Compositions for use in methods for regeneration and regrowth of cartilage following injury - Patents.com

Abstract

Provided is a method for enhancing the regeneration of cartilage following injury, comprising identifying a mammal as experiencing injury to cartilage, and applying to the mammal a composition comprising covalently bonded graphene oxide (GO) and hyaluronic acid (HA), XAV939, and water.The composition of the method can also comprise a surfactant, such as PEG.The composition can be administered topically to a subject to treat the cartilage injury of the subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 243,638, filed September 13, 2021, which is expressly incorporated by reference herein for all purposes.

[0002] Field The disclosure described herein relates to compositions and methods for the treatment of cartilage injury, and in particular, for enhanced cartilage regeneration following cartilage injury. [Background technology]

[0003] background All publications, patents, and patent applications cited within this application are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent application or patent disclosure was specifically and individually indicated to be incorporated by reference in its entirety.

[0004] Cartilage represents an important form of connective tissue in mammals, providing both structural and functional activity within the body. Cartilage is composed of three broad categories, the most widespread forms being hyaline cartilage, commonly found at the ends of bones forming part of joints, elastic cartilage, such as that found in the ear or nose, which provides structure on the body, and fibrocartilage, found at the insertions of ligaments and tendons.

[0005] Although cartilage repair is observed during fetal development or at very young ages, cartilage repair and regeneration following wounding in mature mammals is almost completely absent. Although the prior art has recognized the role of growth factors and wound lesion edge integrity in the repair process, there is an unmet need for therapeutic agents that improve cartilage regeneration and healing following injury.

[0006] The Wnt pathway has been shown to play an important role in dermal fibrosis and scarring. The Wnt pathway is an evolutionarily conserved pathway that controls crucial aspects of cell fate determination, cell polarity, cell migration, neural patterning, and organogenesis during embryonic development. This pathway helps ensure proper tissue development in the embryo and tissue maintenance in the adult. Wnt signaling is involved in the early stages of skin development. Following gastrulation, embryonic cells of the ectoderm and mesoderm differentiate to form the epidermis and dermis, respectively.

[0007] There are at least three distinct Wnt signaling pathways involved in the signaling process, but the canonical (i.e., β-catenin-dependent) Wnt pathway is the best understood. β-catenin is the key effector molecule resulting from canonical Wnt pathway signaling, and its protein levels are controlled through the "destruction complex." In the absence of Wnt signals, the transcriptional activator β-catenin is actively degraded in cells by the action of a protein complex designated the "destruction complex." Within this complex, Axin-1 and -2 form a scaffold with adenomatous polyposis coli, which promotes the phosphorylation of β-catenin by casein kinase 19a and glycogen synthase kinase 3β. The phosphorylated β-catenin is recognized and ubiquitinated, which leads to its proteosomal degradation. Tankryase I and II (TK1 and TK2) are poly(ADP-ribose) polymerases (PARPs) that function to parsylate and destabilize Axin-1 and -2 proteins, thus destabilizing the β-catenin destruction complex. Once the destruction complex is destabilized, this allows β-catenin to be dephosphorylated and subsequently stabilized, accumulating in the cytoplasm and entering the cell nucleus where it interacts with members of the Tcf / Lef family. β-catenin converts Tcf proteins into potent transcriptional activators by recruiting coactivator proteins, thus ensuring efficient activation of Wnt target genes. Once activated by natural ligands of the Wnt family, the Wnt pathway upregulates TNK1 and TNK2 to help destabilize the destruction complex. Studies have shown that TNK1 and TNK2 are crucial regulators of canonical Wnt signaling.

[0008] XAV939 has an IC of 11nM / 4nM in cell-free assays 50XAV939 is a small molecule that selectively inhibits Wnt / β-catenin-mediated transcription through TNK1 and TNK2 inhibition with β-catenin, controls axin levels, and has no effect on CRE, F-κβ, or TGF-β. Topical application of XAV939 in mouse ear punch assays demonstrated that XAV939 significantly increases the rate of wound closure with reduced fibrosis (scarring). However, XAV939 was dissolved in DMSO and used only as a "research tool" compound due to its very low water solubility (<1 μg / mL). The problem with this approach is that the use of DMSO for topical administration may result in undesirable pharmacological activity or side effects. A soluble form of XAV939 suitable for human use is needed for practical and medical use.

[0009] Matrix components including graphene oxide (GO) and hyaluronic acid (HA) have been shown to be effective in both providing a support matrix for XAV939 and enabling the use of XAV939 as a therapeutic agent for wound healing in humans and animals. See, for example, US Patent Publication No. 20210000959, where XAV939 in a GO-HA matrix provides substantial improvements in the rate and quality of wound healing, specifically allowing the tissue to limit wound healing following fibrotic healing pathways that lead to scarring. Additionally, increased cartilage regeneration and healing following acute injury has been reported using administration of XAV939 dissolved in DMSO, for example, using a 2 mm biopsy punch wound created in the center of the cartilage region of C57Bl / CJ mice (Bastakoty, D. et al. 2015, 29(12):4881-4892).

[0010] While any regeneration of cartilage following administration of a therapeutic agent would be beneficial given the limited natural regenerative capacity of cartilage in non-fetal mammals, there is a need in the art for improved compositions for inducing, enhancing or increasing cartilage regeneration following acute injury. Summary of the Invention

[0011] overview The present disclosure provides a method of stimulating regeneration of cartilage in a mammal subject to an injury, comprising optionally identifying the mammal as experiencing injury to cartilage; optionally identifying the location and extent of the injured cartilage; contacting the injured cartilage with an effective amount of a composition comprising a matrix component consisting of a conjugate of graphene oxide (GO) and hyaluronic acid (HA) (GO-HA), where GO and HA are covalently linked via a linker; 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one (XAV939); and water, optionally wherein XAV939 comprises about 0.001% to about 5% by weight of the total composition. In one embodiment, the injured cartilage is acutely injured cartilage. In a further embodiment, the injured cartilage is elastic cartilage. In another embodiment, the composition further comprises a surfactant. In a further embodiment, the surfactant is polyethylene glycol (PEG) or a poloxamer. In yet a further embodiment, the PEG has a molecular weight of about 200 to about 400 daltons. In yet a further embodiment, the PEG is in an amount of about 0.1% to about 20% by weight of the total composition. In another embodiment, the composition further comprises a thickening agent. In a further embodiment, the thickening agent comprises hydroxypropylcellulose (HPC). In another embodiment, the linker comprises 2-25 carbons. In a further embodiment, the linker comprises one or more -CH2CH2O- units. In another embodiment, the linker comprises -R x -R S -R y -(In the formula, R x and R y are each independently selected from the group consisting of -CO-, -COO-, -NH-, -NH-NH-, -NH-NH-CO-, -CS-, -S-, and -O-; R Sis an unsubstituted, saturated or unsaturated, straight chain alkylene group having 2 to 20 backbone carbons. x and R y are each -NH-NH-CO-. In another embodiment, the weight ratio of XAV939 to GO-HA is from about 1:2 to about 2:1. In another embodiment, GO-HA comprises from about 0.001% to about 5% by weight of the total composition.

