TREATMENT OF FIBROSIS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 USC 119(e) to U.S. Provisional Patent Application No. 62/530,061, filed July 7, 2017, the disclosure of which is hereby

incorporated by reference in its entirety.

BACKGROUND

[0002] Fibrosis is often defined as a tissue wound-healing response where the healing process becomes pathogenic resulting in remodeling of the ECM and formation of scar tissue. Despite sub-type, fibrotic disorders have a persistent irritant that sustains pathological remodeling state. In normal injury, the damaged vascular wall activate platelets that release multiple mediators, initiating a cascade of inflammation, white cell recruitment and tissue remodeling. In the setting of chronic injury, the above cascade is perpetual, resulting in ongoing tissue remodeling and excessive ECM deposition.

SUMMARY

[0003] The present disclosure provides methods for inhibiting and/or treating fibrosis using collagen-binding bioconjugates. In certain embodiments, the fibrosis is the result of a fibrotic disease, such as pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, renal fibrosis, liver fibrosis, cirrhosis, cardiac fibrosis, atrial fibrosis, endomyocardial fibrosis, myocardial infarction, glial scar, arthrofibrosis, Crohn's disease, Dupuytren's contracture, keloid, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma, systemic sclerosis and/or adhesive capsulitis.

[0004] Also provided are methods for preventing and/or treating vasculitis. The methods may be used to treat large vessel vasculitis (LVV), medium vessel vasculitis (MVV), small vessel vasculitis (SVV), variable vessel vasculitis (VVV), single-organ vasculitis (SOV), vasculitis associated with systemic disease, and/or vasculitis associated with probable etiology. Non-limiting examples of large vessel vasculitis (LVV) include takayasu arteritis (TAK) and giant cell arteritis (GCA). Non-limiting examples of medium vessel vasculitis (MVV) include polyarteritis nodosa (PAN) and kawasaki disease (KD). Non-limiting examples of small vessel vasculitis (SVV) include antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), microscopic polyangiitis (MP A), granulomatosis with polyangiitis (Wegener's) (GPA), eosinophilic granulomatosis with polyangiitis (Churg- Strauss) (EGPA), immune complex SVV, anti-glomerular basement membrane (anti-GBM) disease, gryoglobulinemic vasculitis (CV), IgA vasculitis (Henoch-Schonlein) (IgAV), and hypocomplementemic urticarial vasculitis (HUV)) (anti-Clq vasculitis). Non-limiting examples of variable vessel vasculitis (VVV) include Behcet's disease (BD) and Cogan's syndrome (CS). Non-limiting examples of single-organ vasculitis (SOV) include cutaneous leukocytoclastic angiitis, cutaneous arteritis, primary central nervous system vasculitis, and isolated aortitis. Non-limiting examples of vasculitis associated with systemic disease include lupus vasculitis, rheumatoid vasculitis, and sarcoid vasculitis. Non-limiting examples of vasculitis associated with probable etiology include hepatitis C virus-associated

cryoglobulinemic vasculitis, hepatitis B virus-associated vasculitis, syphilis-associated aortitis, drug-associated immune complex vasculitis, drug-associated ANCA-associated vasculitis, and cancer-associated vasculitis. Other examples of vasculitis include

antiphospholipid syndrome, Buerger's disease (thromboangiitis obliterans),

cryoglobulinemia, cryopyrin-associated autoinflammatory syndrome (CAPS) (juvenile), goodpastures, localized scleroderma (juvenile), polymyalgia rheumatica, Raynaud's phenomenon, scleroderma, Sjogren's syndrome, and systemic lupus erythematosus.

[0005] In one embodiment, the disease is not acute tubular necrosis, diabetic chronic renal failure, lupus nephritis, renal fibrosis, or acute glomerulonephritis. In one embodiment, the disease is not idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease, asthma, or emphysema.

[0006] Also provided are methods for inhibiting and/or treating fibrosis or vasculitis by administering a collagen-binding bioconjugate in combination another agent. Non-limiting examples of anti-fibrotic agents include predonine, N-acetylcysteine, pirfenidone, nintedanib, corticosteroids, cyclophosphamide, azathioprine, methotrexate, penicillamine, cyclosporine A, FK506, colchicine, IFN-γ and mycophenolate mofetil. Non-limiting examples of agents for treating vasculitis include prednisone, Cyclophosphamide (Cytoxan), methylprednisolone, methotrexate sodium, Medrol (Pak), Medrol, dexamethasone, prednisolone, DexPak, Deltasone, cortisone, Prednisone Intensol, dexamethasone sodium phosphate, Orapred ODT, Trexall, Rheumatrex, methotrexate sodium (PF), Veripred 20, Dexamethasone Intensol, prednisolone sodium phosphate, Pediapred, Millipred, Rayos, Millipred, and DoubleDex

[0007] In one embodiment, the present disclosure provides a method for treating fibrosis or vasculitis in a patient in need thereof, comprising administering to the patient an effective amount of a bioconjugate comprising a glycan bonded to at least one peptide comprising at least one collagen-binding unit.

[0008] The collagen binding unit can bind to any one, or more type of collagen. In certain embodiments, the collagen-binding unit binds to type I or III collagen. In other embodiments, the collagen-binding unit binds to type IV collagen.

[0009] The number of available binding sites for peptide conjugation can also vary depending on the structure of the glycan which is employed, and thus the number of peptides bound to the glycan can vary. In certain embodiments, the bioconjugate comprises from 1 to about 25 peptides, or from about 5 to about 25 peptides, or from about 1 to about 15 peptides, or about 2 peptides, or about 5 peptides, or about 10 peptides, or about 15 peptides per glycan. In certain embodiments, the glycan comprises: a) from about 1 to about 75 percent (%) functionalization, b) from about 5 to about 30 percent (%) functionalization, c) from about 10 to about 40 percent (%) functionalization, d) about 25 percent (%) functionalization, or e) about 30 percent (%) functionalization, wherein the percent (%) functionalization is determined by a percent of disaccharide units on the glycan which are functionalized with peptide. In certain embodiments, the peptide is bound to the glycan via a spacer.

BRIEF DESCRIPTION OF THE FIGURES

[0010] FIG. 1 shows that Compound 1 can target damaged lung tissue.

[0011] FIG. 2 is an illustration showing that Compound 1 reduced inflammatory cell number in lung injury.

DETAILED DESCRIPTION

[0012] 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.

1. Definitions

[0013] 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. [0014] 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.

[0015] 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.

[0016] 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%.

[0017] The following abbreviations used herein have the following meanings.

SPR Surface Plasmon Resonance

TAPS 3-[[l,3-dihydroxy-2-(hydroxymethyl)propan-2- yl]amino]propane-l -sulfonic acid

TES 2-[[l,3-dihydroxy-2-(hydroxymethyl)propan-2- yl]amino]ethanesulfonic acid

Tris 2-Amino-2-hydroxymethyl-propane-l,3-diol

w/w Weight/Weight

w/v Weight/Volume

[0018] As used herein, the term "treating" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, inhibiting, suppressing and/or halting fibrosis, including one or more clinical symptoms of fibrosis.

[0019] 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 bioconjugate, 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; through a catheter) to be treated, for local effect.

[0020] 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 of the material to a patient, taking into consideration the disease or conditions to be treated and the respective route of administration. For example, it is commonly required that such a material be essentially sterile.

[0021] 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 desired tissue or a tissue adjacent to the desired tissue.

[0022] 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.

[0023] As used herein, the term "delivery" refers to approaches, formulations,

technologies, and systems for transporting a pharmaceutical composition in the body as needed to safely achieve its desired therapeutic effect. In some embodiments, an effective amount of the composition is formulated for delivery into the blood stream of a patient (e.g., intravenous delivery).

[0024] As used herein, the term "solution" 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 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.

[0025] As used herein, the term "polymer matrix" or "polymeric agent" refers to a biocompatible polymeric materials. 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(gly colic acid), copolymers of lactic and gly colic acids, or other polymeric agents both natural and synthetic.

[0026] As used herein, the term "absorbable" refers to the ability of a material to be absorbed into the body. In certain embodiments, the polymeric matrix is absorbable, such as, for example collagen, polyglycolic acid, polylactic acid, polydioxanone, and caprolactone. In other embodiments, the polymer is non- absorbable, such as, for example polypropylene, polyester or nylon. [0027] 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 of 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).

2. Treatment of Fibrosis

[0028] In one embodiment, provided herein are bioconjugates and methods for preventing and/or treating fibrosis. Fibrosis is an inflammatory disease in which inflammatory cells migrate into tissue and organs, leading to cellular responses that result in scarring. Fibrosis can occur in many tissues within the body, typically as a result of inflammation or damage. By preventing inflammatory cell extravasation, fibrosis can be attenuated or prevented.

[0029] In one embodiment, the bioconjugates and methods provided herein can be used to prevent and/or treat pulmonary fibrosis. In lungs, types of fibrosis include pulmonary fibrosis such as cystic fibrosis and idiopathic pulmonary fibrosis. Pulmonary fibrosis is a respiratory disease in which scars are formed in the lung tissues, leading to serious breathing problems. Scar formation leads to thickening of the walls, and causes reduced oxygen supply in the blood. As a consequence patients suffer from perpetual shortness of breath.

[0030] In one embodiment, the bioconjugates and methods provided herein can be used to treat liver fibrosis. Liver fibrosis may result from a wide variety of conditions including chronic alcohol exposure, hepatitis B virus (HBV) infection, non-alcoholic fatty liver disease (NAFLD), Nonalcoholic steatohepatitis (NASH), hepatitis C virus (HCV) infection, Wilson's disease, alpha- 1 -antitrypsin deficiency, hemochromatosis, primary biliary cirrhosis, primary sclerosing cholangitis, and autoimmune hepatitis. Chronic HCV is the leading contributor to chronic liver disease and the liver elicits a persistent inflammatory and fibrosis, which is characterized by the formation of fibrous tissue and scarring on the liver. NAFLD and NASH also cause inflammation and fibrosis in the liver.

