Methods and compositions for arthritis

Abstract

The present disclosure provides a pharmaceutical composition for treating and / or reducing a likelihood of developing arthritis. In some embodiments, the pharmaceutical composition comprises a therapeutically-effective amount of curcumin; a therapeutically-effective amount of carvacrol; a therapeutically-effective amount of epigallocatechin-3-gallate; a therapeutically-effective amount of oligomeric proanthocyanidins; a therapeutically-effective amount of piperine; and a pharmaceutically acceptable carrier.

Description

WSGR Docket No. 53126-704.601METHODS AND COMPOSITIONS FOR ARTHRITISCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 690,041, filed on September 3, 2024, which is entirely incorporated herein by reference.BACKGROUND

[0002] Arthritis is a disease that causes damage in a person’s joints. There are several types of arthritis, for example, osteoarthritis, rheumatoid arthritis, gout, ankylosing spondylitis, or psoriatic arthritis. Osteoarthritis (OA) is the most common type of arthritis. OA is debilitating, painful, and chronic, and affects over 32 million American adults. Aging is the greatest risk factor for developing OA. Among individuals with OA, 43% are aged 65 or older, and 88% are aged 45 or older.

[0003] Additional concern exists for older OA patients who are taking medications for concomitant diseases (e.g., heart failure or kidney disease) that can cross-react with other drugs. Furthermore, individuals who suffer a joint injury or trauma are susceptible to developing OA. The OA related structural changes caused by these injuries can be detected as early as 6 months after injury. Meniscal injury can accelerate the onset of OA and 50% of those undergoing meniscectomy may cause radiographic OA 10 to 20 years later.SUMMARY OF THE INVENTION

[0004] In some embodiments, the present disclosure provides a pharmaceutical composition, comprising: a therapeutically-effective amount of curcumin; a therapeutically-effective amount of carvacrol; a therapeutically-effective amount of epigallocatechin-3 -gallate; a therapeutically- effective amount of oligomeric proanthocyanidins; a therapeutically-effective amount of piperine; and a pharmaceutically acceptable carrier.

[0005] In some embodiments, the pharmaceutical composition is in an oral dosage form. In some embodiments, the pharmaceutical composition is in a capsule form. In some embodiments, curcumin is present in the pharmaceutical composition at a level from about 10% (w / w) to about 30% (w / w). In some embodiments, carvacrol is present in the pharmaceutical composition at a level from about 20% (w / w) to about 40% (w / w). In some embodiments, epigallocatechin-3- gallate is present in the pharmaceutical composition at a level from about 2% (w / w) to about 20% (w / w). In some embodiments, oligomeric proanthocyanidins is present in the pharmaceutical composition at a level from about 30% (w / w) to about 60% (w / w). In some embodiments, piperine is present in the pharmaceutical composition at a level from about 10%WSGR Docket No. 53126-704.601(w / w) to about 30% (w / w). In some embodiments, a weight ratio of curcumin / carvacrol / epigall ocatechin-3 -gall ate / oligomeric proanthocyanidins / piperine is about 0.18:0.29:0.07:0.46:0.2.

[0006] In some embodiments, the present disclosure provides a method of treating a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically-effective amount of any of the pharmaceutical compositions as disclosed herein.

[0007] In some embodiments, the condition is tendinopathy, tendonitis / tendinitis, tenosynovitis, tendon injury, acute tendon injury, chronic tendon injury, a tendon condition, a cartilage injury, a ligament injury, a ligament condition, a soft tissue condition, or a joint condition.

[0008] In some embodiments, the condition is arthritis. In some embodiments, the arthritis is osteoarthritis. In some embodiments, the arthritis is rheumatoid arthritis. In some embodiments, the arthritis is gout. In some embodiments, the arthritis is ankylosing spondylitis. In some embodiments, the arthritis is psoriatic arthritis.

[0009] In some embodiments, the administering is oral administration. In some embodiments, the administering comprises administering multiple doses of the therapeutically-effective amount of the pharmaceutical composition. In some embodiments, the administering comprises administering a single dose of the therapeutically-effective amount of the pharmaceutical composition a day for a period of one week. In some embodiments, the administering comprises administering a single dose of the therapeutically-effective amount of the pharmaceutical composition a day for a period of one to four weeks.