[0012] In another aspect, the disclosure provides a method of stimulating regeneration of injured cartilage in a mammal, comprising optionally identifying the mammal as experiencing injury to cartilage; optionally identifying the location and extent of the injured cartilage; contacting the injured cartilage once every 48 hours with an effective amount of a composition comprising a matrix component comprising a conjugate of graphene oxide (GO) and hyaluronic acid (HA) (GO-HA), where GO and HA are covalently linked via a linker; 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one (XAV939); and water, optionally wherein XAV939 comprises about 0.001% to about 5% by weight of the total composition. In one embodiment, the injured cartilage is acutely injured cartilage. In a further embodiment, the injured cartilage is elastic cartilage. In another embodiment, the composition further comprises a surfactant. In a further embodiment, the surfactant is polyethylene glycol (PEG) or a poloxamer. In yet a further embodiment, the PEG has a molecular weight of about 200 to about 400 daltons. In yet a further embodiment, the PEG is in an amount of about 0.1% to about 20% by weight of the total composition. In another embodiment, the composition further comprises a viscosity increasing agent. In a further embodiment, the viscosity increasing agent comprises hydroxypropyl cellulose (HPC). In another embodiment, the linker comprises 2-25 carbons. In a further embodiment, the linker comprises one or more -CH2CH2O- units. In another embodiment, the linker comprises -R x -R S -R y -(In the formula, R x and R y are each independently selected from the group consisting of -CO-, -COO-, -NH-, -NH-NH-, -NH-NH-CO-, -CS-, -S-, and -O-; R Sis an unsubstituted, saturated or unsaturated, straight chain alkylene group having 2 to 20 backbone carbons. x and R y are each -NH-NH-CO-. In another embodiment, the weight ratio of XAV939 to GO-HA is from about 1:2 to about 2:1. In another embodiment, GO-HA comprises from about 0.001% to about 5% by weight of the total composition.

[0013] In another aspect, the disclosure provides a composition for use in a method of stimulating regeneration of injured mammalian cartilage, the composition comprising a matrix component comprising a conjugate of graphene oxide (GO) and hyaluronic acid (HA) (GO-HA), where GO and HA are covalently linked via a linker; 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one (XAV939); and water; optionally, where XAV939 constitutes about 0.001% to about 5% by weight of the total composition; wherein the method optionally comprises identifying the mammal as experiencing injury to cartilage; optionally identifying the location and extent of the injured cartilage; and contacting the injured cartilage with an effective amount of the composition. In one embodiment, the injured cartilage is acutely injured cartilage. In a further embodiment, the injured cartilage is elastic cartilage. In another embodiment, the composition further comprises a surfactant. In a further embodiment, the surfactant is polyethylene glycol (PEG) or a poloxamer. In yet a further embodiment, the PEG has a molecular weight of about 200 to about 400 daltons. In yet a further embodiment, the PEG is in an amount of about 0.1% to about 20% by weight of the total composition. In another embodiment, the composition further comprises a thickening agent. In a further embodiment, the thickening agent comprises hydroxypropylcellulose (HPC). In another embodiment, the linker comprises 2-25 carbons. In a further embodiment, the linker comprises one or more -CH2CH2O- units. In another embodiment, the linker comprises -R x -R S -R y -(In the formula, R x and R y are each independently selected from the group consisting of -CO-, -COO-, -NH-, -NH-NH-, -NH-NH-CO-, -CS-, -S-, and -O-; R Sis an unsubstituted, saturated or unsaturated, straight chain alkylene group having 2 to 20 backbone carbons. x and R y are each -NH-NH-CO-. In another embodiment, the weight ratio of XAV939 to GO-HA is from about 1:2 to about 2:1. In another embodiment, GO-HA comprises from about 0.001% to about 5% by weight of the total composition.

[0014] In another aspect, the disclosure provides a composition for use in a method of stimulating regeneration of injured mammalian cartilage, the composition comprising: a matrix component comprising a conjugate of graphene oxide (GO) and hyaluronic acid (HA) (GO-HA), wherein GO and HA are covalently linked via a linker; 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one (XAV939); and water; wherein XAV939 constitutes about 0.001% to about 5% by weight of the total composition, the method comprising: identifying the mammal as experiencing injury to cartilage; identifying the location and extent of the injured cartilage; and contacting the injured cartilage with an effective amount of the composition once every 48 hours. In one embodiment, the injured cartilage is acutely injured cartilage. In a further embodiment, the injured cartilage is elastic cartilage. In another embodiment, the composition further comprises a surfactant. In a further embodiment, the surfactant is polyethylene glycol (PEG) or a poloxamer. In yet a further embodiment, the PEG has a molecular weight of about 200 to about 400 daltons. In yet a further embodiment, the PEG is in an amount of about 0.1% to about 20% by weight of the total composition. In another embodiment, the composition further comprises a viscosity increasing agent. In a further embodiment, the viscosity increasing agent comprises hydroxypropyl cellulose (HPC). In another embodiment, the linker comprises 2-25 carbons. In a further embodiment, the linker comprises one or more -CH2CH2O- units. In another embodiment, the linker comprises -R x -R S -R y -(In the formula, R x and R y are each independently selected from the group consisting of -CO-, -COO-, -NH-, -NH-NH-, -NH-NH-CO-, -CS-, -S-, and -O-; R Sis an unsubstituted, saturated or unsaturated, straight chain alkylene group having 2 to 20 backbone carbons. x and R y are each -NH-NH-CO-. In another embodiment, the weight ratio of XAV939 to GO-HA is from about 1:2 to about 2:1. In another embodiment, GO-HA comprises from about 0.001% to about 5% by weight of the total composition.

[0015] In some or any of the foregoing embodiments, XAV939 comprises about 0.001% to about 5% by weight of the combined XAV939, GO-HA, and water.