[0031] Cirrhosis is fibrosis in the liver in which the liver does not function properly due to long-term damage. Typically, the disease comes on slowly over months or years. Early on, there are often no symptoms. As the disease worsens, a person may become tired, weak, itchy, have swelling in the lower legs, develop yellow skin, bruise easily, have fluid buildup in the abdomen, or develop spider-like blood vessels on the skin. The fluid build-up in the abdomen may become spontaneously infected. Other complications include hepatic encephalopathy, bleeding from dilated veins in the esophagus or dilated stomach veins, and liver cancer. Hepatic encephalopathy results in confusion and possibly unconsciousness. Cirrhosis can result in liver dysfunction. The following symptoms or features are direct consequences of liver dysfunction and thus can also be treated or ameliorated by the presently disclosed compositions and methods.

[0032] It has been shown that that direct interaction between hepatic stellate cells (HSCs) and tumor cells promotes tumor growth via multiple mechanisms. Therefore, targeting HSCs to lessen or eliminate their tumor-supporting role presents a potential therapeutic strategy to prevent, inhibit or treat hepatocellular carcinoma (HCC). In certain embodiments, provided is a method of preventing or inhibiting the development of hepatocellular carcinoma (HCC) in a patient in need thereof, comprising administering to the patient an effective amount of a bioconjugate as described herein. In certain embodiments, the development of hepatocellular carcinoma (HCC) is a result of liver cirrhosis. In certain embodiments, the method comprises inhibiting hepatic stellate cell proliferation and/or fibrotic phenotype transition. In certain embodiments, the bioconjugate is administered locally to the liver, such as during a transcatheter arterial chemoembolization (TACE) procedure.

[0033] Targeting HSCs can also lessen or eliminate other disorders. Spider angiomas or spider nevi are vascular lesions consisting of a central arteriole surrounded by many smaller vessels and occur due to an increase in estradiol. Palmar erythema is a reddening of palms at the thenar and hypothenar eminences also as a result of increased estrogen. Gynecomastia, or increase in breast gland size in men that is not cancerous, is caused by increased estradiol and can occur in up to two thirds of patients. Hypogonadism, a decrease in sex hormones manifest as impotence, infertility, loss of sexual drive, and testicular atrophy, can result from primary gonadal injury or suppression of hypothalami c/pituitary function. Hypogonadism is associated with cirrhosis due to alcoholism and hemochromatosis. Liver size can be enlarged, normal, or shrunken in people with cirrhosis.

[0034] In one embodiment, the bioconjugates and methods provided herein can be used to prevent and/or treat renal fibrosis. Renal fibrosis can result from acute or sustained injury to the kidney. The injury can lead to excessive deposition of extracellular matrix. Over time, this can result in kidney failure, requiring patients to undergo dialysis or kidney transplant.

[0035] Ascites, accumulation of fluid in the peritoneal cavity, gives rise to flank dullness. This can be visible as increase in abdominal girth. Fetor hepaticus is a musty breath odor resulting from increased dimethyl sulfide. Jaundice is yellow discoloration of the skin and mucous membranes due to increased bilirubin. In addition, liver cirrhosis increases resistance to blood flow and higher pressure in the portal venous system, resulting in portal hypertension.

[0036] In one embodiment, the bioconjugates and methods provided herein can be used to prevent and/or treat fibrosis in the heart. Fibrosis in the heart is present in the form of atrial fibrosis, endomyocardial fibrosis, or myocardial infarction. Glial scar is fibrosis in the brain. Other types of fibrosis include, without limitation, arthrofibrosis (knee, shoulder, other joints), Crohn's disease (intestine), Dupuytren's contracture (hands, fingers), keloid (skin), mediastinal fibrosis (soft tissue of the mediastinum), myelofibrosis (bone marrow),

Peyronie's disease (penis), nephrogenic systemic fibrosis (skin), progressive massive fibrosis (lungs), retroperitoneal fibrosis (soft tissue of the retroperitoneum), scleroderma/systemic sclerosis (skin, lungs), and some forms of adhesive capsulitis (shoulder).

[0037] It is contemplated that the compositions and methods of the present disclosure are suitable for preventing and/or treating any of these diseases or symptoms or features associated with these diseases. Development of fibrosis involves stimulated cells laying down connective tissue, including collagen and glycosaminoglycans. The bioconjugates of the present disclosure can interact with the collagen or glycosaminoglycans and thus disrupt the formation of such excessive connective tissue. The bioconjugates can also protect the endothelial barrier. This can be by interacting with exposed extracellular matrix due to microvascular injury. Protecting the endothelial barrier prevents inflammatory cells from extravasating into the tissue to cause the excessive ECM deposition that leads to the fibrotic tissue. Accordingly, the bioconjugates can prevent, inhibit, delay, and/or reverse fibrosis. [0038] In certain embodiments, the fibrosis is post ischemic, post infectious, or idiopathic (e.g., renal, hepatic, cardiac, pulmonary). See, e.g., Guerrot, D., et al. Fibrogenesis & tissue repair 5.Suppl 1 (2012): S15, and Yamaguchi, I, et al. Nephron Experimental Nephrology 120.1 (2012): e20-e31. In certain embodiments, the fibrosis is retroperitoneal. In certain embodiments, the fibrosis is dermal (e.g., scleroderma). See, e.g., Maurer, B., et al. Annals of the rheumatic diseases (2013): annrheumdis-2013.

[0039] In one embodiment, the disease is not acute tubular necrosis, diabetic chronic renal failure, lupus nephritis, renal fibrosis, or acute glomerulonephritis. In one embodiment, the disease is not idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease, asthma, or emphysema.

[0040] In one embodiment, provided herein is a use of the bioconjugate(s) disclosed herein for the prevention or treatment of fibrosis. In one embodiment, provided herein is a use of the bioconjugate(s) disclosed herein for the preparation of a medicament for the prevention or treatment of fibrosis. In one embodiment, provided herein is a use of the bioconjugate(s) disclosed herein for the prevention or treatment of liver fibrosis. In one embodiment, provided herein is a use of the bioconjugate(s) disclosed herein for the prevention or treatment of pulmonary fibrosis. In one embodiment, the bioconjugate comprises heparin and from about 5 to about 10, or about 7, peptides, wherein the peptides comprise at least one sequence of RRANAALKAGELYKSILY (SEQ ID NO: ), RRANAALKAGELYKSILYGSG (SEQ ID NO:), GQLYKSILY (SEQ ID NO:), or GQLYKSILYGSGSGSRR (SEQ ID NO: ). In one embodiment, the peptide(s) are bound to the heparin or other glycan via a hydrazide- carbonyl linkage. In one embodiment, the bioconjugate comprises heparin and from about 5 to about 20% functionalization with peptides, wherein the peptides comprise at least one sequence of RRANAALKAGELYKSILY (SEQ ID NO: ), RRANAALKAGELYKSILYGSG (SEQ ID NO:), GQLYKSILY (SEQ ID NO: ), or GQLYKSILYGSGSGSRR (SEQ ID NO: ). In one embodiment, the peptide(s) are bound to the heparin via a hydrazide-carbonyl linkage. In one embodiment, the bioconjugate comprises heparin and from about 5 to about 20% functionalization with peptides, wherein the peptides comprise at least one sequence of: GQLYKSILY (SEQ ID NO: ), GQLYKSILYGSGSGSRR (SEQ ID NO: ),

CPGRVMHGLHLGDDEGPC (SEQ ID NO: ), CVWLWEQC (SEQ ID NO: ), or

WREPSFCALS (SEQ ID NO: ), or an amino acid sequence having one, two, or three amino additions, deletions and/or substitutions each therefrom. [0041] In one embodiment, provided herein is a method for preventing or treating liver fibrosis or pulmonary fibrosis in a patient in need thereof, comprising administering to the patient an effective amount of a bioconjugate comprising a glycan bonded to about 5, about 6, about 7, or about 8 peptides comprising GQLYKSILYGSGSGSRR (SEQ ID NO: ) or GQLYKSILY (SEQ ID NO: ). In one embodiment, provided herein is a use of the bioconjugate(s) disclosed herein for the prevention or treatment of liver fibrosis or pulmonary fibrosis in a patient in need thereof. In one embodiment, an effective amount of a

bioconjugate comprising a glycan bonded to about 5, about 6, about 7, or about 8 peptides comprising GQLYKSILYGSGSGSRR (SEQ ID NO: ) is administered.

[0042] Also provided herein are methods for preventing and/or treating vasculitis.

