mimic osteoarthritis (OA). FBS was used to inactivate the trypsin. Plugs then rinsed with HBSS + PIC for storage. See, e.g., Poole AR, et al. J Histochem Cytochem 1980; 28(7):621-35.

Macroscale COF: Rheometer The following protocol is based on the method described in Schmidt et al. Boundary lubrication of articular cartilage: Role of synovial fluid constituents, Arthritis & Rheumatism 56, 882-891, 2007.

A 3 mm diameter hole was cut in the middle of the 7 mm plug to create an annulus with an effective radius of 2. 6 mm. This value was used in all COF calculations.

The articular surface of the cartilage samples were treated with Synvisc alone, 0.3 mg/ml mLub for 5-10 minutes alone or then washed and then treated with 4 mg/ml Synvisc (commercial HA). The concentrations of the solutions were chosen based on the

concentration of these molecules in synovial fluid. The cartilage annulus was glued to a 20 mm flat geometry head as close to the center as possible. A clean glass slide was taped to the bottom plate of the rheometer. The plug was lowered to almost touching the glass slide and then the software program was run. The plug was surrounded by HBSS + PIC solution. The rheometer head compressed the cartilage plug until it reached 50 N and then sat at equilibrium for 60 minutes. The cartilage plug was then rotated at an angular velocity of 0.08726 rad/sec for 2 minutes. Torque and normal force were recorded throughout the run. See e.g., FIG. 6 Typical normal force and torque graphs during macroscale coefficient of friction testing using a rheometer.

Static coefficient of friction (COF) was calculated by taking the maximum torque during the first 10 degrees (~2sec) and normal force into this equation. (R = effective radius of 2.6 mm)

T

The kinetic COF was calculated by averaging the COF calculated from the second rotation.

The goal was to bring down the COF of a trypsin treated plug to match the COF of the WT cartilage. FIG. 7 shows total static and kinetic coefficient of friction (COF) data from rheometer. The rheometer data was of cartilage on glass. The static and kinetic COF values were calculated for each treatment group. In static COF, there is statistical difference between the trypsin treated plug and the WT, mLubl5 + Synvisc, and mLubl5 + HA treatments. In kinetic friction, there is statistical difference between the mLub 15 +Synvisc treatment and the trypsin, Synvisc, and mLublO + Synvisc treatments. Standard error bars are shown. ( p<0.05 for both static and kinetic COF differences). Each treatment group had a different n value (ranging from n=9 to 12). Thus, trypsin treated plugs that were treated with mLubl5 then Synvisc had significantly lower kinetic COF than the wild type, whereas trypsin treated plugs that were treated with mLub 10 then Synvisc had higher COF than the wild type. FIG. 8 shows fluorescent staining images of cryosections of cartilage with mLub probed with streptavidin and DAPI for nuclei. The left image represents a cartilage sample that did not go through the compression and shear movements on the rheometer while the right image was cyro- sectioned after the rheometer test. FIG. 8 shows that there is still mLub present after compression and shear movement. Arrows point to the mLub covered cartilage surface.

AFM

Contact AFM was used with a 2 μιη diameter borosilicate sphere tip cantilever probe. The stiffness was precalibrated by the manufacturers to be around 0.6 N/m. The lateral force calibration constant was calculated using Varenberg' s improved wedge calibration method (2003). The cantilever deflection sensitivity and adhesion force was calculated using the software on a force vs distance curve on a glass slide in PBS. A silicon calibration grating sample with 54'44" slopes was used for lateral calibration. The slope was scanned and the trace and retrace friction graph was recorded for values to be used for the calibration calculations. Samples were scanned in 50 um square sections. Speed was 1.5 ln/sec. The set point

(normal force) was varied to be 1, 2, 4, and 5 mV (34.38 - 171.88 nN) for each area on the cartilage sample. 5 different areas were scanned on each cartilage spot.

Using another software tool, RMS roughness was calculated . An adhesion force test was taken to calculate the average elasticity/stiffness of the surface as well as the average adhesion force.