[0010] In some embodiments, the method increases expression of CITED2, reduces expression of iNOS / NOS2, and decreases expression of MMP-13. In some embodiments, the method further comprises assaying the subject for modulation of gene expression of CITED2, iNOS, NOS2 and / or MMP-13. In some embodiments, the treating comprises mitigating histologic features of arthritis. In some embodiments, the treating comprises increasing the subject’s arthritis pain threshold and / or improving the subject’s OARSI score.

[0011] In some embodiments, the present disclosure provides a method of treating arthritis in a subject in need thereof, the method comprising administering to the subject a therapeutically- effective amount of any of the pharmaceutical compositions disclosed herein. In some embodiments, the arthritis comprises osteoarthritis, rheumatoid arthritis, gout, ankylosing spondylitis, or psoriatic arthritis. In some embodiments, the arthritis is osteoarthritis.

[0012] In some embodiments, the present disclosure provides a method of reducing a likelihood of developing arthritis in a subject in need thereof, the method comprising administering to theWSGR Docket No. 53126-704.601 subject a therapeutically-effective amount of any of the pharmaceutical compositions as disclosed herein.

[0013] In some embodiments, the arthritis is osteoarthritis. In some embodiments, the arthritis is rheumatoid arthritis. In some embodiments, the arthritis is gout. In some embodiments, the arthritis is ankylosing spondylitis. In some embodiments, the arthritis is psoriatic arthritis. In some embodiments, the administering is oral administration. In some embodiments, the administering comprises administering multiple doses of the therapeutically-effective amount of the pharmaceutical composition. In some embodiments, the administering comprises administering a single dose of the therapeutically-effective amount of the pharmaceutical composition a day for a period of one week. In some embodiments, the administering comprises administering a single dose of the therapeutically-effective amount of the pharmaceutical composition a day for a period of one to four weeks.

[0014] In some embodiments, the method increases expression of CITED2, reduces expression of iNOS / NOS2, and decreases expression of MMP-13. In some embodiments, the method further comprises assaying the subject for modulation of gene expression of CITED2, iNOS, NOS2 and / or MMP-13. In some embodiments, the treating comprises mitigating histologic features of arthritis. In some embodiments, the treating comprises increasing the subject’s arthritis pain threshold and / or improving the subject’s OARSI score.INCORPORATION BY REFERENCE

[0015] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIGS. 1A-1E show histopathology (Safranin O staining) images in the articular cartilage of the rats that had been subjected to sham surgery (FIG. 1A), subjected to destabilization of the medial meniscus (DMM) surgery and administered placebo (FIG. IB), subjected to DMM surgery and administered piperine (FIG. 1C), subjected to DMM surgery and administered C'- CEO (FIG. ID), or subjected to DMM surgery and administered C'-CEO+P (FIG. IE).

[0017] FIG. 2A shows Osteoarthritis Research Society International (OARSI) score for characterizing structural progression of OA based on cartilage damage. FIG. 2B shows Von Frey assay (assessment of OA-related pain) which measures paw withdrawal threshold to mechanical stimuli.WSGR Docket No. 53126-704.601

[0018] FIGS. 3A-3D show mRNA expression of CITED2 (FIG. 3A), matrix metalloproteinase- 13 or MMP-13 (FIG. 3B), iNOS (Inducible nitric oxide synthase) (FIG. 3C), and NOS2 (nitric oxide synthase 2) (FIG. 3D), analyzed by real-time PCR.

[0019] FIG. 4 illustrates a working model of the pharmacologic action of C'-CEO+P.

[0020] FIGS. 5A-5F show histopathology (Safranin O staining) images in the articular cartilage of the rats that had been subjected to sham surgery (FIG. 5 A), subjected to DMM surgery and administered placebo (FIG. 5B), subjected to DMM surgery and administered piperine (FIG. 5C), subjected to DMM surgery and administered C'-CEO50° / o(FIG. 5D), subjected to DMM surgery and administered C'-CEO50° / o+P (FIG. 5E), or subjected to DMM surgery and administered C'-CEO100%(FIG. 5F).

[0021] FIG. 6A shows Osteoarthritis Research Society International (OARSI) score for characterizing structural progression of OA based on cartilage damage. FIG. 6B shows Von Frey assay (assessment of OA-related pain) which measures paw withdrawal threshold to mechanical stimuli.