Vasculitis is defined by inflammation of the blood-vessel wall and forms the pathological foundation of a diverse group of individual disease entities. Vasculitis is one of the intractable pathological conditions commonly observed in autoimmune diseases, and many cases thereof are refractory to conventionally-used therapeutic methods such as steroids and immunosuppressants. In the vasculitis syndrome, inflammation occurs in arteries of various sizes, and fever, pain in muscles and joints, vascular occlusion, skin ulcer, and mononeuritis multiplex may develop. The methods may be used to treat large vessel vasculitis (LVV), medium vessel vasculitis (MVV), small vessel vasculitis (SVV), variable vessel vasculitis (VVV), single-organ vasculitis (SOV), vasculitis associated with systemic disease, and/or vasculitis associated with probable etiology. Non-limiting examples of large vessel vasculitis (LVV) include takayasu arteritis (TAK) and giant cell arteritis (GCA). Non-limiting examples of medium vessel vasculitis (MVV) include polyarteritis nodosa (PAN) and kawasaki disease (KD). Non-limiting examples of small vessel vasculitis (SVV) include antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), microscopic polyangiitis (MP A), granulomatosis with polyangiitis (Wegener's) (GPA), eosinophilic granulomatosis with polyangiitis (Churg-Strauss) (EGPA), immune complex SVV, anti- glomerular basement membrane (anti-GBM) disease, gryoglobulinemic vasculitis (CV), IgA vasculitis (Henoch-Schonlein) (IgAV), and hypocomplementemic urticarial vasculitis (HUV)) (anti-Clq vasculitis). Non-limiting examples of variable vessel vasculitis (VVV) include Behcet's disease (BD) and Cogan's syndrome (CS). Non-limiting examples of single-organ vasculitis (SOV) include cutaneous leukocytoclastic angiitis, cutaneous arteritis, primary central nervous system vasculitis, and isolated aortitis. Non-limiting examples of vasculitis associated with systemic disease include lupus vasculitis, rheumatoid vasculitis, and sarcoid vasculitis. Non-limiting examples of vasculitis associated with probable etiology include hepatitis C virus-associated cryoglobulinemic vasculitis, hepatitis B virus-associated vasculitis, syphilis-associated aortitis, drug-associated immune complex vasculitis, drug- associated ANCA-associated vasculitis, and cancer-associated vasculitis. Other examples of vasculitis include antiphospholipid syndrome, Buerger's disease (thromboangiitis obliterans), cryoglobulinemia, cryopyrin-associated autoinflammatory syndrome (CAPS) (juvenile), goodpastures, localized scleroderma (juvenile), polymyalgia rheumatica, Raynaud's phenomenon, scleroderma, Sjogren's syndrome, and systemic lupus erythematosus. It is contemplated that the bioconjugates and methods disclosed herein can be used to inhibiting and/or treating vasculitis.

[0043] In one embodiment, provided herein are methods for preventing and/or treating vessel vasculitis. In one embodiment, provided herein are methods for preventing and/or treating small vessel vasculitis, including antineutrophil cytoplasmic antibody (ANCA)- associated vasculitis (AAV), microscopic polyangiitis (MP A), granulomatosis with polyangiitis (Wegener's) (GPA), eosinophilic granulomatosis with polyangiitis (Churg- Strauss) (EGPA), immune complex SVV, anti-glomerular basement membrane (anti-GBM) disease, gryoglobulinemic vasculitis (CV), IgA vasculitis (Henoch-Schonlein) (IgAV), and/or hypocomplementemic urticarial vasculitis (HUV)) (anti-Clq vasculitis). Such diseases affect small vessels (e.g., very small arteries, arterioles, capillaries, and small veins). In one embodiment, the bioconjugate comprises heparin and from about 5 to about 10, or about 7, peptides, wherein the peptides comprise at least one sequence of

RRANAALKAGELYKSILY (SEQ ID NO: ), RR AN A ALK AGEL YK SIL YGS G (SEQ ID NO:), GQLYKSILY (SEQ ID NO: ), or GQLYKSILYGSGSGSRR (SEQ ID NO: ). In one embodiment, the peptide(s) are bond to the heparin or other glycan via a hydrazide-carbonyl linkage. In one embodiment, the bioconjugate comprises heparin and from about 5 to about 20% functionalization with peptides, wherein the peptides comprise at least one sequence of RRANAALKAGELYKSILY (SEQ ID NO: ), RR AN A ALK AGEL YK SIL YGS G (SEQ ID NO: ), GQLYKSILY (SEQ ID NO: ), or GQLYKSILYGSGSGSRR (SEQ ID NO: ). In one embodiment, the peptide(s) are bound to the heparin via a hydrazide-carbonyl linkage. In one embodiment, the bioconjugate comprises heparin and from about 5 to about 20%

functionalization with peptides, wherein the peptides comprise at least one sequence of: GQLYKSILY (SEQ ID NO: ), GQLYKSILYGSGSGSRR (SEQ ID NO: ),

CPGRVMHGLHLGDDEGPC (SEQ ID NO: ), CVWLWEQC (SEQ ID NO: ), or WREPSFCALS (SEQ ID NO: ), or an amino acid sequence having one, two, or three amino additions, deletions and/or substitutions each therefrom.

Combination Therapy

[0044] In some embodiments, the compositions of the present disclosure can be used in combination with a second agent useful for preventing or treating fibrosis. Accordingly, in one embodiment, a combination, composition, package or kit is provided that includes any composition of the present disclosure and one or more such second agent. In one

embodiment, any treatment method of the present disclosure further includes administration of one or more such second agent.

[0045] The second agent can be any pharmaceutical or biologic agent that is useful for preventing, treating or otherwise ameliorating symptoms of fibrosis. Non-limiting examples include steroids such as predonine, reducing agents such as N-acetylcysteine, antifibrotic drugs such as pirfenidone and nintedanib, immunosuppressive drugs such as corticosteroids, cyclophosphamide, azathioprine, methotrexate, penicillamine, and cyclosporine A and FK506, and other agents like colchicine, IFN-γ and mycophenolate mofetil.

[0046] In some embodiments, the compositions of the present disclosure can be used in combination with a second agent useful for preventing or treating vasculitis. Accordingly, in one embodiment, a combination, composition, package or kit is provided that includes any composition of the present disclosure and one or more such second agent. In one

embodiment, any treatment method of the present disclosure further includes administration of one or more such second agent.

[0047] The second agent can be any pharmaceutical or biologic agent that is useful for preventing, treating or otherwise ameliorating symptoms of vasculitis. Non-limiting examples include prednisone, Cyclophosphamide (Cytoxan), methylprednisolone, methotrexate sodium, Medrol (Pak), Medrol, dexamethasone, prednisolone, DexPak, Deltasone, cortisone, Prednisone Intensol, dexamethasone sodium phosphate, Orapred ODT, Trexall, Rheumatrex, methotrexate sodium (PF), Veripred 20, Dexamethasone Intensol, prednisolone sodium phosphate, Pediapred, Millipred, Rayos, Millipred, and DoubleDex.

3. Bioconjugates

[0048] As used herein, the term "bioconjugate" refers to a conjugate that comprises a glycan and one or more synthetic peptides covalently bonded thereto, where the peptides comprise at least one collagen-binding unit. The glycan portion can be made synthetically or derived from animal sources. The synthetic peptides can be covalently bonded directly to the glycan or via a linker. For methods of conjugating the peptides as described herein to a glycan, see, e.g., US 2013/0190246, US 2012/0100106, US 2011/0020298, and

WO2016/168743, the disclosures of which are incorporated herein by reference in their entirety. In one embodiment, the molecular weight range for the bioconjugate is from about 13 kDa to about 1.2 MDa, or from about 500 kDa to about 1 MDa, or from about 20 kDa to about 90 kDa, or from about 10 kDa to about 70 kDa.

[0049] As used herein, the term "glycan" refers to a compound having a large number of monosaccharides linked glycosidically. In certain embodiments, the glycan is a

glycosaminoglycan (GAG), 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 (Dex), chondroitin, chondroitin sulfate (CS), dermatan, dermatan sulfate (DS), heparan sulfate, heparin (Hep), keratin, keratan sulfate, and hyaluronic acid (HA), including derivatives thereof. In one embodiment, the molecular weight of the glycan is a key parameter in its biological function. 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 another embodiment, the glycan molecular weight is about 46 kDa. In another 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). In some embodiments, the glycan is unmodified.

[0050] In one embodiment, the glycan is heparin (Hep). Heparin is a highly sulfated glycosaminoglycan, is widely used as an injectable anticoagulant, and has the highest negative charge density of any known biological molecule. Heparin is a naturally occurring anticoagulant produced by basophils and mast cells. Native heparin is a polymer with a molecular weight ranging from 3 to 30 kDa, although the average molecular weight of most commercial heparin preparations is in the range of 12 to 15 kDa. Heparin is a member of the glycosaminoglycan family of carbohydrates (which includes the closely related molecule heparan sulfate) and consists of a variably sulfated repeating disaccharide unit. The most common disaccharide unit is composed of a 2-O-sulfated iduronic acid and 6-O-sulfated, N- sulfated glucosamine, IdoA(2S)-GlcNS(6S).

[0051] As used herein, the terms "bound", "bonded" and "covalently bonded" can be used interchangeably, and refer to the sharing of one or more pairs of electrons by two atoms. In one embodiment, the peptide is bonded to the glycan. In one embodiment, the peptide is covalently bonded to the glycan, with or without a linker. In one embodiment the peptide is covalently bonded to the glycan via a linker. In one embodiment the peptide is directly bonded to the glycan.

[0052] In one embodiment, the bioconjugates of the disclosure bind, either directly or indirectly to collagen, ECM, and/or endothelium. 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. 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.

Collagen-Binding Peptides

[0053] "Collagen-binding peptides" are peptides comprising 1 to about 120 amino acids having one or more collagen-binding units (or sequences). 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):

el 000335. In one embodiment, the peptide, or the collagen-binding unit of the peptide, binds to collagen with a dissociation constant (Kd) 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.

[0054] Collagen-binding peptide can bind to one or more of collagen type I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, or XIV. In some embodiments, the collagen-binding peptides bind to type IV collagen, which can be intact, cleaved or degraded. In some embodiments, the collagen-binding peptides bind to type I or III collagen, which can be intact, cleaved or degraded.

[0055] In certain embodiments, the peptides comprise up to about 50 amino acids, or from about 10 to about 50, or from about 10 to about 40, or from about 10 to about 30, or from about 10 to about 20, or from about 15 to about 50, or from about 15 to about 40, or from about 15 to about 30, or from about 15 to about 20. the peptide comprises up to about 50, or about 40, or about 30, or about 20 amino acids. In certain embodiments, the peptides comprise up to about 50, or about 40, or about 30, or about 20 amino acids. In certain embodiments, the peptides comprise up to about 50, or about 40, or about 30, or about 20, or about 15, or about 10 amino acids.