The friction measurements were taken on 50 micron square sections of the cartilage sample at a speed of 50 μνη/ εο. The area was scanned with a 5V normal load applied (around 120 nN). The normal load (V) was converted to normal force (N) by multiplying by the deflection sensitivity and spring constant. While observing the friction force plots during data analysis, areas of the surface where the controller was not detecting the surface were observed. In order to take these areas out of the data analysis we programmed data analysis code areas with the presence of fibers and no controller interference. These areas were then used for analysis. The average area taken from each sample area was 971 squared microns with a standard deviation of 280 squared microns. The friction voltage signal was averaged for both the trace and retrace scan and then the difference between the two divided by two. This value was then converted to friction force by multiplying by the calibration constant. Averaged friction force divided by normal force to calculate coefficient of friction.

Using the same areas taken for friction measurements, surface RMS roughness was calculated using the topography data as shown in FIG. 9, which represent samples with roughness values near the values of those shown in FIG. 10.

FIG. 10 shows friction coefficient, roughness and adhesion force on cartilage surface. Friction coefficient and roughness values of selected areas of cartilage surfaces (n = 9) (p > 0.05) are shown. Standard error bars are shown. Friction was measured at selected areas within a 50 by 50 micron area where clear fibers are imaged and no AFM controller interference. This was done on 5 areas of each sample and averaged. COF was calculated by dividing the measured friction force by normal force. Adhesion values of a small subset of cartilage samples (n=2)(p < 0.05) are shown. Standard error bars are shown.

Fluorescent Imaging

Cartilage plugs (after treatment with trypsin then mLub 15 and rinsed) were frozen in O.C.T. compound and sectioned at 10 um thickness. The sections were allowed to dry overnight before being fixed in 4% paraformaldehyde. The sections were briefly rinsed and then fluorescent Streptavidin and DAPI were added and incubated for 30 minutes. The sectioned were rinsed and then imaged with a fluorescent microscope. Images are shown in FIG. 11.

Toluidine Blue Staining

Samples cryo- sectioned the same way were also stained with 0.04% Toluidine Blue and counterstained with 0.05% Fast Green FCF dye. This was done to show the proteoglycan depleting effect of the trypsin treatment. Images are shown in FIG. 11.

In Vivo Residency Time

Dunkin Hartley guinea pigs were chosen as a model due to their development of spontaneous osteoarthritis. The animals were allowed to grow to 4 months before testing. The patellar tendon was located on the hind legs of the animals for injection. Sterile injections of 100

of 0.5 mg/ml mLub in PBS were injected into the synovial fluid behind the patellar tendon. The other knee was injected with 100 ul PBS for a control. After 6 hours, 1 and 2 weeks, the animals were sacrificed and the femoral condyles were harvested and prepared in O.C.T. compound for cryo-sectioning. Before this study, a 50 mg/ml Coomassie Brilliant Blue G-250 solution was injected for visualization to confirm the procedure was correct. 15 μιη sections were stained with the same protocol as stated previously.

FIG. 12 shows fluorescent imaging of the articular cartilage at 6 hours, 1 week and 2 weeks after in vivo injection with PBS in the top row and mLubl5 is on the bottom row. FIG. 13 shows trypsin treated cartilage. Cell nuclei are stained with DAPI. No red fluorescence is observed.

FIG. 14 shows trypsin treated cartilage with mLubl5 treatment. Cell nuclei are stained with DAPI. Fluorescence is observed from streptavidin bound to the biotinylated mLub showing that mLub is bound at the surface of cartilage.

FIG. 15 shows 10X magnification or FIG. 14. Trypsin treated cartilage with mLubl5 treatment.

Example 6: In Vivo Efficacy of mLub

Dunkin Hartley guinea pigs were chosen as a model due to their development of spontaneous osteoarthritis. The animals were allowed to grow to 4 months before testing. The patellar tendon was located on the hind legs of the animals for injection. Sterile injections of 100 μΐ_^ of 0.5 mg/ml mLub 15 in PBS were injected into the synovial fluid behind the patellar tendon. The other knee was injected with 100 ul PBS for a control. The injections were performed 3 times at one-week intervals. The animals were sacrificed 12 weeks after the first injection.

Histology: Femoral and tibial condyles were harvested and sagittal sections were taken and scored blindly (by Alison Bendele, (HistoTox Labs, Boulder, CO)) for cartilage degeneration in three different zones (inner, middle, and outer zones) of each section .