[0016] In some or any of the foregoing embodiments, the GO-HA comprises from about 0.001% to about 5% by weight of the combined XAV939, GO-HA, and water. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows the distance between opposing cartilage endpoints in mice following 30 days of treatment of biopsy punch wounds with XAV939 in saline and DMSO versus 15 days of treatment with GO-HA and ELU42. [Diagram 2] FIG. 2 shows the distance between opposing cartilage endpoints in mice following 30 days of treatment with XAV939 in DMSO and 15 days of treatment with ELU42 in biopsy punch wounds. [Diagram 3] FIG. 3 shows the distance between opposing cartilage endpoints in mice following 15 days of treatment with GO-HA and 15 ELU42. [Figure 4] FIG. 4 shows the distance between opposing cartilage endpoints in rabbits after 21 days of saline and 21 days of ELU42 treatment of biopsy punch wounds. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description The present disclosure provides novel methods of administration of the compositions of the present disclosure to induce improved cartilage regeneration and healing.

[0019] It should be understood that the description of the compounds, compositions, formulations, and methods of treatment described herein includes "comprising," "consisting," and "consisting essentially of" embodiments. In some embodiments, for all compositions described herein, and all methods of using the compositions described herein, the composition can either include the recited components or steps, or "consist essentially of" the recited components or steps. When a composition is described as "essentially consisting of" the recited components, the composition contains the recited components and can contain other components that do not substantially affect the basic and novel properties (in some embodiments, the condition being treated), but does not contain any other components other than the explicitly recited components that substantially affect the basic and novel properties (in some embodiments, the condition being treated); or, if the composition contains additional components other than the recited components that substantially affect the basic and novel properties (in some embodiments, the condition being treated), the composition does not contain the additional components in a concentration or amount sufficient to substantially affect the basic and novel properties (in some embodiments, the condition being treated). When a method is described as "consisting essentially of" recited steps, the method contains the recited steps and may contain other steps that do not substantially affect the basic and novel properties (in some embodiments, the condition being treated), but the method does not contain any other steps other than those explicitly recited that substantially affect the basic and novel properties (in some embodiments, the condition being treated). As a non-limiting example, when a composition is described as "consisting essentially of" a component, the composition may further contain any amount of a pharma- ceutically acceptable carrier, vehicle, or diluent, as well as other such components that do not substantially affect the basic and novel properties (in some embodiments, the condition being treated).

[0020] As used herein, "alkyl" refers to a straight-chain or branched hydrocarbon. Alkyl can be straight-chain, branched, cyclic, or a combination thereof, and can contain, for example, 1 to 60 carbon atoms. Examples of alkyl groups include, but are not limited to, ethyl, ethyl, propyl, isopropyl, cyclopropyl, butyl isomers (e.g., n-butyl, iso-butyl, tert-butyl, etc.), cyclobutyl isomers (e.g., cyclobutyl, methylcyclopropyl, etc.), pentyl isomers, cyclopentane isomers, hexyl isomers, cyclohexane isomers, and the like.

[0021] As used herein, the term "straight chain alkyl" refers to a chain of carbon and hydrogen atoms (eg, ethane, propane, butane, pentane, hexane, etc.).

[0022] As used herein, the term "branched alkyl" refers to a chain of carbon and hydrogen atoms without double or triple bonds containing forks, branches, and / or splits in the chain. "Branched" refers to the branching of the carbon chain, while "substituted" refers to the presence of non-carbon / non-hydrogen atoms in the moiety.

[0023] The term "cycloalkyl" as used herein refers to a fully saturated monocyclic or polycyclic hydrocarbon ring system. When composed of two or more rings, the rings may be joined together in a fused, bridged or spiro-connected manner. Cycloalkyl groups may be unsubstituted, substituted, branched and / or unbranched. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. When substituted, the substituent(s) may be alkyl or may be selected from those set forth above for substitution of alkyl groups, unless otherwise indicated. Unless otherwise specified (e.g., substituted cycloalkyl groups, heterocyclyl, cycloalkoxy groups, halocycloalkyl, cycloalkylamine, thiocycloalkyl, and the like), alkyl groups contain only carbon and hydrogen atoms.

[0024] As used herein, the term "heteroalkyl" refers to an alkyl group in which one or more carbon atoms are independently replaced by one or more heteroatoms (e.g., oxygen, sulfur, nitrogen, phosphorus, silicon, or combinations thereof). The alkyl group containing the non-carbon substituent(s) can be a straight chain alkyl, branched alkyl, cycloalkyl (e.g., cycloheteroalkyl), or combinations thereof. The non-carbon can be at a terminal position (e.g., 2-hexanol) or can be part of the alkyl group (e.g., diethyl ether).

[0025] As used herein, the term "alkoxy," alone or in combination, refers to the group --O--alkyl.

[0026] As used herein, "alkenyl," used alone or in combination, means a straight or branched chain hydrocarbon having at least two carbon atoms containing at least one carbon-carbon double bond.

[0027] As used herein, "alkynyl," used alone or in combination, means a straight or branched chain hydrocarbon having at least two carbon atoms containing at least one carbon-carbon triple bond.

[0028] As used herein, "amine" or "amino" is represented by the formula NA1A2A3, where A1, A2, and A3 can be independently hydrogen or an optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, or heteroaryl group as described herein. In certain embodiments, amine refers to any of NH2, NH(alkyl), NH(aryl), N(alkyl)2, N(alkyl)(aryl), and N(aryl)2.

[0029] As used herein, "acute injury" refers to injury or wound caused by, among others, trauma, chemical toxicity, burn, frostbite, acute ischemia, and reperfusion injury.Exemplary trauma injuries include, among others, surgical trauma and blunt trauma (e.g., gunshot wound, knife wound, etc.).The composition can be applied onto or injected into the affected tissue to promote the vascularization, repair, and regeneration of such damaged tissue.

[0030] "Regeneration" as used herein means the growth of destroyed or devitalized tissue from remnants. It is the body's attempt to repair itself and, in terms of cartilage, refers to the migration or replication of chondrocytes or the conversion of progenitor cells into chondrocytes, which results in the organization of said chondrocytes to form hyaline, elastic or fibrocartilage.

[0031] As used herein, a "thickener" is a substance that increases the viscosity of a liquid without substantial alteration to the chemical or biological properties of the liquid.