[0056] A non-limiting example of collagen-binding units that bind type IV collagen is TLTYTWS (SEQ ID NO: ) which binds specifically to MMP 2 and 9-degraded basement membrane type IV collagen. Likewise, TLTYTWSGSG (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.

[0057] In various embodiments, the peptides that bind to type I or III collagen include 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: ), GLRSK SKKFRRPDIQ YPD ATDEDIT SUM (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: ), GQLYKSILYGSGSGSRR (SEQ ID NO: ), RRANAALKAGQLYKSILY (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.

[0058] In one embodiment, peptides that bind to type I or III collagen include an amino acid sequence RRANAALKAGELYKSILY (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.

[0059] In one embodiment, peptides that bind to type I or III collagen include an amino acid sequence GELYKSILY (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.

[0060] In one embodiment, peptides that bind to type I or III collagen include an amino acid sequence GQLYKSILY (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.

[0061] In one embodiment, peptides that bind to type I or III collagen include an amino acid sequence GQLYKSILYGSGSGSRR (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.

[0062] In one embodiment, peptides that bind to type I or III collagen include an amino acid sequence RRANAALKAGQLYKSILY (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.

[0063] In certain embodiments, the peptide comprises an amino acid sequence that has at least about 80%, or at least about 83%, or at least about 85%, or at least about 90%, or at least about 95%), or at least about 98%>, or at least about 100%> sequence identity with the collagen- binding domain(s) of the von Willebrand factor (vWF) or a platelet collagen receptor as described in Chiang, T.M., et al. J. Biol. Chem., 2002, 277: 34896-34901, Huizinga, E.G. et al., Structure, 1997, 5: 1147-1156, Romijn, R.A., et al., J. Biol. Chem., 2003, 278: 15035- 15039, and Chiang, et al., Cardio. & Haemato. Disorders-Drug Targets, 2007, 7: 71-75, each incorporated herein by reference. A non-limiting example is WREPSFCALS (SEQ ID NO: ), derived from vWF.

[0064] 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: ), β AUKCP WHL YTTH YCF TBi p (SEQ ID NO: ), β AUKCP WHL YTH YCF T (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), WTCSGDEYTWHC (SEQ ID NO: ), WTCVGDHKTWKC (SEQ ID NO: ),

Q WHC TTRFPHH YCL YG (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.

[0065] 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).

[0066] 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, RRANAALKAGELYKSILYGC (SEQ ID NO: ),

MIVIELGTNPLKS SGIENGAFQGMKK (SEQ ID NO: ), KELNLVY (SEQ ID NO: ), DARKSEVQK (SEQ ID NO: ), HVWMQAP (SEQ ID NO: ), HWGSLRA (SEQ ID NO: ) (see, Hendra Wahyudi et al., J Control Release. 2016, 240, 323-331), and

GKWH[CTTKFPHHYC]LYBip-CONH2, where Bip is biphenylalanine (see, Wei Chen et al., JACC Cardiovasc Imaging. 2013, 6(3): 373-384).

[0067] 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) ₄ K ₂ K (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 AP SLRP APPPI S GGGYR (SEQ ID NO: ).

[0068] Similarly for a collagen-binding peptide, a peptide derived 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., SILYbiotin) 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.

[0069] In one embodiment, the peptides comprise one or more collagen-binding units which binds any one or more of collagen type I, III or IV. In one embodiment, the peptide binds to type I collagen with a dissociation constant (Kd) 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 (Kd) 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 (Kd) 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 (Kd) 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.

[0070] In any of the embodiments described herein, the peptide collagen-binding unit, comprises any amino acid sequence described in the preceding paragraphs or an amino acid sequence having at least about 80%, or at least about 83%, or at least about 85%, or at least about 90%), or at least about 95%>, or at least about 98%>, or at least about 100%> homology to any of these amino acid sequences. In various embodiments, the peptide components of the bioconjugates 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.

[0071] As used herein, the term "sequence identity" refers to a level of amino acid residue or nucleotide identity between two peptides or between two nucleic acid molecules. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. A peptide (or a polypeptide or peptide region) has a certain percentage (for example, at least about 60%, or at least about 65%, or at least about 70%), or at least about 75%, or at least about 80%>, or at least about 83%>, or at least about 85%), or at least about 90%, or at least about 95%, or at least about 98%> or at least about 99%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. It is noted that, for any sequence ("reference sequence") disclosed in this application, sequences having at least about 60%), or at least about 65%>, or at least about 70%, or at least about 75%, or at least about 80%), or at least about 83%, or at least about 85%, or at least about 90%, or at least about 95%), or at least about 98% or at least about 99% sequence identity to the reference sequence are also within the disclosure. Likewise, the present disclosure also includes sequences that have one, two, three, four, or five substitution, deletion or addition of amino acid residues or nucleotides as compared to the reference sequences.

[0072] 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 1.

Table 1

[0073] The bioconjugates of the present disclosure can include a glycan and at least one peptide comprising a collagen-binding unit. It is contemplated that any glycan can be utilized in the various embodiments described herein, including, but not limited to, alginate, chondroitin, chondroitin sulfate, dermatan, dermatan sulfate, heparan, heparan sulfate, heparin, dextran, dextran sulfate, and hyaluronan, or a derivative thereof. The glycan can be naturally occurring or chemically derivatized, such as, but not limited to, partially N- desulfated derivatives, partially O-desulfated derivatives, and/or partially O- carboxymethylated derivatives.

[0074] In one embodiment, the glycan is dermatan sulfate. In certain embodiments, 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 another 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).

[0075] In another embodiment, the glycan is heparin. Various molecular weights for the heparin can be used in the bioconjugates described herein, such as from a single disaccharide unit of about 650-700 Da to about 50 kDa. In some embodiments, the heparin is from about 10 to about 20 kDa. In some embodiments, the heparin is up to about 15, or about 16, or about 17, or about 18, or about 19, or about 20 kDa. In certain embodiments, the heparin may be oxidized under conditions that do not cleave one or more of the saccharide rings (see, e.g., Wang, et al. Biomacromolecules 2013, 14(7):2427-2432). In one embodiment, the heparin may include heparin derivatives, such as, but not limited to partially N- and/or partially O- desulfated heparin derivatives, partially O-carboxymethylated heparin derivatives, or a combination thereof. In certain embodiments, the heparin is non-oxidized heparin (i.e., does not contain oxidatively cleaved saccharide rings) and does not contain aldehyde functional groups. Heparin derivatives and/or methods for providing heparin derivatives, such as partially N-desulfated heparin and/or partially O-desulfated heparin (i.e., 2-0 and/or 6-0- desulfated heparin) are known in the art (see, e.g., Kariya et al., J. Biol. Chem., 2000, 275:25949-5958; Lapierre, et al. Glycobiology, 1996, 6(3):355-366). It is also contemplated that partially O-carboxymethylated heparin (or any glycan) derivatives, such as those which could be prepared according to Prestwich, et al. (US 2012/0142907; US 2010/0330143), can be used to provide the bioconjugates disclosed herein.

[0076] The peptide(s) can be bonded to the glycan directly or via a linker. In some embodiments, the linker can be any suitable bifunctional linker, e.g., Ν-[β- maleimidopropionic acid]hydrazide (BMPH), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), and the like. In any of the various embodiments described herein, the sequence of the peptide may be modified to include a glycine-cysteine (GC) attached to the C-terminus of the peptide and/or a glycine-cysteine-glycine (GCG) attached to the N-terminus to provide an attachment point for a glycan or a glycan-linker conjugate. In certain embodiments, the linker is N-[P-maleimidopropionic acidjhydrazide (BMPH). In certain embodiments, the linker is 3-(2-pyridyldithio)propionyl hydrazide (PDPH). In some embodiments, the peptide to linker ratio is from about 1 : 1 to about 5: 1. In other embodiments, the peptide to linker ratio is from about 1 : 1 to about 10: 1. In other embodiments, the peptide to linker ratio is from about 1 : 1 to about 2: 1, or from about 1 : 1 to about 3 : 1, or from about 1 : 1 to about 4: 1, or from about 1 : 1 to about 5 : 1 , or from about 1 : 1 to about 6 : 1 , or from about 1 : 1 to about 7: 1, or from about 1 : 1 to about 8: 1, or from about 1 : 1 to about 9: 1. In one embodiment, the peptide linker ratio is about 1 : 1. In one embodiment, the peptide linker ratio is about 2: 1. In one embodiment, the peptide linker ratio is about 3 : 1. In one embodiment, the peptide linker ratio is about 4: 1. In one embodiment, the peptide linker ratio is about 5: 1. In one embodiment, the peptide linker ratio is about 6: 1. In one embodiment, the peptide linker ratio is about 7: 1. In one embodiment, the peptide linker ratio is about 8: 1. In one embodiment, the peptide linker ratio is about 9: 1. In one embodiment, the peptide linker ratio is about 10: 1.