FIGS. 16-17 show medial femur and tibial cartilage degeneration scores for three different zones of cartilage samples taken from guinea pigs treated with mLub 15 or a control, showing that treatment with mLub 15 protected articular cartilage in vivo. FIGS. 18-19 show % collagen degeneration measured in cartilage samples taken from guinea pigs treated with mLubl5 (or a control) across a population of guinea pigs having varying degrees of severity of osteoarthritis. A decrease in collagen degeneration was observed in guinea pigs treated with mLubl5 compared to the control.

This example demonstrates in vivo efficacy of mLubl5 in the protection of articular cartilage and attenuation of osteoarthritis.

Claims

What is claimed is:

1. A bioconjugate comprising: a) a glycan; b) from about 3 to about 30 collagen-binding peptides having at least one collagen- binding domain; and c) from about 1 to about 10 hyaluronic acid-binding peptide(s) having at least one hyaluronic acid-binding domain; wherein the ratio of collagen-binding peptides to hyaluronic acid-binding peptide(s) is about 3: 1 or about 4: 1, and wherein the peptides are covalently bonded to the glycan.

2. The bioconjugate of claim 1, wherein the glycan is alginate, dextran, dextran sulfate, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, heparan, heparan sulfate, heparin, keratin, keratan sulfate, or hyaluronic acid.

3. The bioconjugate of claim 1, wherein the glycan is dermatan sulfate.

4. The bioconjugate of claim 1, wherein the glycan is heparin.

5. The bioconjugate of claim 1, wherein the glycan is chondroitin sulfate.

6. The bioconjugate of claim 1, wherein the peptides are covalently bonded to the glycan via a linker.

7. The bioconjugate of claim 1, wherein the collagen-binding peptides exhibit binding affinity to one or more of collagen types I, II, III, or IV.

8. The bioconjugate of claim 1, wherein the collagen-binding peptides comprise at least one collagen-binding domain which binds to type I collagen and at least one collagen-binding domain that binds to type II collagen.

9. The bioconjugate of claim 1, wherein the collagen-binding peptides comprise an amino acid sequence selected from the group consisting of: i) WYRGRL (SEQ ID NO: ), RRANAALKAGELYKS ILY (SEQ ID NO: ),

RRANAALKAGELYKCILY (SEQ ID NO: ), RLDGNEIKR (SEQ ID NO: ),

AHEEISTTNEGVMGC (SEQ ID NO: ), NGVFKYRPRYFLYKHAYFYPPLKRFPVQ (SEQ ID NO: ), CQDSETRTFY (SEQ ID NO: ), TKKTLRT (SEQ ID NO: ),

GLRSKSKKFRRPDIQYPDATDEDITSHM (SEQ ID NO: ), SQNPVQP (SEQ ID NO: ), SYIRIADTNIT (SEQ ID NO: ), KELNLVYT (SEQ ID NO: ), GELYKSILY (SEQ ID NO: ), GELYKCILY (SEQ ID NO: ) and GSITTIDVPWNV (SEQ ID NO: ), or ii) any peptide sequence comprising a sequence having at least about 80% sequence identity to the amino acid sequence of i).

10. The bioconjugate of claim 1, wherein the collagen-binding peptide comprises

WYRGRL (SEQ ID NO: ), RRANAALKAGELYKS ILY (SEQ ID NO: ),

RRANAALKAGELYKCILY (SEQ ID NO: ), RLDGNEIKR (SEQ ID NO: ),

AHEEISTTNEGVMGC (SEQ ID NO: ), NGVFKYRPRYFLYKHAYFYPPLKRFPVQ (SEQ ID NO: ), CQDSETRTFY (SEQ ID NO: ), TKKTLRT (SEQ ID NO: ),

GLRS KS KKFRRPDIQ YPD ATDEDITSHM (SEQ ID NO: ), SQNPVQP (SEQ ID NO: ),

SYIRIADTNIT (SEQ ID NO: ), KELNLVYT (SEQ ID NO: ), GELYKSILY (SEQ ID NO: ), GELYKCILY (SEQ ID NO: ) or GSITTIDVPWNV (SEQ ID NO: ).