[0032] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, cosolvents, complexing agents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are not biologically or otherwise undesirable. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the composition. In addition, various adjuvants as commonly used in the art can be included. These and other such compounds are described in the literature, for example, in the Merck Index (Merck & Company, Rahway, NJ), which describes the considerations for the inclusion of various ingredients in pharmaceutical compositions (for example, Gilman et al. (Eds.), 2010, Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 12th Ed., The McGraw-Hill Companies).

[0033] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the compounds provided herein, which are not biologically or otherwise undesirable. In many cases, the compounds provided herein may form acid salts and / or base salts due to the presence of amino and / or carboxyl groups or groups similar thereto. Pharmaceutically acceptable acid addition salts may be formed with inorganic and organic acids. Inorganic acids from which salts may be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Organic acids from which salts may be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc. Pharmaceutically acceptable base addition salts may be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, etc., with the ammonium, potassium, sodium, calcium, and magnesium salts being particularly preferred. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, such as, in particular, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, etc. Many such salts are known in the art, as described in WO 87 / 05297.

[0034] A "therapeutically effective amount" or "pharmaceutical effective amount" of a compound provided herein is an amount that is sufficient to achieve a desired effect, and may vary according to the nature and severity of the disease state and the efficacy of the compound. A "therapeutically effective amount" is also intended to include one or more of the compositions of the present disclosure to result in increased regeneration of cartilage following acute injury. The combination of compounds and / or compositions is preferably a synergistic combination. Synergy occurs when the effect of the compounds administered in combination is greater than the additive effect of the compounds administered alone as a single agent, as described in the art (e.g., Chou, 2010, Canc. Res. 70(2):440-446). In general, synergy is most clearly demonstrated at suboptimal concentrations of the compounds. It will be recognized that different concentrations may be used for the prevention of active disease than for its treatment. This amount may further depend on the height, weight, sex, age and medical history of the patient.

[0035] The term "mammal" is used in its normal biological sense, i.e., it specifically includes humans, cows, horses, dogs, and cats, but also includes many other species.

[0036] In one embodiment of the present disclosure, a composition for treating acutely injured cartilage is provided, comprising a matrix component comprising a conjugate of graphene oxide (GO) and hyaluronic acid (HA), where GO and HA are covalently linked via a linker; XAV939; and water. The covalently linked GO and HA are also referred to herein as GO-HA conjugate or simply GO-HA.

[0037] XAV939 is a potent tankyrase inhibitor with the chemical name 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one. The structure of XAV939 is shown below:

[0038] [ka]

[0039] Graphene oxide (GO) as used herein refers to the oxidized form of graphene, which is a single-layer form of graphite. GO can be obtained by treating graphite with strong oxidizing agents. GO contains carbon, oxygen, and hydrogen in varying amounts depending on the method by which it is made. It can be hundreds of nanometers, up to a few micrometers in its planar direction, and can be about 0.7-1.2 nanometers thick. GO can contain various oxygen-containing moieties, such as oxygen epoxide groups, carboxylic acids (-COOH), and phenols, when prepared using sulfuric acid (e.g., Hummers method). An example of a GO structure is shown below.

[0040] [ka]

[0041] Hyaluronic acid (HA) is an anionic, highly hydrophilic, non-sulfated glycosaminoglycan that occurs naturally throughout the human body. It can be several thousand carbohydrate units long and can bind water to form a gel with a firm, viscous quality. An example of the structure of HA is provided below:

[0042] [ka]

[0043] In the compositions of the present disclosure, GO and HA are covalently linked to form a matrix component (i.e., a carrier), which can serve to solubilize XAV939 as well as provide other simultaneous benefits to wound healing. Covalent linkage can be achieved by using a linker (or linker moiety). In some embodiments, the linker can include 2-25 carbons. In some embodiments, the linker is linear. In other embodiments, the linker is branched. The linker can be saturated or unsaturated.

[0044] In some embodiments, the linker is C2-C 25 It may include alkylene groups, where the carbons and hydrogens in the alkylene group may be replaced by oxygen or other atoms or groups, such as hydroxy, carboxy, amino, alkyl, alkoxy, alkenyl, alkynyl, nitro, etc. In some embodiments, the linker may include one or more -CH2CH2O- units.

[0045] In some embodiments, the linker is -R x -R s -R y -(In the formula, R x and R y are each independently selected from the group consisting of -CO-, -COO-, -H-, -HH-, -HH-CO-, -CS-, -S-, and -O-; R s (also referred to in this application as a spacer group) includes a linear alkylene group, which may be unsubstituted or substituted, saturated or unsaturated, having 2 to 20 backbone carbons. x and R y Both are * -HH-CO-( * indicates the end of the linker distal to Rs).

[0046] In some embodiments of the composition, the weight ratio of XAV939 to GO-HA can be about 1:100 to 100:1, such as about 1:2 to about 2:1. In some embodiments, in the GO-HA conjugate, the weight ratio of GO:HA can be about 1:1 to about 1:20, or about 1:6 to about 1:10.

[0047] Other non-ionic hydrophilic materials such as copolymers of PEG and PPG (polypropylene glycol), e.g., poloxamers, can also be used. In one example, Poloxamer-188 (having an average molecular weight of about 8400 Daltons) can be used.

[0048] In some embodiments, the composition further comprises, in addition to XAV939, a pharmaceutical carrier, excipient, compound(s), or material that allows the composition to be presented in a semi-solid aqueous gel form that can be administered topically. For example, carboxymethylcellulose can be used as a gel-forming agent. However, other cellulose derivatives such as microcrystalline cellulose, as well as polysaccharides such as alginate, agarose, tragacanth, guar gum, and xantham gum are also suitable as gel-forming agents. The gel can be made thicker and / or firmer, if necessary, by the addition of a relatively elastic gel-forming material such as cross-linked fibrous proteins, for example gelatin or collagen cross-linked with formaldehyde. In some embodiments, the composition can be in the form of a cream, which can include excipients suitable for cream formulations, such as paraffin oil, petrolatum, wax, organic esters such as cetyl palmitate, etc.

[0049] In some embodiments, the composition of the present disclosure further comprises a thickening agent for the desired viscosity of the composition for skin delivery.For example, the thickening agent can comprise hydroxypropylcellulose (HPC) or xanthan gum.HPC can make the composition into a smooth film for easy application.It also reduces evaporation, allowing wounds to remain moist longer, a factor that has been shown to improve healing and result in reduced scarring.Various grades of HPC are available according to molecular weight or viscosity of HPC aqueous solution at a particular concentration.