[0022] FIGS. 7A-7D show mRNA expression of CITED2 (FIG. 7A), matrix metalloproteinase- 13 or MMP-13 (FIG. 7B), iNOS (Inducible nitric oxide synthase) (FIG. 7C), and NOS2 (nitric oxide synthase 2) (FIG. 7D), analyzed by real-time PCR.

[0023] FIG. 8 illustrates a working model of the pharmacologic action of C'-CEO+P.DETAILED DESCRIPTION

[0024] The present disclosure provides compositions and methods to treat and / or reduce a likelihood of developing arthritis. Non-limiting examples of arthritis include osteoarthritis, rheumatoid arthritis, gout, ankylosing spondylitis, or psoriatic arthritis. In some embodiments, the arthritis is osteoarthritis (OA).

[0025] Many diseases, such as arthritis, involve multiple pathogenic factors that are insufficiently treated with drugs designed against single targets. A drug cocktail or a multi-drug therapy can be used to target multiple pathogenic factors. The present disclosure provides a composition of compounds designed to hit multiple targets involved with arthritis pathogenesis and disease progression. The composition provided herein offers a more effective treatment by exerting its therapeutic effect on tissues in the whole joint.

[0026] A screening strategy is used to identify botanical compounds with favorable safety profiles that induces the expression of a molecular target, a chondroprotective transcriptional regulator known as CITED2 (CBP / p300 Interacting Transactivator with ED-rich tail 2) and suppresses the expression of signaling pathways critically involved in the pathogenesis ofWSGR Docket No. 53126-704.601 arthritis (e.g., osteoarthritis or OA). The screening strategy disclosed herein is based on multiple facets of OA. A pool of botanical -based compounds that have biological and pharmacological anti-OA potential (e.g., suppressing inflammatory and / or pain cytokines or mediators, proteolytic enzymes, oxidative stress products, and anti-apoptosis) and pain relief are screened and a botanical formulation is identified. The identified formulation comprises curcumin [C'], carvacrol [C], epigallocatechin gallate (EGCG) [E], and oligomeric proanthocyanidins (OPC) [O], and can be referred to as C'-CEO.

[0027] In some embodiments, the C'-CEO formulation is: (i) efficacious on mitigating arthritis (e.g., OA) pathology and arthritis (e.g., OA)-related pain; and (ii) safe in a rat model of arthritis (e.g., OA) induced by destabilization of the medial meniscus (DMM). A pilot study using a DMM-induced canine model of OA showed that while daily oral administration of the effective dosage of C'-CEO in rats resulted in a clinically meaningful improvement in gait without adverse effects, the dosage was not sufficient for improving OA pathology in large animals. In some embodiments, increasing the dosage allows C'-CEO to reach effective dose in large animals.

[0028] The present disclosure provides formulations that can improve the effectiveness of C'- CEO. In some embodiments, the formulations provided herein comprise piperine (P). Piperine is a naturally occurring and safe compound. In some embodiments, piperine increases the potency of the C'-CEO composition. In some embodiments, piperine increases the bioavailability, e.g., of curcumin and / or EGCG, and enhances the effect of C'-CEO on expression of genes that are critical for chondroprotection and arthritis (e.g., OA) pathogenesis in vitro.

[0029] In some embodiments, piperine enhances the effect of C'-CEO on expression of genes that are critical for arthritis (e.g., OA) pathogenesis in vivo. In some embodiments, piperine increases the bioavailability of curcumin by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more, as compared to C'-CEO formulation without piperine. In some embodiments, piperine increases the bioavailability of EGCG by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, or more, as compared to C'-CEO formulation without piperine.

[0030] In some embodiments, the present disclosure provides a pharmaceutical composition for treating or reducing a likelihood of developing arthritis (e.g., OA). In some embodiments, the pharmaceutical composition comprises a plurality of compounds (or pharmaceutical compounds). In some embodiments, the plurality of compounds comprises curcumin [C'],WSGR Docket No. 53126-704.601 carvacrol [C], epigallocatechin gallate (EGCG) [E], oligomeric proanthocyanidins (OPC) [O], and piperine [P], In some embodiments, the pharmaceutical composition comprises one or more of pharmaceutically acceptable carrier, stabilizer, diluent, dispersing agent, suspending agent, thickening agent, solubilizing agent, buffer preservative, surface active agent, and / or excipient.