[0077] Depending on the desired properties of the bioconjugate, the total number of peptides bonded to the glycan can be varied. In certain embodiments, the total number of peptides present in the bioconjugate is from about 1 or 2 to about 160, or from about 10 to about 160, or from about 20 to about 160, or from about 30 to about 160, or from about 40 to about 160, or from about 40 to about 150, or from about 40 to about 140, or from about 10 to about 120, or from about 20 to about 110, or from about 20 to about 100, or from about 20 to about 90, or from about 30 to about 90, or from about 40 to about 90, or from about 50 to about 90, or from about 50 to about 80, or from about 60 to about 80, or about 10, or about 20, or about 30, or about 40, or about 50, or about 60, or about 70, or about 80, or about 90, or about 100, or about 110, or about 120. In certain embodiments, the bioconjugate comprises less than about 50 peptides. In various embodiments , the bioconjugate comprises from about 5 to about 40 peptides. In some embodiments, the bioconjugate comprises from about 10 to about 40 peptides. In other embodiments, the bioconjugate comprises from about 5 to about 20 peptides. In various embodiments, the bioconjugate comprises from about 4 to about 18 peptides. In certain embodiments, the bioconjugate comprises less than about 20 peptides. In certain embodiments, the bioconjugate comprises less than about 18 peptides. In certain embodiments, the bioconjugate comprises less than about 15 peptides. In certain embodiments, the bioconjugate comprises less than about 10 peptides. In certain

embodiments, the bioconjugate comprises about 20 peptides. In certain embodiments, the bioconjugate comprises about 40 peptides. In certain embodiments, the bioconjugate comprises about 18 peptides. In certain embodiments, the bioconjugate comprises from about 5 to about 40, or from about 10 to about 40, or from about 5 to about 20, or from about 4 to about 18, or about 10, or about 11, or about 18, or about 20 peptides. In certain embodiments, the bioconjugate comprises from about 1 to about 10, or from about 2 to about 10, or from about 2 to about 8, or from about 5 to about 10, or about 5, or about 6, or about 7, or about 8 peptides.

[0078] In any of the embodiments described herein, the number of peptides per glycan is an average, where certain bioconjugates in a composition may have more peptides per glycan and certain bioconjugates have less peptides per glycan. Accordingly, in certain

embodiments, the number of peptides as described herein is an average in a composition of bioconjugates. For example, in certain embodiments, the bioconjugates are a composition where the average number of peptides per glycan is about 5. In other embodiments, the average number of peptides per glycan is about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20, or about 25, or about 30. In certain embodiments, the number of peptides per glycan may be described as a "percent (%) functionalization" based on the percent of disaccharide units which are functionalized with peptide on the glycan backbone. For example, the total number of available disaccharide units present on the glycan can be calculated by dividing the molecular weight (or the average molecular weight) of a single disaccharide unit (e.g., about 550-800 Da, or from about 650-750 Da) by the molecular weight of the glycan (e.g., about 25 kDa up to about 70 kDa, or even about 100 kDa). For example, in some embodiments, the number of available disaccharide units present on the glycan is from about 10 to about 80, or from about 10 to about 70, or from about 15 to about 70, or from about 20 to about 70, or from about 30 to about 70, or from about 35 to about 70, or from about 40 to about 70, or from about 10 to about 50, or from about 20 to about 50, or from about 25 to about 50, or from about 10 to about 30, or from about 15 to about 30, or from about 20 to about 30, or about 15, or about 20, or about 25, or about 30, or about 35, or about 40, or about 45, or about 50, or about 55, or about 60, or about 65, or about 70.

[0079] Therefore, in certain embodiments, the glycan comprises from about 1 to about 50, or from about 5 to about 30% functionalization, or about 25% functionalization, wherein the percent (%) functionalization is determined by a percent of disaccharide units on the glycan which are functionalized with peptide. In some embodiments, the percent (%)

functionalization of the glycan is from about 1% to about 50%, or from about 3% to about 40%), or from about 5% to about 30%, or from about 10% to about 20%, or about 1%, or about 2%), or about 5%, or about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or about 55%, or about 60%, or about 65%, or about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 100%.

[0080] In one embodiment, the bioconjugate used in the methods described above comprises heparin and from about 5 to about 10, or about 7, peptides, wherein the peptides comprise at least one sequence of RRANAALKAGELYKSILY (SEQ ID NO: ),

RR AN A ALK AGEL YK S IL YGS G (SEQ ID NO: ), GQLYKSILY (SEQ ID NO: ), or GQLYKSILYGSGSGSRR (SEQ ID NO: ) and are bound to the heparin via a hydrazide- carbonyl linkage. In certain embodiments, the heparin is unfractionated heparin (UFH) or low molecular weight heparin (LMWH).

[0081] In one embodiment, the bioconjugate used in the methods described above comprises heparin and from about 5 to about 20% functionalization with peptides, wherein the peptides comprise at least one sequence of RRANAALKAGELYKSILY (SEQ ID NO: ) RR AN A ALK AGEL YK S IL YGS G (SEQ ID NO: ), GQLYKSILY (SEQ ID NO: ), or GQLYKSILYGSGSGSRR (SEQ ID NO: ) and are bound to the heparin via a hydrazide- carbonyl linkage.

[0082] In one embodiment, the bioconjugate used in the methods described above comprises heparin and from about 5 to about 20% functionalization with peptides, wherein the peptides comprise at least one sequence of: GQLYKSILY (SEQ ID NO: ),

GQLYKSILYGSGSGSRR (SEQ ID NO: ), CPGRVMHGLHLGDDEGPC (SEQ ID NO: ), CVWLWEQC (SEQ ID NO: ), or WREPSFCALS (SEQ ID NO: ), or an amino acid sequence having one, two, or three amino additions, deletions and/or substitutions each therefrom. In certain embodiments, the bioconjugate comprises at least two different collagen-binding units.

[0083] 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., SILYbiotin) 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., collagen-binding affinity).

[0084] Therefore, in some embodiments, peptides are bound to glycans, such as dermatan sulfate, by utilizing oxidation chemistry to cleave one or more of the saccharide ring within the glycan backbone in order to provide aldehyde binding sites on the glycan. The aldehyde binding sites are then used to conjugate the peptides (e.g., via a -C(0)-NH-N=C bond).

[0085] In some embodiments, the peptides can be covalently bound to glycan via a -C(O)- NH-NH-C(O)- (i.e. a hydrazide-carbonyl) linkage. Here, the peptides are bound to the glycan via a hydrazide-carbonyl linkage, where a carbonyl group of the hydrazide-carbonyl is an exocyclic carbonyl group present on the glycan. The exocyclic carbonyl group may be present on the native glycan, or alternatively, the glycan can be modified to include such a functional group. Such methods are further detailed below. It is contemplated that the beneficial effects exhibited by the bioconjugates as disclosed herein (such as increased binding affinity) is at least partially due to the glycan not containing oxidatively cleaved saccharide rings.

[0086] Accordingly, in certain embodiments, the peptides as described herein further comprise a hydrazide moiety for conjugation to the peptide. The hydrazide group can be bound to the peptide(s) at any suitable point of attachment, such as for example, the C- terminus, the N-terminus or via a side chain on an amino acid. For example, when a peptide is bound to the glycan via a side chain of an amino acid of the peptide, the side chain is glutamic acid or aspartic acid. The hydrazide can be formed between a hydrazine (-NHNH2) bound to a carbonyl group present on an amino acid in the peptide sequence (e.g., a C- terminal carbonyl group) or to a spacer, if present.

[0087] In certain embodiments, the peptide(s) are bonded to the glycan (or the linker, if present) via a spacer. As used herein, the term "spacer" is intended to refer to an optional portion of the bioconjugate which links the peptide (or binding unit) to either the linker, when present, or the glycan (can be bound directly). In any of the embodiments described herein, any one or more of the peptides may have a linear or branched spacer sequence comprising from 1 to about 15 amino acids. In one embodiment, the spacer comprises one or more, or from 1 to 10, or from 1 to 5, or from 1 to 3, amino acids. It is contemplated that any amino acid, natural or unnatural, can be used in the spacer sequence, provided that the spacer sequence does not significantly interfere with the intended binding of the peptide. The amino acids are, in some instances, non-polar amino acids, such as alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. In certain embodiments, the amino acids are selected from the group consisting of glycine, alanine, arginine and serine.

[0088] Exemplary spacers include, but are not limited to, short sequences comprising from one to five glycine units (e.g., G, GG, GGG, GGGG, or GGGGG), optionally comprising cysteine (e.g., GC, GCG, GSGC, or GGC) and/or serine (e.g., GSG, SGG, or GSGSG), or from one to five arginine units (e.g., R, RR, RRR, etc.). In one embodiment, the spacer is selected from the group consisting of glycine (G), glycine-glycine (GG), and glycine-serine- glycine(GSG). The spacer may also comprise non-amino acid moieties, such as polyethylene glycol (PEG), 6-aminohexanoic acid, succinic acid, or combinations thereof, with or without an additional amino acid spacer.

[0089] In certain embodiments, the spacer comprises more than one binding site (where the spacer may be linear or branched) such that more than one peptide sequence can be bound thereto, thus creating a branched construct. In addition, since the peptide can be bound to the glycan via a terminal or non-terminal amino acid moiety, the peptide will be branched when bound to the glycan via a non-terminal amino acid moiety. The binding sites on the spacer can be the same or different, and can be any suitable binding site, such as an amine or carboxylic acid moiety, such that a desired peptide sequence can be bound thereto (e.g. via an amide bond). Thus in certain embodiments, the spacer contains one or more lysine, glutamic acid or aspartic acid residues. In certain embodiments, the spacer comprises from 2 to 6 amino acids, or 3 or 4 amino acids. In certain embodiments, the spacer comprises one or more amino acid sequences of the formula KXX, where each X is independently a natural or unnatural amino acid. Specific examples of spacers which can be used alone or in combination to make branched constructs include, but are not limited to, KRR, KKK, (K)nGSG, and (KRR) _n -KGSG, where n is 0 to 5, or 1, 2, 3, 4, or 5.