[0050] In some embodiments of the composition, XAV939 may comprise about 0.001% to about 5% by weight of the total composition (including water). In some embodiments, XAV939 may comprise about 0.01% to about 2% by weight, about 0.02% to about 1% by weight, or about 0.05% to about 0.5% by weight of the total composition. In some embodiments, GO-HA may comprise about 0.001% to about 5% by weight of the total composition. In some embodiments, GO-HA may comprise about 0.01% to about 2% by weight, about 0.02% to about 1% by weight, or about 0.05% to about 0.5% by weight of the total composition. In some or any of the above embodiments, the weight percentage of XAV939 is based on the total of XAV939, GO-HA, and water. In some or any of the above embodiments, the weight percentage of GO-HA is based on the total of XAV939, GO-HA, and water.

[0051] XAV939 is uniformly dispersed in a viscous suspension that is stable for several months at room temperature. In some embodiments, the composition further comprises a surfactant that enhances the miscibility or solubility of hydrophobic substances in water. In some examples, the surfactant can be a non-ionic hydrophilic material such as polyethylene glycol (PEG). The PEG can have a number average molecular weight of about 100 to about 10,000 daltons, or about 200 to about 4000 daltons, e.g., about 200 to about 1000 daltons, about 200 to about 800 daltons, about 200 to about 500 daltons, about 200 to about 400 daltons, about 300 to about 400 daltons, about 350 to about 450 daltons, about 200 daltons, about 250 daltons, about 300 daltons, about 350 daltons, about 400 daltons, about 450 daltons, about 500 daltons, about 550 daltons, about 600 daltons, about 650 daltons, about 700 daltons, about 750 daltons, about 800 daltons, about 850 daltons, about 900 daltons, about 950 daltons, about 1000 daltons, etc. In some embodiments, PEG may be present in the composition in an amount of about 0.1 to about 20% by weight of the total composition, for example, PEG may be about 0.2% to about 10% by weight, or about 0.5% to about 10% by weight, or about 1% to about 10% by weight of the total composition.

[0052] Provided is the composition, wherein the composition is a liposome formulation. Provided is the composition, wherein the composition comprises: a matrix component comprising a conjugate of graphene oxide (GO) and hyaluronic acid (HA) (GO-HA), wherein GO and HA are covalently linked via a linker; XAV939; water; and butylene glycol. In some or any embodiment, the composition comprises: a matrix component comprising a conjugate of graphene oxide (GO) and hyaluronic acid (HA) (GO-HA), wherein GO and HA are covalently linked via a linker; XAV939; water; and polysorbate 20. In some or any embodiment, the composition comprises: a matrix component comprising a conjugate of graphene oxide (GO) and hyaluronic acid (HA) (GO-HA), wherein GO and HA are covalently linked via a linker; XAV939; water; and phosphatidylcholine. In some or any of the embodiments, the composition comprises a matrix component comprising a conjugate of graphene oxide (GO) and hyaluronic acid (HA) (GO-HA) (wherein GO and HA are covalently linked via a linker); XAV939; water; and ethanol. In some or any of the embodiments, the composition comprises a matrix component comprising a conjugate of graphene oxide (GO) and hyaluronic acid (HA) (GO-HA) (wherein GO and HA are covalently linked via a linker); XAV939; water; and hydroxypropyl cellulose. Any of the preceding embodiments in this paragraph may be combined with each other; in particular, the liposomal formulation comprises XAV939, butylene glycol, polysorbate 20, phosphatidylcholine, ethanol, GO-HA, water, and hydroxypropyl cellulose. In some or any of the embodiments, the liposomal formulation is used to carry out any of the embodiments described herein, including those in the claims.

[0053] In the compositions described herein, other pharmaceutical or therapeutic compounds may be included in addition to XAV939. In other words, the composition in which XAV939 is present may also serve as a base dispersion medium that can disperse other pharmaceutical or therapeutic agents, especially those that are hydrophobic, for example, for local administration to acutely damaged cartilage. These agents may include anti-fibrotic compounds such as pirfenidone, halofuginone, nintedanib, tocilizumab, rilonacept, anti-cancer agents, anti-inflammatory agents, analgesics, antibiotics, etc.

[0054] In one aspect, the present disclosure provides a method for stimulating the regeneration of acutely damaged cartilage, comprising contacting the cartilage with an effective amount of the composition described herein.It is contemplated that acutely damaged cartilage includes, but is not limited to, those resulting from surgical wounds (such as lacerations, abrasions, cuts, scratches, or punctures caused by knives, scalpels, or other sharp or blunt objects) that disrupt cartilage structure and function, or those resulting from physical impacts (such as knives, bullets, or other sharp or blunt objects) in non-surgical settings that disrupt cartilage structure and function.The present disclosure contemplates acutely damaged cartilage due to excessive (low or high) temperature, such as burns, ionizing radiation, chemotherapy, or unexpected acute injury resulting from accidents or accidents.

[0055] In some embodiments, the method of treatment may include delivering a second drug or therapeutic agent to the acutely injured cartilage, including one or more of corticosteroids, cytotoxic drugs, antibiotics, antiseptics, nicotine, antiplatelet drugs, NSAIDs, colchicine, anticoagulants, vasoconstrictors or immunosuppressants, growth factors, antibodies, proteases, protease inhibitors, antimicrobial peptides, adhesion peptides, hemostatic agents, live cells, honey, or nitric oxide. These therapeutic agents may be delivered as a separate dosage form from the compositions described herein, or may be included as an additional component of the compositions described herein and therefore delivered together with XAV939.

[0056] The composition(s) of the present disclosure described herein can be administered by topically applying the composition(s) onto the acutely injured cartilage. When the composition is contained in a medical device described herein, including a substrate such as a patch or pad, the medical device can be fixed to the acute injury site so that the composition contacts the cartilage.

[0057] In the method of preparing the composition for use in the method of the present disclosure, the spacer group can be an unsubstituted or substituted, saturated or unsaturated, linear alkylene group having 2-20 backbone carbons. By way of example and not limitation, the reagent for derivatizing HA can be selected from the following:

[0058] [ka]

[0059] (In the formula, R 1 and R 2 can be independently -COHNH2, -SH, -H2, -OH, or other nucleophiles, and n is an integer, e.g., 1-20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.). In some embodiments, the reagent for derivatizing HA can be a dihydrazide, such as adipic acid dihydrazide.