[0031] In some embodiments, the pharmaceutical composition mitigates structural progression of arthritis (e.g., OA). In some embodiments, the pharmaceutical composition improves arthritis (e.g., OA)-related symptoms of pain and joint function. In some embodiments, the pharmaceutical composition has no or minimal adverse effect on the subject.

[0032] In some embodiments, a pharmaceutical composition disclosed herein is suspended in a solution, e.g., a saline solution. In some embodiments, a pharmaceutical composition disclosed herein comprises one or more additional active ingredients such as antimicrobial agents, antiinflammatory agents, or anesthetics.

[0033] In some embodiments, the pharmaceutical composition facilitates administration of the compounds to a subject. In some embodiments, the pharmaceutical composition can be administered by various forms and routes including, for example, injection, peri-tissue injection, intra-tissue injection, intratendinous injection, intraarticular, intramuscular, intravascular, subcutaneous, infusion, intravenous, enteral, oral, buccal, sublingual, rectal, parenteral, ocular, and topical administration. In some embodiments, the pharmaceutical composition can be administered via local injection into the subject. In some embodiments, the pharmaceutical composition can be administered via local injection into a tissue of a subject. In some embodiments, the pharmaceutical composition can be administered via local injection into a joint of a subject. In some embodiments, the pharmaceutical composition can be administered via local injection into a tendon of a subject. In some embodiments, the pharmaceutical composition can be administered via a biocompatible substrate, e.g., an implant, scaffold, vesicle, or liposome.

[0034] In some embodiments, the pharmaceutical composition can be administered in a local or systemic manner, for example, via injection of the pharmaceutical composition directly into an organ, optionally in a depot or sustained release formulation. In some embodiments, the pharmaceutical composition can be provided in the form of a rapid release formulation, in the form of an extended release formulation, or in the form of an intermediate release formulation. In some embodiments, a rapid release form can provide an immediate release. In some embodiments, an extended release formulation can provide a controlled release or a sustained delayed release.WSGR Docket No. 53126-704.601

[0035] In some embodiments, parenteral injections can be formulated for bolus injection or continuous infusion. In some embodiments, the pharmaceutical composition can be in a form suitable for parenteral injection as a sterile suspension, solution, or emulsion in oily or aqueous vehicles, and can contain formulatory agents, such as suspending, stabilizing, or dispersing agents. In some embodiments, a pharmaceutical composition for parenteral administration includes aqueous solutions of the active compounds in water-soluble form. Suspensions of the active compounds can be prepared as oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. The suspension can also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. In some embodiments, the pharmaceutical composition can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0036] In some embodiments, the pharmaceutical composition can be formulated using one or more physiologically-acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations that can be used pharmaceutically. An excipient can fill a role as simple and direct as being an inert filler, or an excipient as used herein can be part of a pH stabilizing system or coating to ensure delivery of the ingredients safely to a target tissue. Formulations can be modified depending upon the route of administration chosen. Pharmaceutical compositions comprising a compound described herein can be manufactured, for example, by mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or compression processes.

[0037] In some embodiments, the pharmaceutical composition can include at least one pharmaceutically-acceptable carrier, diluent, or excipient and compounds described herein as free-base or pharmaceutically-acceptable salt form. In some embodiments, the pharmaceutical composition can include crystalline forms (or polymorphs), and active metabolites of these compounds having the same type of activity.

[0038] Non-limiting examples of pharmaceutically-acceptable excipients may comprise granulating agents, binding agents, lubricating agents, disintegrating agents, sweetening agents, glidants, anti -adherents, anti-static agents, surfactants, anti-oxidants, gums, coating agents, coloring agents, flavoring agents, coating agents, plasticizers, preservatives, suspending agents, emulsifying agents, anti-microbial agents, plant cellulosic material and spheronization agents, and any combination thereof.WSGR Docket No. 53126-704.601

[0039] Non-limiting examples of pharmaceutically-acceptable liquid carriers include water, saline solution, Ringer’s solution, glycerol, ethanol, and dextrose solution. The pH of the carrier or solution can be from about 5 to about 8, and can be from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the compound or a pharmaceutically-acceptable salt thereof, where the matrices are in the form of shaped articles, such as films, liposomes, microparticles, and microcapsules.