[0090] Such constructs can provide peptides having more than one unit of the formula PnL, where at least one P is a collagen-binding unit, L is a spacer and n is an integer from 2 to about 10, or from 2 to 8, or from 2 to 6, or from 2 to 5, or from 2 to 4, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10. For example, the spacer L can be an amino acid sequence such as KGSG (SEQ ID NO: ), KKGSG (SEQ ID NO: ), K2KGSG (SEQ ID NO: ), or KKKGSG (SEQ ID NO: ), etc., where peptides can be bound to the N-terminus and the side chain nitrogen, providing 2, 3, and 4 binding sites, respectively. A schematic of these spacers bound to eptides is shown in the table below.

4. Synthesis of Bioconjugates

[0091] The peptides used in the method described herein (i.e., the collagen-binding peptide) may be purchased from a commercial source or partially or fully synthesized using methods well known in the art (e.g., chemical and/or biotechnological methods). In certain embodiments, the peptides are 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.

[0092] In certain embodiments, the peptides are covalently bonded to the glycan directly (i.e., without a linker). In such embodiments, the bioconjugates 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. See, e.g., Hermanson G.T., Bioconjugate Techniques, Academic Press, pp. 169-186 (1996), incorporated herein by reference. As shown in Scheme 1, the glycan (e.g., chondroitin sulfate "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.

[0093] 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) 0Γ 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.

[0094] As shown in Scheme 1, in one embodiment, the peptides are covalently bonded to the glycan (e.g., chondroitin sulfate "CS") by reacting an aldehyde function of the oxidized glycan (e.g., "ox-CS") with 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. Synthesis of CS-BMPH-Peptide,

"BMPH-CS"

[0095] Another example is illustrated in Scheme 2, the peptides as described herein can be covalently bound to the glycan (e.g., heparin) 1A through a carboxylic acid moiety to provide a bioconjugate IB as disclosed herein. As is typical in peptide coupling reactions, an activating agent may be used to facilitate the reaction. Suitable coupling agents (or activating agents) are known in the art and include for example, carbodiimides (e.g., Ν,Ν'- dicyclohexylcarbodiimide (DCC), Ν,Ν'-dicyclopentylcarbodiimide, Ν,Ν'- diisopropylcarbodiimide (DIC), l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-t- butyl-N-methylcarbodiimide (BMC), N-t-butyl-N-ethylcarbodiimide (BEC), l,3-bis(2,2- dimethyl-l,3-dioxolan-4-ylmethyl)carbodiimide (BDDC), etc.), anhydrides (e.g., symmetric, mixed, or cyclic anhydrides), activated esters (e.g., phenyl activated ester derivatives, p- hydroxamic activated ester, hexafluoroacetone (HFA), etc.), acylazoles (acylimidazoles using CDI, acylbenzotriazoles, etc.), acyl azides, acid halides, phosphonium salts (HOBt, PyBOP, HOAt, etc.), aminium/uronium salts (e.g., tetramethyl aminium salts, bispyrrolidino aminium salts, bispiperidino aminium salts, imidazolium uronium salts, pyrimidinium uronium salts, uronium salts derived from N,N,N'-trimethyl-N'-phenylurea, morpholino-based

aminium/uronium coupling reagents, antimoniate uronium salts, etc.), organophosphorus reagents (e.g., phosphinic and phosphoric acid derivatives), organosulfur reagents (e.g., sulfonic acid derivatives), triazine coupling reagents (e.g., 2-chloro-4,6-dimethoxy-l,3,5- triazine, 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4 methylmorpholinium chloride, 4-(4,6- dimethoxy-l,3,5-triazin-2-yl)-4 methylmorpholinium tetrafluoroborate, etc.), pyridinium coupling reagents (e.g., Mukaiyama' s reagent, pyridinium tetrafluoroborate coupling reagents, etc.), polymer-supported reagents (e.g., polymer-bound carbodiimide, polymer- bound TBTU, polymer-bound 2,4,6-trichloro-l,3,5-triazine, polymer-bound HOBt, polymer- bound HOSu, polymer-bound IIDQ, polymer-bound EEDQ, etc.), and the like (see, e.g., El- Faham, et al. Chem. Rev., 2011, 111(11): 6557-6602; Han, et al. Tetrahedron, 2004, 60:2447-2467).

[0096] In one embodiment, the peptide coupling reaction proceeds via an activated glycan intermediate by reacting a carboxylic acid moiety of the glycan with a coupling agent (e.g., a carbodiimide reagent, such as but not limited to, Ν,Ν'-dicyclohexylcarbodiimide (DCC), Ν,Ν'-diisopropylcarbodiimide (DIC), l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), etc.) to form an O-acylisourea intermediate. Suitable coupling agents can be purchased from commercial sources. Contacting the O-acylisourea intermediate with the desired peptide yields the bioconjugate. The glycan can be contacted with activating agent prior to, or in the presence of, the peptide. In some embodiments, the reaction is carried out in the presence of N-hydroxysuccinimide (NHS) or derivatives thereof. In certain embodiments, the peptide sequence can comprise a reactive moiety (e.g., a hydrazide functional group) to aid in the coupling reaction with the glycan, or O-acylisourea intermediate thereof. In some embodiments, the peptide sequence includes one or more amino acid residues that act as a spacer between the binding unit and the terminal amino acid (e.g., a terminating glycine) or reactive moiety (i.e., hydrazide functional group). For example, a serine-glycine (SG), glycine-serine-glycine (GSG) or glycine-serine-glycine- serine-glycine (GSGSG) spacer may be added to provide an attachment point for the glycan. In addition, in certain instances where one or more amino acids in the peptides contain reactive functional groups (e.g., carboxylic acid side chains), standard protecting group chemistry may be used to protect one or more side chains facilitate the coupling reaction. In addition, non-amino acid spacers may also be employed alone, or in combination with amino acid spacers (e.g., aminohexanoic acid).

[0097] In certain embodiments, the bioconjugates are derived from modified glycan derivatives (e.g., heparin) (Scheme 3). Various glycan derivatives suitable for use in the bioconjugates described herein are known in the art, such as partially N-desulfated heparin and partially O-desulfated heparin (i.e., 2-0 and/or 6-O-desulfated heparin, see, e.g., Kariya et al., J. Biol. Chem., 2000, 275:25949-5958; Lapierre, et al. Glycobiology, 1996, 6(3):355- 366). Exemplary methods are shown below in Scheme 3. As shown in Scheme 3, glycan (e.g., heparin) 1A can be reacted with a suitable desulfating agent, such as for example, a base (e.g., NaOH) or a silylating reagent (e.g., N,0-bis(trimethylsilyl)acetamide (BTSA), N- methyl-N-(trimethylsilyl)trifluoro acetamide (MTSTFA), etc.) to provide one or more desulfated glycan derivative(s) 2A. As is apparent to one of skill in the art, the glycan derivative 2A can be tailored depending on the reagents and reaction conditions employed, such that partial, complete or a mixture of desulfated glycan derivative(s) 2A can be obtained. The desulfated glycan derivative(s) 2A can then be reacted with peptide, optionally in the presence of a coupling agent, as described above for Scheme 2, under typical peptide coupling reaction conditions to provide bioconjugate 2B. In addition, as shown in Scheme 3, glycan derivatives having at least one hydroxyl group (e.g., 6-O-desulfated heparin) can be converted to an O-carboxymethylated glycan derivative(s) (e.g., 6-0- carboxymethylated heparin) 2C (see, e.g., Prestwich, et al. in US 2012/0142907 and US 2010/0330143).

Reaction of 2C with peptide, optionally in the presence of a coupling agent as described above for Scheme 2 under typical peptide coupling reaction conditions can provide bioconjugates 2D and/or 2E. Scheme 3. Alternative Synthesis of Bioconjugates

peptide-NHNH ₂

[0098] In contrast to Schemes 1 and 2, Scheme 3 shows the synthesis of bioconjugates known in the art. As shown in Scheme 2, the glycan (e.g., chondroitin sulfate "CS") is 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. The peptides are then covalently bonded to the glycan (e.g., chondroitin sulfate "CS") by reacting an aldehyde function of the oxidized glycan (e.g., "ox-CS") with N-[P-maleimidopropionic acid]hydrazide (BMPH) to form a 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.

5. Compositions

[0099] 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. 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 provided in accordance with the present disclosure are formulated as a solution for delivery into a patient in need thereof. Diluent or carriers employed in the compositions can be selected so that they do not diminish the desired effects of the bioconjugate. Examples of suitable compositions include aqueous solutions, for example, a solution in isotonic saline, 5% glucose. Other well-known pharmaceutically acceptable liquid carriers such as alcohols, glycols, esters and amides, may be employed. In certain embodiments, the composition further comprises one or more excipients, such as, but not limited to ionic strength modifying agents, solubility enhancing agents, sugars such as mannitol or sorbitol, pH buffering agent, surfactants, stabilizing polymer, preservatives, and/or co-solvents.

[0100] In certain embodiments, a polymer matrix or polymeric material is employed as a pharmaceutically acceptable carrier. The polymeric material described herein may comprise natural or unnatural polymers, for example, such as sugars, 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(gly colic acid), copolymers of lactic and gly colic acids, or other polymeric agents both natural and synthetic. In certain embodiments, the compositions provided herein is formulated as films, gels, foams, or and other dosage forms.

[0101] Suitable ionic strength modifying agents include, for example, glycerin, propylene glycol, mannitol, glucose, dextrose, sorbitol, sodium chloride, potassium chloride, and other electrolytes.

[0102] 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.

[0103] 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 the viscosity of the pharmaceutically acceptable carrier. Suitable polymeric materials include, but are not limited to: ionic and non-ionic water soluble polymers; hyaluronic acid and its salts, chondroitin sulfate and its salts, dextrans, gelatin, chitosans, gellans, other bioconjugate 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; gellan gums; alginate and sodium alginate; chitosans; vinyl polymers;

hydrophilic polymers such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers, and polyvinylalcohol; carboxyvinyl polymers or crosslinked acrylic acid polymers such as the "carbomer" family of polymers, e.g., carboxypolyalkylenes that may be obtained commercially under the CarbopolTM trademark; and various other viscous or viscoelastomeric substances. 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.