[0060] In some embodiments, the method of preparing the composition of the present disclosure includes obtaining GO-HA (e.g., by the method described above), dissolving the GO-HA conjugate in water to obtain an aqueous GO-HA solution, and adding XAV939 to the aqueous GO-HA solution to form a mixture. In some examples, this is accomplished by first dissolving XAV939 in a non-ionic hydrophilic polymer, such as PEG-400 (i.e., PEG 400, having an average molar mass of about 400), and then adding the XAV939 solution to the aqueous GO-HA conjugate solution.

[0061] The present disclosure arises from the novel and unexpected finding of significantly increased regeneration of cartilage following acute injury in mammals, both in the amount of re-growth and in the quality of cartilage regeneration and re-growth.While increased regeneration of cartilage following administration of a Wnt inhibitor such as XAV939 is known in the art, as is the benefit of providing XAV939 in a matrix of GO-HA, the significant increase in cartilage regeneration of injured cartilage with timely administration of XAV939 in a matrix including GO-HA is both novel and unexpected.

[0062] The reduced rate of administration of the compositions of the present disclosure compared to administration of active XAV939 dissolved in DMSO demonstrated increased cartilage regeneration following acute cartilage injury in animal models recognized by the art as providing a reduced rate of healing (see, e.g., Sullivan SR, 2004, Plast. Reconstr. Surg. 113:953-60), and provided significantly improved amounts of cartilage regrowth and improved quality of cartilage production in half the time compared to XAV939 in DMSO. EXAMPLES

[0063] The following examples are provided for the purpose of illustrating certain aspects of the description herein and should not be construed as limiting the invention in any way.

[0064] Example 1: Preparation of a wound care composition. The procedure for preparing the composition is disclosed in US Patent No. 11,369,685 B2. Briefly, graphene oxide (1.25 g, 250 mL of 5 mg / mL GO dispersion in deionized water, supplied by Goographene Inc.) was added to 250 mL of deionized ultrapure water and stirred for 5 minutes. Sodium hydroxide pellets (3 g (0.075 moles; supplied by Sigma-Aldrich) were added to the mixture in small amounts of solid over 30 minutes. Once the addition was complete, it was stirred at room temperature for 1 hour. The solution was then sonicated for 30 minutes and then chloroacetic acid (3.54 g, 0.0375 moles; supplied by Alfa Aesar) was added in small solid portions over 20 minutes. The reaction mixture was then stirred at room temperature for 18 hours. The reaction mixture was acidified with hydrochloric acid (7 mL, 12 N). The solution was then transferred to a centrifuge tube and centrifuged at 5,000 rpm for 15 minutes. The aqueous layer was then decanted and additional deionized ultrapure water (approximately 30 mL) was added to the tube before centrifuging again. This process was repeated three times. Methanol (approximately 30 mL) was added to the precipitated modified graphene oxide remaining in the centrifuged tube and centrifuged at 5,000 rpm for 15 minutes. This process was repeated three times. Once the methanol was decanted, the tube was placed under vacuum at room temperature for 48 hours for drying. The product was then acidified with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (52 mg, 0.00027 moles, supplied by Alfa) while maintaining the pH with 0.1 N hydrochloric acid. The GO-HA was derivatized by the addition of adipic dihydrazide (17 mg, 0.00027 moles; supplier: Alfa Aesar) and then added dropwise at room temperature to a separate mixture containing adipic dihydrazide (17 mg, 0.00027 moles; supplier: Alfa Aesar) in 5 mL of deionized ultrapure water. Once the addition was complete, it was stirred at room temperature for 18 hours. The solution was then subjected to dialysis (MWC=3500) for 24 hours and lyophilized to yield GO-HA, which is further referred to herein.

[0065] 11 mg of GO-HA was dissolved in 11 mL of ultrapure water to create an effective concentration of 1 mg / mL. The solution was sonicated for 10 minutes. XAV939 (11 mg; supplier: APEBIO) was added to PEG-400 (0.5 mL) and subjected to sonication for 30 minutes. The XAV939 solution was added dropwise to the GO-HA and vigorously stirred for 5 minutes. The combined solution was then subjected to sonication for 1 hour and then stirred at room temperature for 18 hours. Hydroxypropylcellulose (0.2 g; supplier: Sigma-Aldrich) was added in small portions at room temperature with vigorous stirring. Once the addition was complete, the solution was stirred at room temperature for 24 hours to form a viscous solution of GO-HA / XAV939 complex. The complex was used at a final concentration of 1.0 mg / mL of XAV939 in 1.0 mg / mL GO-HA and 0.182 g / mL of hydroxypropylcellulose (ELU42).

[0066] Example 2: Cartilage regrowth in mice following administration of compounds of the present disclosure. C57Bl / 6J mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and maintained by PPY. C57Bl / 6J mice (at least 3 months old) were anesthetized with 3-5% isoflurane in O2 administered using the Tabletop Laboratory Animal Anesthesia System (VetEquip Inc., Pleasanton, CA, USA). 2 mm biopsy punch wounds were made in the center of the cartilage region of each ear using a disposable biopsy punch (Acuderm Inc., Fort Lauderdale, FL, USA) as previously described (Rai, MF et al, 2021, Arthritis Rheum., 64:2300-2310). Ears were treated topically daily for 30 days with 5 μL / ear of XAV939 (Selleck Chemicals, S1180, Houston, TX, USA) dissolved at 5 μM in dimethyl sulfoxide (DMSO) or a control solution of DMSO, resulting in a daily administration of 7.81 μg of XAV939.

[0067] After 30 days of treatment, ears were imaged using a Nikon Coolpix 8700 digital camera (Nikon Corporation, Japan). To quantify wound closure, ears were removed following mouse sacrifice, fixed, and placed on slides for examination using a digital camera attached to a Micromaster Inverted microscope (Thermo Fisher Scientific, Waltham, MA, USA). Fixation was by immersion in 10% buffered formalin for 24 hours, sliced ​​longitudinally across the injury, and then embedded in paraffin for sectioning. Tissue sections were then stained with Trichome blues and imaged and analyzed using an Olympus DP71 microscope camera (Olympus America, Center Valley, PA, USA).