[0040] In some embodiments, the pharmaceutical composition can be produced by various methods in any quantity. Methods for the preparation of compositions comprising the compounds include formulating the compounds with one or more inert, pharmaceutically- acceptable excipients or carriers to form a solid, semi-solid, or liquid composition. Solid compositions include, for example, powders, tablets, dispersible granules, capsules, cachets, and suppositories. Liquid compositions include, for example, solutions in which a compound is dissolved, emulsions comprising a compound, or a solution containing liposomes, micelles, nanoparticles, vesicles, microvesicles, or nanovesicles comprising a compound as disclosed herein. Semi-solid compositions include, for example, gels, suspensions, and creams. The compositions can be in liquid solutions or suspensions, solid forms suitable for solution or suspension in a liquid prior to use, or as emulsions. These compositions can also contain minor amounts of nontoxic, auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and other pharmaceutically-acceptable additives.

[0041] In some embodiments, a dosage form suitable for use in the present disclosure includes feed, food, pellet, lozenge, liquid, elixir, aerosol, inhalant, spray, powder, tablet, pill, capsule, gel, geltab, nanosuspension, nanoparticle, microgel, suppository troches, aqueous or oily suspensions, ointment, patch, lotion, dentifrice, emulsion, creams, drops, dispersible powders or granules, emulsion in hard or soft gel capsules, syrups, phytoceuticals, nutraceuticals, and any combination thereof.

[0042] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of curcumin [C'J), a therapeutically effective amount of carvacrol [C], a therapeutically effective amount of epigallocatechin gallate (EGCG) [E], a therapeutically effective amount of oligomeric proanthocyanidins (OPC) [O], a therapeutically effective amount of piperine [P], and a pharmaceutically acceptable carrier. The pharmaceutical composition disclosed herein may be referred to as C'-CEO+P.

[0043] In some embodiments, the pharmaceutical composition is in an oral dosage form. In some embodiments, the pharmaceutical composition is in a capsule form.WSGR Docket No. 53126-704.601

[0044] In some embodiments, curcumin is present in the pharmaceutical composition at a level from about 10% (w / w) to about 15% (w / w), from about 10% (w / w) to about 20% (w / w), from about 10% (w / w) to about 30% (w / w), from about 15% (w / w) to about 20% (w / w), from about 15% (w / w) to about 30% (w / w), or from about 20% (w / w) to about 30% (w / w).

[0045] In some embodiments, carvacrol is present in the pharmaceutical composition at a level from about 20% (w / w) to about 30% (w / w), from about 20% (w / w) to about 40% (w / w), or from about 30% (w / w) to about 40% (w / w).

[0046] In some embodiments, epi gallocatechin-3 -gallate is present in the pharmaceutical composition at a level from about 2% (w / w) to about 50% (w / w), from about 2% (w / w) to about 10% (w / w), from about 2% (w / w) to about 15% (w / w), from about 2% (w / w) to about 20% (w / w), from about 5% (w / w) to about 10% (w / w), from about 5% (w / w) to about 15% (w / w), from about 5% (w / w) to about 20% (w / w), from about 10% (w / w) to about 15% (w / w), from about 10% (w / w) to about 20% (w / w), or from about 15% (w / w) to about 20% (w / w).

[0047] In some embodiments, oligomeric proanthocyanidins is present in the pharmaceutical composition at a level from about 30% (w / w) to about 40% (w / w), from about 30% (w / w) to about 50% (w / w) from about 30% (w / w) to about 60% (w / w), from about 40% (w / w) to about 50% (w / w), from about 40% (w / w) to about 60% (w / w), or from about 50% (w / w) to about 60% (w / w).

[0048] In some embodiments, piperine is present in the pharmaceutical composition at a level from about 10% (w / w) to about 15% (w / w), from about 10% (w / w) to about 20% (w / w), from about 10% (w / w) to about 30% (w / w), from about 15% (w / w) to about 20% (w / w), from about 15% (w / w) to about 30% (w / w), or from about 20% (w / w) to about 30% (w / w).

[0049] In some embodiments, a weight ratio of curcumin / carvacrol / epi gallocatechin-3 -gallate / oligomeric proanthocyanidins / piperine is about (0.1-0.3):(0.15-0.4):(0.01-0.2):(0.35-0.6):(0.1- 0.3). In some embodiments, a weight ratio of curcumin / carvacrol / epigallocatechin-3-gallate / oligomeric proanthocyanidins / piperine is about 0.18:0.29:0.07:0.46:0.2.