[0104] 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). Typically, non-acidic lubricity enhancing agents, such as a neutral or basic agent are employed in order to facilitate achieving the desired pH of the formulation.

[0105] 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(gly colic acid), copolymers of lactic and gly colic acids, or other polymeric agents both natural and synthetic.

[0106] Suitable pH buffering agents for use in the anti-adhesion compositions herein 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.

[0107] 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 alance (HLB) in the range of 12.4 to 13.2 and are acceptable for ophthalmic use, such as TritonXl 14 and tyloxapol.

[0108] 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 polyelectrolyte 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.

[0109] 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.

[0110] 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.

[0111] In some embodiments, preservatives are added to the composition to prevent microbial contamination during use. Suitable preservatives added to the anti-adhesion 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 ophthalmic compositions contain 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 ophthalmic compositions do not contain a preservative and are referred to as "unpreserved". In some embodiments, the unit dose compositions are sterile, but unpreserved.

[0112] In some embodiments, separate or sequential administration of the bioconjugate and other agent is necessary to facilitate delivery of the composition into the patient. 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. [0113] Any effective regimen for administering the bioconjugate can be used. For example, the bioconjugate can be administered as a single dose, as an infusion, 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.

[0114] Exemplary compositions for use with the bioconjugates for catheter-based delivery may comprise: a) a bioconjugate as described herein; b) a pharmaceutically acceptable carrier; c) a polymer matrix; d) a pH buffering agent to provide a pH in the range of about pH 4 to about pH 8; and e) a water soluble lubricity enhancing agent in the concentration range of about 0.25% to about 10% total formula weight or any individual component a), b), c), d) or e), or any combinations of a), b), c), d) or e).

6. Pharmaceutical Formulations

[0115] Formulations 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.

[0116] 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. [0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] Exemplary formulations may comprise: a) bioconjugate as described herein; b) pharmaceutically acceptable carrier; c) polymer matrix; and d) pH buffering agent to provide a pH in the range of about pH 4 to about pH 8, wherein said solution has a viscosity of from about 3 to about 30 cps for a liquid 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.

[0123] Alternatively, exemplary formulations may comprise: a) 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.

[0124] In certain embodiments, the bioconjugate, or a composition comprising the same, is lyophilized prior to, during, or after, formulation. In certain embodiments, the bioconjugate, or a composition comprising the same, is lyophilized in a pharmaceutical formulation comprising a bulking agent, a lyoprotectant, or a mixture thereof. In certain embodiments, the lyoprotectant is sucrose. In certain embodiments, the bulking agent is mannitol. In certain embodiments, the bioconjugate, or a composition comprising the same, is lyophilized in a pharmaceutical formulation comprising mannitol and sucrose. Exemplary pharmaceutical formulations may comprise about 1-20% mannitol and about 1-20% sucrose. The pharmaceutical formulations may further comprise one or more buffers, including but not limited to, phosphate buffers. Accordingly, also provided herein is a lyophilized composition comprising a bioconjugate or composition comprising the same as described herein.

7. Dosing

[0125] 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, a dose ranges from about 0.0001 mg to about 10 mg. 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 about 10 mg per dose. In other illustrative embodiments, effective doses can be about 1 μg, about 10 μg, about 25 μg, about 50 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg, about 200 μg, about 250 μg, about 275 μg, about 300 μg, about 350 μg, about 400 μg, about 450 μg, about 500 μg, about 550 μg, about 575 μg, about 600 μg, about 625 μg, about 650 μg, about 675 μg, about 700 μg, about 800 μg, about 900 μg, 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 100 mg, or about 100 mg to about 30 grams. In certain embodiments, the dose is from about 0.01 mL to about 10 mL.

[0126] In some embodiments, the compositions are packaged in multidose form.

Preservatives are thus required to prevent microbial contamination during use. In certain embodiments, suitable preservatives as described above can be added to the compositions. In some embodiments, the composition contains a preservative. In certain embodiments the preservatives are employed at a level of from about 0.001% to about 1.0% w/v. In some embodiments, the unit dose compositions are sterile, but unpreserved.

[0127] In one embodiment, an effective amount of a composition comprising a bioconjugate and pharmaceutically acceptable carrier is administered to a patient in need to treating a fibrotic disease, for instance, without limitation.

[0128] The administration of the bioconjugates are typically administered locally by injection to the site of fibrosis or vasculitis. A skilled practitioner will typically be able to readily determine how to administer based on the condition sought to be treated.

EXAMPLES

EXAMPLE 1

Synthesis of Bioconjugates

Materials

[0129] A suitable reaction buffer is prepared (e.g., 2-(N-morpholino)ethanesulfonic acid (MES)) with an appropriate concentration of a chaotropic agent, such as butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, or urea (e.g., from about 5 M to about 10 M urea). The final pH is adjusted to a pH of from about 4.5 to about 6 with 1 N HC1.

[0130] Peptide (e.g., RRAN A ALK AGEL YK SIL YGS G- HNH2 (SEQ ID NO: ) (MW = 2253 Da, structure shown below)) is dissolved in reaction buffer to 3 mg/mL. The peptide solution is typically freshly prepared prior to the coupling reaction.

[0131] Biotinylated peptide (e.g., biotinRRANAALKAGELYKSILYGSG-NHNH ₂ (SEQ ID NO: ) (MW = 2479 Da)) is dissolved in reaction buffer to 3 mg/mL. The resulting labeled peptide solution is typically freshly prepared prior to the coupling reaction. [0132] Glycan (e.g., heparin (MWavg = 16 kDa)) is dissolved in reaction buffer to 20 mg/mL and either stored at -20 °C or prepared freshly prior to the coupling reaction.

[0133] EDC (l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) is dissolved to 75 mg/mL in reaction buffer immediately before adding to the glycan.

Conjugation— Heparin Containing Bioconjugate (100 mg)

[0134] Heparin was activated by adding l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (59.3 mg or 0.79 mL dissolved at 75 mg/mL in water) in a 50 molar excess to heparin. The starting materials were reacted at room temperature for about 5 minutes. The labeled peptide was then added to the activated heparin in a 1 : 1 molar ratio (heparimlabeled peptide) (15.3 mg or 5.1 mL at 3 mg/mL in reaction buffer). The reaction mixture was then shaken for about 5 minutes at room temperature. While shaking, the peptide was added in a 1 :7 molar ratio (Hep:peptide) (97.3 mg or 32.4 mL at 3 mg/mL in reaction buffer). The components were then allowed to react for about 2 hours at room temperature while shaking. After the allotted time, the reaction was quenched by raising the pH to 8 with 0.5 M NaOH (approximately 4.5 mL) for about 30 minutes at room temperature while shaking.

[0135] The resulting bioconjugate was purified via diafilter (Spectrum - MidiKros mPES 10 K hollow tube filter) using 5 column volumes (CVs) of reaction buffer (approximately 250 mL), followed by 10 CVs of water (approximately 500 mL) at a flow rate of 35 mL/min with TMP at approximately 15 psi. The retentate (i.e., final product) was then frozen at -80 °C. Optionally, the product is lyophilized to dryness.

[0136] Additional bioconjugates were synthesized in a similar manner. For example, the following method is used to synthesize a bioconjugate, Compound 1 (Cpdl).

[0137] A suitable reaction buffer was prepared (e.g., 2-(N-morpholino)ethanesulfonic acid (MES)) with an appropriate concentration of a chaotropic agent, such as butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, or urea (e.g., from about 5 M to about 10 M urea). The final pH is adjusted to a pH of from about 4.5 to about 6 with 1 N HC1. Hydrize functionahzed peptide GQLYKSILYGSGSGSRR-NHNH2 (SEQ ID NO: ) was dissolved in reaction buffer to 3 mg/mL. The peptide solution was freshly prepared prior to the coupling reaction. [0138] Biotinylated peptide (e.g., biotin GQL YK SIL YGS GS GSRR- HNH2 (SEQ ID NO: ) (MW = 1526 Da) was dissolved in reaction buffer to 3 mg/mL. The resulting biotin-labeled peptide solution was freshly prepared prior to the coupling reaction. Glycan (e.g., heparin (MWavg = 16 kDa)) was dissolved in reaction buffer to 20 mg/mL and either stored at - 20 °C or prepared freshly prior to the coupling reaction.

[0139] EDC (l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) was dissolved to 75 mg/mL in reaction buffer immediately before adding to the glycan. Heparin was activated by adding l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (59.3 mg or 0.79 mL dissolved at 75 mg/mL in water) in a 50 molar excess to heparin. The starting materials were reacted at room temperature for about 5 minutes. The biotin-labeled peptide was then added to the activated heparin in a 1 : 1 molar ratio (heparimlabeled peptide) (15.3 mg or 5.1 mL at 3 mg/mL in reaction buffer). The reaction mixture was then shaken for about 5 minutes at room temperature. While shaking, unlabeled peptide was added in a 1 :7 molar ratio (Hep:peptide) (97.3 mg or 32.4 mL at 3 mg/mL in reaction buffer). The components were then allowed to react for about 2 hours at room temperature while shaking. After the allotted time, the reaction was quenched by raising the pH to 8 with 0.5 M NaOH (approximately 4.5 mL) for about 30 minutes at room temperature while shaking.