[0068] For administration of ELU42, STZ-induced type I diabetic C57Bl / 6J mice were used. Diabetes-induced mice aged 8 to 12 weeks with serum glucose levels between 270 and 478 mg / dL were started in the study. Mice were anesthetized with 3-5% isoflurane in O2 administered using a Tabletop Laboratory Animal Anesthesia System (VetEquip Inc., Pleasanton, CA, USA). A 2 mm biopsy punch wound was made in the center of the cartilage region of each ear using a disposable biopsy punch (Acuderm Inc., Fort Lauderdale, FL, USA) as previously described (Rai, MF et al, 2021, Arthritis Rheum., 64:2300-2310). Ears were topically treated with 50 μL of ELU42 or GO-HA control solution prepared as described in Example 1 containing 1 mg / mL XAV939 (n=6) every other day for 15 days. For quantification of wound closure, ears were excised after sacrificing mice, placed on glass slides (Denville Scientific, South Plainfield, NJ, USA), and imaged using a Nikon Coolpix 8700 mounted on a Micromaster Inverted microscope (Thermo Fisher Scientific, Waltham, MA, USA). Excised ears were fixed in 10% buffered formalin for 24 hours, cut longitudinally across the injury, and embedded in paraffin blocks. Sections of embedded tissues were stained with Trichome blues and imaged and analyzed using an Olympus DP71 microscope camera (Olympus America, Center Valley, PA, USA).

[0069] The distance between opposing cartilage endplates was measured in four separate groups of animals following treatment of biopsy punch wounds: XAV939 in DMSO daily for 30 days, DMSO (as control) daily for 30 days, ELU42 every other day for 15 days, and GO-HA (as control) every other day for 15 days. Figure 1 shows the distance between opposing cartilage endpoints between 30-day treatment of biopsy punch wounds with XAV939 in DMSO, 15-day treatment of biopsy punch wounds with GO-HA, 30-day treatment of biopsy punch wounds with saline, and 15-day treatment of biopsy punch wounds with ELU42; here, administration of ELU42 significantly reduced cartilage regrowth in half the time compared to administration of a lower dose of the active ingredient XAV939 (ANOVA). * p<0.05, unpaired t-test ** p<0.001) quantitative improvement, and significant differences were observed between ELU42 and DMSO (ANOVA ** p<0.0001). A highly significant improvement was observed for ELU42 compared to GO-HA (unpaired t-test). * p<0.05). Additionally, the nature and characteristics of cartilage regrowth in the wounds were observed to be improved in XAV939 in DMSO and ELU42 compared to DMSO and GO-HA controls, respectively. Figure 2 shows a direct comparison of the distance between opposing cartilage endpoints of biopsy punch wounds treated with XAV939 in DMSO for 30 days and ELU42 for 15 days. Figure 3 shows a direct comparison of the distance between opposing cartilage endpoints of biopsy punch wounds treated with GO-HA for 15 days and ELU42 for 15 days.

[0070] Example 3: Cartilage regrowth in rabbit ears following administration of compounds of the present disclosure. For testing compositions of the present disclosure for increased cartilage regeneration following cartilage wounding in rabbits; exemplary compositions of the present disclosure were formulated as part of a phospholipid as follows.

[0071] XAV939 (0.09 wt%) was suspended in 2 mL (4.6 wt%) butylene glycol and 2 mL (4.6 wt%) polysorbate 20 and heated to 60° C. for 10 min with stirring. The mixture was then placed in an ultrasonic bath and sonicated for 30 min. Phosphatidylcholine (1.2 wt%) was slowly dissolved in 2 mL (4.6 wt%) ethanol. Once dissolved, the solution was added to the XAV939 / butylene glycol / polysorbate 20 mixture, stirred at room temperature for 5 min, and then subjected to sonication for 30 min. After 30 min, the solution was added to a previously made solution of GO-HA (0.09 wt%) in water (84%) and this new solution was sonicated for 30 min. After sonication, hydroxypropylcellulose (0.75 wt%) was added to the mixture and the mixture was stirred at room temperature for 24 h before use to provide a liposomal formulation of ELU42.

[0072] New Zealand White rabbits were purchased from Western Oregon Rabbit Company. Animals were anesthetized using either inhaled isoflurane (1-5% Iso / 2.0L O2) or a cocktail of ketamine / dilazine administered intramuscularly for the minimum amount of time possible to complete the biopsy procedure described herein. Prior to surgery, the ventral hair of each ear was thoroughly removed with a razor or other appropriate method (e.g., depilatory), and the biopsy site was surgically scrubbed with chlorhexidine and alcohol. Wounds of 6 mm size were created via punch biopsy using a disposable biopsy punch, with the wounds extending throughout the entire ear. A maximum total of eight wounds were created per animal, and the excised tissue was discarded. Following wound creation, the wound bed was cleaned with sterile saline and / or gauze to remove any foreign bodies / loose tissue debris, if necessary.

[0073] The wounds were dosed every other day for 21 days with either 0.3 mL of saline or 0.3 mL of a 1 mg / mL liposomal formulation of ELU42. On each dosing day, the wound surface was cleaned and any blood clots, excess exudate, dressing debris, test article residue, or tissue accumulated on the wound bed was removed using sterile materials (i.e., sterile gauze moistened with saline), but care was taken to avoid disturbing the wound surface. The test article was applied directly to the wound site evenly over the designated wound site in a thin layer using a sterile applicator. After each dosing, the wound site was covered / bandaged according to the study protocol or as recommended by the attending veterinarian. Images were taken on each dosing day after the wound had been thoroughly cleaned and prior to dose administration on the dosing day. Each image was taken directly in front of the wound to ensure accurate measurements and included a ruler with the appropriate identifier.

[0074] Figure 4 shows a direct comparison of the distance between opposing cartilage endpoints of biopsy punch wounds through rabbit ears following 21 days of treatment with saline and a liposomal formulation of ELU42. As can be seen, ELU42 has a statistically significant improvement in cartilage regeneration compared to saline (ANOVA). * p<0.05, unpaired t test).

[0075] Although specific embodiments of the present invention have been described in the foregoing description, it should be understood that other embodiments are possible within the scope of the present invention and are intended to be included herein. It will be apparent to those skilled in the art that modifications and adjustments to this invention, not shown, are possible without departing from the spirit of the invention, as demonstrated through the exemplary embodiments. The present invention should therefore be considered limited only by the scope of the appended claims.

Claims

1. A pharmaceutical composition for treating cartilage disorders, the pharmaceutical composition comprising: a matrix component comprising a conjugate of graphene oxide (GO) and hyaluronic acid (HA) (GO-HA), where GO and HA are covalently bonded via a linker; 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one (XAV939); and water Including; Optionally, wherein XAV939 comprises about 0.001% to about 5% by weight of the total pharmaceutical composition; said treatment comprising stimulating the regeneration of injured mammalian cartilage; The cartilage is mammalian cartilage. Pharmaceutical compositions.