11. The bioconjugate of claim 1, wherein the collagen-binding peptides comprise

WYRGRL.

12. The bioconjugate of claim 1, wherein the hyaluronic acid-binding peptide(s) comprise an amino acid sequence selected from the group consisting of: i) GAHWQFNALTVR (SEQ ID NO: ), GDRRRRRMWHRQ (SEQ ID NO: ), GKHLGGKHRRSR (SEQ ID NO: ), RGTHHAQKRRS (SEQ ID NO: ), RRHKSGHIQGSK (SEQ ID NO: ), SRMHGRVRGRHE (SEQ ID NO: ), RRRAGLTAGRPR (SEQ ID NO: ), RYGGHRTS RKW V (SEQ ID NO: ), RSARYGHRRGVG (SEQ ID NO: ),

GLRGNRRVFARP (SEQ ID NO: ), SRGQRGRLGKTR (SEQ ID NO: ),

DRRGRS S LPKLAGP VEFPDRKIKGRR (SEQ ID NO: ), RMRRKGRVKHWG (SEQ ID NO: ), RGGARGRHKTGR (SEQ ID NO: ), TGARQRGLQGGWGPRHLRGKDQPPGR (SEQ ID NO: ), RQRRRDLTRVEG (SEQ ID NO: ),

S TKDHNRGRRN VGP VS RS TLRDPIRR(S EQ ID NO: ), RRIGHQVGGRRN (SEQ ID NO: ), RLES R A AGQRR A (SEQ ID NO: ), GGPRRHLGRRGH (SEQ ID NO: ),

VS KRGHRRT AHE (SEQ ID NO: ), RGTRSGSTR (SEQ ID NO: ), RRRKKIQGRSKR (SEQ ID NO: ), RKSYGKYQGR (SEQ ID NO: ), KNGRYSISR (SEQ ID NO: ),

RRRCGQKKK (SEQ ID NO: ), KQKIKHVVKLK (SEQ ID NO: ), KLKSQLVKRK (SEQ ID NO: ), RYPISRPRKR (SEQ ID NO: ), KVGKSPPVR (SEQ ID NO: ), KTFGKMKPR (SEQ ID NO: ), RIKWSRVSK (SEQ ID NO: ) and KRTMRPTRR (SEQ ID NO: ); or ii) any peptide sequence comprising a sequence having at least about 80% sequence identity to the amino acid sequence of i).

13. The bioconjugate of claim 1, wherein the hyaluronic acid-binding peptide(s) comprise GAHWQFNALTVR (SEQ ID NO: ), GDRRRRRMWHRQ (SEQ ID NO: ),

GKHLGGKHRRSR (SEQ ID NO: ), RGTHHAQKRRS (SEQ ID NO: ), RRHKSGHIQGSK (SEQ ID NO: ), SRMHGRVRGRHE (SEQ ID NO: ) , RRRAGLTAGRPR (SEQ ID NO: ), RYGGHRTS RKW V (SEQ ID NO: ), RSARYGHRRGVG (SEQ ID NO: ),

GLRGNRRVFARP (SEQ ID NO: ), SRGQRGRLGKTR (SEQ ID NO: ),

DRRGRS S LPKLAGP VEFPDRKIKGRR (SEQ ID NO: ), RMRRKGRVKHWG (SEQ ID NO: ), RGGARGRHKTGR (SEQ ID NO: ), TGARQRGLQGGWGPRHLRGKDQPPGR (SEQ ID NO: ), RQRRRDLTRVEG (SEQ ID NO: ),

STKDHNRGRRNVGPVSRSTLRDPIRR (SEQ ID NO: ), RRIGHQVGGRRN (SEQ ID NO: ), RLES R A AGQRR A (SEQ ID NO: ), GGPRRHLGRRGH (SEQ ID NO: ),

VS KRGHRRT AHE (SEQ ID NO: ), RGTRSGSTR (SEQ ID NO: ), RRRKKIQGRSKR (SEQ ID NO: ), RKSYGKYQGR (SEQ ID NO: ), KNGRYSISR (SEQ ID NO: ),

RRRCGQKKK (SEQ ID NO: ), KQKIKHVVKLK (SEQ ID NO: ), KLKSQLVKRK (SEQ ID NO: ), RYPISRPRKR (SEQ ID NO: ), KVGKSPPVR (SEQ ID NO: ), KTFGKMKPR (SEQ ID NO: ), RIKWSRVSK (SEQ ID NO: ) or KRTMRPTRR (SEQ ID NO: ).