[0140] The resulting Compound 1 was purified via diafilter (Spectrum - MidiKros mPES 10 K hollow tube filter) using 5 column volumes (CVs) of reaction buffer (approximately 250 mL), followed by 10 CVs of water (approximately 500 mL) at a flow rate of 35 mL/min with TMP at approximately 15 psi. The retentate, which is the final product was then frozen at - 80 °C. Optionally, the final product was lyophilized to dryness. Compound 1 includes about 5 peptides conjugated with heparin.

EXAMPLE 2

Fibrillogenesis Assay

[0141] The following method is used to assess the effect of the bioconjugates disclosed herein on fibrillogenesis. Collagen solutions were prepared by diluting 2 mg/mL collagen in HCl to 1 mg/mL in 2X TES buffer (60 mM TES, 60mM Na ₂ HP0 ₄ , 300 mM NaCl) and kept on ice. Test samples containing bioconjugate were dissolved to a final concentration of 0.6 mg/mL in IX phosphate buffered saline (PBS) solutions and also kept on ice. Test samples were added to collagen in a ratio of 1 : 1 (volume:volume) such that the final collagen concentration was 0.5 mg/mL. Collagen test solutions were then thoroughly mixed by vortexing.

[0142] Fibrillogenesis was measured by monitoring the turbidity (absorbance at 313 nm) of the collagen test solutions during incubation at 37 °C. Samples were pipetted into a 96- well plate at 100 μΐ ννεΐΐ. A microplate reader was held at 37 °C, and turbidity was monitored every minute for up to 60 minutes.

EXAMPLE 3

Collagen-Binding Plate Assay

[0143] The following method is used to assess the binding affinity of bioconjugates disclosed herein for collagen. Similar assays are employed to assess binding affinity of other targets (e.g., hyaluronic acid, ICAM, VCAM, selectin).

[0144] Collagen-binding of bioconjugate variants is compared by a plate-assay, in which collagen is coated on 96-well plates. Collagen is coated on high-bind plates at 50 μg/mL in 0.02 N acetic acid for 1 hour at room temperature. Unbound collagen is rinsed with IX PBS pH 7.4. Plates are then blocked in 1% milk in IX PBS solution for 1 hour at room temperature.

[0145] Bioconjugate variants containing biotinlyated peptides are dissolved to a final concentration of 1 mg/mL in 1% milk in IX PBS pH 7.4. From this solution, a 10X serial dilution was performed. Molecules are then incubated on the blocked collagen-coated plates and incubated for 15 minutes at room temperature. Plates are then rinsed 3 times with IX PBS in 1% BSA and 0.2% Tween20.

[0146] Bound molecules are detected by streptavidin-HRP, which is diluted 1 :500 in IX PBS with 1% BSA and 0.2% Tween20 and incubated 200 uL/well for 20 minutes at room temperature. Streptavidin solution is rinsed from the plates 3 times with IX PBS with 0.2% Tween20. TMB Substrate solution is then added to each well 100 μL/well for 15 minutes at room temperature, and the color evolution was stopped with 100 μL sulfuric acid solution (0.16 M). Absorbance in the well is then measured at 450 nm and binding affinity is plotted in a dose-response by absorbance vs. concentration. EXAMPLE 4

Fibrosis Model

[0147] The bioconjugates and compositions comprising the same as described herein can be tested for efficacy in fibrosis models known in the art see, e.g., Sadasivan, S.K.,

Fibrogenesis Tissue Repair, 2015, 8, 1.

[0148] Precision-cut liver slices of 150 μπι thickness can be obtained from female

C57BL/6 J mice. The slices can be cultured for 24 hours in media containing a cocktail of 10 nM each of TGF-β, PDGF, 5 μΜ each of lysophosphatidic acid and sphingosine-1 -phosphate and 0.2 μg/ml of lipopolysaccharide along with 500 μΜ of palmitate and are analyzed for triglyceride accumulation, stress and inflammation, myofibroblast activation and extracellular matrix (ECM) accumulation. Incubation with the cocktail resulted in increased triglyceride accumulation, a hallmark of steatosis. The levels of Acta2, a hallmark of myofibroblast activation and the levels of inflammatory genes (IL-6, TNF-a and C-reactive protein) can be measured. In addition, this treatment may result in measurable levels of ECM markers - collagen, lumican and fibronectin.

[0149] This provides the experimental conditions required to induce fibrosis associated with steatohepatitis using physiologically relevant inducers. The system captures various aspects of the fibrosis process like steatosis, inflammation, stellate cell activation and ECM accumulation and serves as a platform to study the liver fibrosis in vitro and to screen bioconjugates for antifibrotic activity.

EXAMPLE 5

The Miles Assay - Vascular Leakage

[0150] In the Miles Assay, vascular barrier function is measured by extravasation of Evans blue dye from the vasculature into tissues. Evans blue binds to albumin, which cannot cross the endothelial barrier in a healthy animal. If the vascular barrier is compromised, then the blue dye will extravate from the vessels into tissues. Tissues can then be isolated, and the amount of blue dye in the tissue can be extracted and quantified by spectrophotometry.

[0151] Vascular leakage or endothelial barrier dysfunction can be initiated by a variety of agents, including lipopolysaccharide (LPS). Mice are IV injected with LPS. An agent designed to protect the endothelial barrier, such as a bioconjugate or a composition comprising the same as described herein, are then also IV injected. Next, Evans blue dye is injected into the animals. After approximately 1 hour, the animals are sacrificed and tissues including lung, brain, and intestines are harvested. The tissues are weighed, and the blue dye is extracted from the tissues with formic acid. The blue dye is then quantified by measuring its absorbance with a spectrophotometer and normalizing to the tissue weight.

[0152] It is contemplated that the bioconjugates or composition comprising the same will decrease vascular leak as determined by a reduced amount of blue dye that is found in tissues following initiation of vascular leak with a compound such as LPS.

EXAMPLE 6

Renal Ischemia Reperfusion

[0153] Rats are subjected to an acute kidney injury by clamping the renal pedicle for 30-45 minutes. Prior to or after clamp removal, the rats are IV injected with a bioconjugate or a composition comprising the same as described herein. After 24 hours, the serum creatinine of the rats is measured. It is contemplated that treatment with bioconjugates will decrease the 24 hour serum creatinine compared to animals that do not receive treatment, and closer to the levels of serum creatinine measured prior to injury. Early protection from damage caused by acute kidney injury is expected to then translate to inhibition of chronic fibrotic kidney disease.

EXAMPLE 7

Compound 1 Targets Damaged Lung Tissue

[0154] Imaging study was performed to determine if the bioconjugate Compound 1 as described in Example 1 could target the damaged lung tissue. C57B/L6 mice were dosed with 15 U/kg bleomycin via IV for either 1 or 5 days. Following the bleomycin dosing, animals were dosed IV with 10 mg/kg Compound 1 that had a fluorescent label (e.g.,

Compound 1-CF633). Two to three hours following treatment dosing, the animals were sacrificed and lungs were isolated and fixed with 10% formalin. Fixed lungs were sectioned and imaged with a fluorescent microscope to determine presence of Compound 1 in the lungs.

[0155] It was discovered that fluorescent signal from the Compound 1 (Cpdl) was stronger in the lungs that received 5 days of bleomycin injury compared to those that received a single day of bleomycin treatment. A representative image of one lobe of a lung can be seen in FIG. 1. The fluorescent signal (white) was due to the fluorescently labeled

Compound 1 (e.g., Compound 1-CF633). Strong Compound 1 signal was apparent following 5 days of injury.

EXAMPLE 8 Lung Injury

[0156] The following method is used to determine whether Compound 1 could affect lung injury. C57B/L6 mice were dosed with 15 U/kg bleomycin via IV for either 1 or 5 days. Following the bleomycin dosing, animals were dosed IV with 10 mg/kg Compound 1 daily for a total of 10 days. A positive control group utilized animals receiving oral pirfenidone 2X per day at 100 mg/kg/dose both during the bleomycin injury phase and 10 days post bleomycin. Animals were sacrificed 2-3 hours following the last treatment dose. Bronchio- alveolar lavage (BAL) fluid was collected and the inflammatory cell count in the BAL was determined.

[0157] It was discovered that both 1 and 5 days of bleomycin caused increase in leukocyte counts in the BAL fluid (FIG. 2). Both pirfenidone and Compound 1 reduced the leukocyte cell number in the BAL fluid compared to the PBS control treatment.

EXAMPLE 9 Cytokine Assay

[0158] The following method is used to access cytokines in the lung tissue to determine whether Compound 1 could affect lung injury. C57B/L6 mice were dosed with 15 U/kg bleomycin via IV for either 1 or 5 days. Following the bleomycin dosing, mice were dosed IV with 10 mg/kg Compound 1 daily for a total of 10 days. A positive control group utilized mice that received oral Pirfenidone 2X per day at 100 mg/kg/dose both during the bleomycin injury phase and 10 days post bleomycin injury. The mice were sacrificed 2-3 hours following the last treatment dose. Lungs were collected and frozen with liquid nitrogen and then stored at -80 °C. Lysis buffer was added to each lung and the tissue was homogenized using a bead disruptor. Tissue homogenate supernatant was collected and tested for the presence of cytokines (e.g., IL-6, IL-lb, and MCP-1) using commercially available ELISA kits. EXAMPLE 10

Compound 1 Targets Damaged Liver Tissue

[0159] The following method is used to determine if the bioconjugate Compound 1 could target the damaged liver tissue. Liver damage was induced in mice either through CCL4 injections or by a high fat diet. At certain time points in the course of disease, the mice were dosed IV with Compound 1 with a fluorescent label. Two to three hours following dosing, the mice were sacrificed and the liver was isolated and fixed with 10 % formalin. Fixed liver tissues were sectioned and imaged with a fluorescent microscope to determine presence of Compound 1 in the damaged liver compared to healthy liver.