[0050] In some embodiments, the pharmaceutical composition provided herein addresses multiple facets of the arthritis (e.g., OA) disease. In some embodiments, the pharmaceutical composition provided herein (e.g., C'-CEO+P) induces expression of CITED2 (Cbp / p300 Interacting Transactivator with ED Rich Tall 2). CITED2 is a stress-responsive transcriptional regulator and a novel molecular target for OA, and plays a critical role in chondroprotection, at least in part, by suppressing the expression of critical proteolytic enzymes for cartilage destruction such as matrix metalloproteinases (e.g., matrix metalloproteinase- 13 or MMP-13) in articular joints. In some embodiments, the pharmaceutical composition provided hereinWSGR Docket No. 53126-704.601 suppresses expression of one or more pro-inflammatory mediators, oxidative stress mediators, and / or catabolic effectors which play critical roles in arthritis (e.g., OA) pathogenesis and pain. In some embodiments, the pharmaceutical composition provided herein suppresses expression of inducible nitric oxide synthase / nitric oxide synthase 2 (iNOS / NOS2). In some embodiments, the pharmaceutical composition provided herein suppresses expression of MMP-13.

[0051] In some embodiments, the pharmaceutical composition provided herein can relieve arthritis (e.g., OA) pain. In some embodiments, the pharmaceutical composition provided herein can exert a synergetic effect on suppressing critical inflammatory mediator related to arthritis (e.g., OA) pain, e.g., iNOS / NOS2, NFKB, IL-ip, or INF a. iNOS / NOS2 can catalyze the synthesis of nitric oxide (NO), a major inflammatory mediator that promotes cartilage destruction, cellular injury, and arthritis (e.g., OA)-related pain.

[0052] In some embodiments, the pharmaceutical composition provided herein induces expression of CITED2 and suppresses proteolytic enzymes such as matrix metalloproteinases (e.g., MMP-1, MMP-3, MMP-9, and / or MMP-13) (e.g., by C and / or E). In some embodiments, the pharmaceutical composition provided herein induces expression of CITED2 and suppresses metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5) (e.g., by E). In some embodiments, the pharmaceutical composition provided herein induces expression of CITED2 and suppresses both proteolytic enzymes and ADAMTS5.

[0053] In some embodiments, the pharmaceutical composition provided herein suppresses inflammatory and / or pain cytokines or mediators, for example, iNOS / NOS2 (e.g., by C', C, E, O, and / or P); PGE2 and COX2 (e.g., by C', C, and / or E); NF-KB, IL-ip, TNF-a, IL-12, and IL- 17 (e.g., by C and / or E); and CCR-2. In some embodiments, the pharmaceutical composition provided herein suppresses oxidative stress products, for example ROS (e.g., by C, E, and / or O), lipid peroxidation (e.g., by C'), and glutathione (e.g., by C). In some embodiments, the pharmaceutical composition provided herein increases SOD activity (e.g., by E and / or O). In some embodiments, the pharmaceutical composition provided herein increases anti-oxidant (e.g., GPx, GR, and / or GST) activity (e.g., by O). In some embodiments, the pharmaceutical composition provided herein suppresses chondrocyte apoptosis, for example suppressing Caspase-3 (e.g., by C and / or O). In some embodiments, the pharmaceutical composition provided herein mitigates OA progression, at least in part, by suppressing pathways that contribute to the development of OA.

[0054] In some embodiments, the pharmaceutical composition provided herein can mitigate arthritis (e.g., OA) disease progression. In some embodiments, the pharmaceutical composition provided herein can mitigate arthritis (e.g., OA) related structure damage. In some embodiments,WSGR Docket No. 53126-704.601 the pharmaceutical composition mitigates the arthritis (e.g., OA) related structural progression and relieves arthritis (e.g., OA) symptom.

[0055] In some embodiments, piperine enhances bioavailability of C'-CEO components, such as curcumin and / or EGCG. In some embodiments, piperine exerts a synergetic effect in inducing the chondroprotective transcriptional regulator CITED2 and suppressing expression of MMP- 13. In some embodiments, piperine exerts a synergetic effect in mitigating the arthritis (e.g., OA) related structural progression and relieving arthritis (e.g., OA) symptom.