14. The bioconjugate of claim 1, wherein the hyaluronic acid-binding peptide(s) comprise GAHWQFNALTVR (SEQ ID NO: ).

15. The bioconjugate of claim 1, wherein the collagen-binding domain binds to collagen with a dissociation constant (¾) of less than about 1 mM.

16. The bioconjugate of claim 1, wherein the collagen-binding domain binds to type I collagen, type II collagen or type III collagen with a dissociation constant (¾) of less than about 1 mM.

17. The bioconjugate of claim 1, wherein the hyaluronic acid-binding domain binds to hyaluronic acid with a dissociation constant (¾) of less than about 1 mM.

18. The bioconjugate of claim 1, wherein the collagen-binding peptides comprise up to 40 amino acids.

19. The bioconjugate of claim 1, wherein the collagen-binding peptides comprise up to 25 amino acids.

20. The bioconjugate of claim 1, wherein the hyaluronic acid-binding peptide(s) comprise up to 40 amino acids.

21. The bioconjugate of claim 1, wherein the hyaluronic acid-binding peptide(s) comprise up to 25 amino acids.

22. The bioconjugate of claim 1, comprising about 15 collagen-binding peptides.

23. The bioconjugate of claim 1, comprising about 5 hyaluronic acid-binding peptides.

24. A pharmaceutical composition comprising the bioconjugate of any one of claims 1-23.

25. A method of treating and/or preventing degradation of a hyaluronic acid rich tissue in a patient comprising administering to a patient in need thereof the bioconjugate of any one of claims 1-23 or the pharmaceutical composition of claim 24 and a viscosupplement.

26. The method of claim 25, wherein the hyaluronic acid rich tissue is the skin.

27. A method of treating and/or preventing cartilage degeneration in a patient comprising administering to a patient in need thereof the bioconjugate of any one of claims 1-23 or the pharmaceutical composition of claim 24 and a viscosupplement.

28. The method of claim 27, wherein the bioconjugate of any one of claims 1-23 or the pharmaceutical composition of claim 24 and a viscosupplement are administered into a synovial cavity of the patient.

29. A method of treating and/or preventing vitreous humor degeneration in a patient comprising administering to a patient in need thereof the bioconjugate of any one of claims 1- 23 or the pharmaceutical composition of claim 24.

30. The method of claim 29, wherein the method further comprises administering a viscosupplement.

31. A method of treating and/or preventing nucleus pulposus degeneration in a patient comprising administering to a patient in need thereof the bioconjugate of any one of claims 1- 23 or the pharmaceutical composition of claim 24.

32. The method of claim 31, wherein the method further comprises administering a viscosupplement.

33. A method of decreasing scar formation in a patient comprising administering to a patient in need thereof the bioconjugate of any one of claims 1-23 or the pharmaceutical composition of claim 24 and a viscosupplement.

34. The method of any of claims 25-28, 30, 32 or 33, wherein the viscosupplement comprises sodium hyaluronate.

35. A bioconjugate comprising: a) chondroitin sulfate; b) WYRGRL (SEQ ID NO: ); and c) GAHWQFNALTVRGG (SEQ ID NO: ); wherein the ratio of WYRGRL (SEQ ID NO: ) and GAHWQFNALTVRGG (SEQ ID NO: ) is about 3: 1, and WYRGRL (SEQ ID NO: ) and GAHWQFNALTVRGG (SEQ ID NO: ) are covalently bonded to the chondroitin sulfate.

36. The bioconjugate of claim 35, wherein WYRGRL (SEQ ID NO: ) and

GAHWQFNALTVRGG (SEQ ID NO: ) are covalently bonded to the chondroitin sulfate via N-[P-maleimidopropionic acid]hydrazide (BMPH).