2. The pharmaceutical composition of claim 1 , wherein the damaged cartilage is acutely damaged cartilage.

3. The pharmaceutical composition of claim 2, wherein the damaged cartilage is elastic cartilage.

4. The pharmaceutical composition described in claim 1, wherein the pharmaceutical composition further contains a surfactant.

5. The pharmaceutical composition described in claim 4, wherein the surfactant is polyethylene glycol (PEG) or poloxamer.

6. The pharmaceutical composition of claim 5, wherein the surfactant is PEG, and the PEG has a molecular weight of about 200 to about 400 daltons.

7. The pharmaceutical composition of claim 5, wherein the PEG is present in an amount of about 0.1% to about 20% by weight of the total pharmaceutical composition.

8. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition further comprises a thickener.

9. The pharmaceutical composition of claim 8, wherein the thickener comprises hydroxypropyl cellulose (HPC).

10. The pharmaceutical composition of claim 1, wherein the linker contains 2 to 25 carbons.

11. The linker comprises one or more —CH ２ CH ２ The pharmaceutical composition of claim 1, comprising an O-unit.

12. The linker according to claim 1, wherein the linker is -R ｘ -R Ｓ -R ｙ - (wherein, R ｘ and R ｙ are each independently selected from the group consisting of —CO—, —COO—, —NH—, —NH—NH—, —NH—NH—CO—, —CS—, —S—, and —O—; R Ｓ is an unsubstituted, saturated or unsaturated, straight-chain alkylene group having 2 to 20 backbone carbons.

13. R ｘ and R ｙ The pharmaceutical composition of claim 12, wherein each is -NH-NH-CO-.

14. The pharmaceutical composition of claim 1, wherein the weight ratio of XAV939 to GO-HA is from about 1:2 to about 2:

1.

15. The pharmaceutical composition described in claim 1, wherein the GO-HA constitutes from about 0.001% by weight to about 5% by weight of the total pharmaceutical composition.

16. The pharmaceutical composition of claim 1 formulated for administration once every 48 hours.

17. 17. The pharmaceutical composition of claim 16, wherein the damaged cartilage is acutely damaged cartilage.

18. 17. The pharmaceutical composition of claim 16, wherein the damaged cartilage is elastic cartilage.

19. The pharmaceutical composition of claim 16, wherein the pharmaceutical composition further comprises a surfactant.

20. The pharmaceutical composition of claim 19, wherein the surfactant is polyethylene glycol (PEG) or poloxamer.

21. The pharmaceutical composition of claim 20, wherein the surfactant is PEG, and the PEG has a molecular weight of about 200 to about 400 daltons.

22. The pharmaceutical composition of claim 20, wherein the PEG is in an amount of about 0.1% to about 20% by weight of the total pharmaceutical composition.

23. The pharmaceutical composition of claim 16, wherein the pharmaceutical composition further comprises a thickener.

24. The pharmaceutical composition of claim 23, wherein the thickener comprises hydroxypropyl cellulose (HPC).

25. The pharmaceutical composition of claim 16, wherein the linker contains 2 to 25 carbons.

26. The linker according to claim 2, wherein the linker is one or more —CH ２ CH ２ 17. The pharmaceutical composition of claim 16, comprising an O-unit. Claim 27: The linker is -R ｘ -R Ｓ -R ｙ - (wherein, R ｘ and R ｙ are each independently selected from the group consisting of —CO—, —COO—, —NH—, —NH—NH—, —NH—NH—CO—, —CS—, —S—, and —O—; R Ｓ is an unsubstituted, saturated or unsaturated, straight-chain alkylene group having 2 to 20 backbone carbons.

28. R ｘ and R ｙ The pharmaceutical composition of claim 27, wherein each is -NH-NH-CO-.

29. The pharmaceutical composition of claim 16, wherein the weight ratio of XAV939 to GO-HA is from about 1:2 to about 2:

1.

30. The pharmaceutical composition described in claim 16, wherein the GO-HA constitutes from about 0.001% by weight to about 5% by weight of the total pharmaceutical composition.

31. The pharmaceutical composition of claim 1 or 16, administered in combination with an additional medicinal or therapeutic agent selected from the group consisting of anti-fibrotic compounds, anti-cancer agents, anti-inflammatory agents, analgesics, corticosteroids, cytotoxic agents, antibiotics, antiseptics, nicotine, antiplatelet agents, NSAIDs, colchicine, anticoagulants, vasoconstrictors or immunosuppressants, growth factors, antibodies, proteases, protease inhibitors, antimicrobial peptides, adhesion peptides, hemostatic agents, live cells, honey, and nitric oxide.

32. The pharmaceutical composition of claim 31, wherein the anti-fibrotic compound is selected from the group consisting of pirfenidone, halofuginone, nintedanib, tocilizumab, and rilonacept.

33. A pharmaceutical composition comprising 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one (XAV939), the pharmaceutical composition is a liposome composition, and the pharmaceutical composition comprises XAV939, a matrix component comprising a conjugate of graphene oxide (GO) and hyaluronic acid (HA) (GO-HA), water, and a phospholipid, wherein the GO and HA are covalently bonded via a linker; Pharmaceutical compositions.

34. The pharmaceutical composition of claim 33, wherein the phospholipid is phosphatidylcholine.

35. The pharmaceutical composition of claim 33, wherein the pharmaceutical composition further comprises butylene glycol.

36. The pharmaceutical composition described in claim 33, wherein the pharmaceutical composition further contains polysorbate 20.

37. The pharmaceutical composition of claim 33, wherein the pharmaceutical composition comprises butylene glycol, polysorbate 20, phosphatidylcholine, ethanol, GO-HA, water, and hydroxypropyl cellulose.

38. The pharmaceutical composition of claim 33, administered in combination with other medicinal or therapeutic agents selected from the group consisting of antifibrotic compounds, anticancer agents, anti-inflammatory agents, analgesics, antibiotics, corticosteroids, cytotoxic agents, antiseptics, nicotine, antiplatelet agents, NSAIDs, colchicine, anticoagulants, vasoconstrictors or immunosuppressants, growth factors, antibodies, proteases, protease inhibitors, antimicrobial peptides, adhesion peptides, hemostatic agents, live cells, honey, and nitric oxide.

39. The pharmaceutical composition of claim 38, wherein the anti-fibrotic compound is selected from the group consisting of pirfenidone, halofuginone, nintedanib, tocilizumab, and rilonacept.

40. A pharmaceutical composition according to claim 33 or 37 for stimulating cartilage regeneration in a mammal.

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