[0056] In some embodiments, the pharmaceutical composition provided herein exerts a more profound effect on relieving arthritis (e.g., OA)-related pain compared with C'-CEO. In some embodiments, the pharmaceutical composition provided herein is safe for long-term use. No toxicity was detected in rats treated with C'-CEO+P.

[0057] In some embodiments, the pharmaceutical composition provided herein exhibits an enhanced therapeutic effect on disease modification using a reduced dosage of C'-CEO, e.g., a reduction of at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60%.

[0058] In some embodiments, oral administration of 50% of the effective dosage of C'-CEO in rats together with piperine, i.e., C'-CEO+P, significantly mitigates OA cartilage damage and OA-pain-related behavior without any observed adverse effects in DMM-induced OA in rats.

[0059] In some embodiments, the compounds (e.g., C', C, E, O, and P) can be present in a pharmaceutical composition or unit dosage form in an amount of from about 0.1 picomole (pmol) to about 100 pmol, about 0.1 nanomole (nmol) to about 100 nmol, about 0.1 micromole (pmol) to about 100 pmol, about 0.1 millimole (mmol) to about 100 mmol, about 0.1 mole (mol) to about 100 mol; about 0.1 picogram (pg) to about 100 pg, about 0.1 nanogram (ng) to about 100 ng, about 0.1 microgram (pg) to about 100 pg, about 0.1 milligram (mg) to about 100 mg; about 0.1 picomolar (pM) to about 100 pM, about 0.1 nanomolar (nM) to about 100 nM, about 0.1 micromolar (pM) to about 100 pM, about 0.1 millimolar (mM) to about 1 mM; or about 0.1 mg / kg to about 100 mg / kg.

[0060] In some embodiments, the compounds (e.g., C', C, E, O, and P) can be present in a pharmaceutical composition or unit dosage form in an amount of about 1 nmol, about 2 nmol, about 3 nmol, about 4 nmol, about 5 nmol, about 6 nmol, about 7 nmol, about 8 nmol, about 9 nmol, about 10 nmol, about 11 nmol, about 12 nmol, about 13 nmol, about 14 nmol, about 15 nmol, about 16 nmol, about 17 nmol, about 18 nmol, about 19 nmol, or about 20 nmol.

[0061] In some embodiments, the compounds (e.g., C', C, E, O, and P) can be present in a pharmaceutical composition or unit dosage form in an amount of about 1 nano mole / L (nM),WSGR Docket No. 53126-704.601 about 2 nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8 nM, about 9 nM, about 10 nM, about 11 nM, about 12 nM, about 13 nM, about 14 nM, about 15 nM, about 16 nM, about 17 nM, about 18 nM, about 19 nM, or about 20 nM.

[0062] In some embodiments, the compounds (e.g., C', C, E, O, and P) can be administered to a subject in a dose amount of at least about 0.1 mg / kg, at least about 0.5 mg / kg, at least about 1 mg / kg, at least about 2 mg / kg, at least about 3 mg / kg, at least about 4 mg / kg, at least about 5 mg / kg, at least about 6 mg / kg, at least about 7 mg / kg, at least about 8 mg / kg, at least about 9 mg / kg, at least about 10 mg / kg, at least about 15 mg / kg, at least about 20 mg / kg, at least about 30 mg / kg, at least about 35 mg / kg, at least about 40 mg / kg, at least about 45 mg / kg, or at least about 50 mg / kg. In some embodiments, the compounds (e.g., C', C, E, O, and P) can be administered to a subject in a dose amount of at most about 50 mg / kg, at most about 40 mg / kg, at most about 30 mg / kg, at most about 20 mg / kg, at most about 10 mg / kg, at most about 9 mg / kg, at most about 8 mg / kg, at most about 7 mg / kg, at most about 6 mg / kg, at most about 5 mg / kg, at most about 4 mg / kg, at most about 3 mg / kg, at most about 2 mg / kg, or at most about 1 mg / kg.

[0063] In some embodiments, the compounds (e.g., C', C, E, O, and P) can be administered to a subject in a dose amount of about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, or about 50 mg / kg.

[0064] In some embodiments, the subject treated with the pharmaceutical composition has increased level of drug components (C', C, E, and / or O) in blood and synovial fluid. In some embodiments, the level of drug components (C', C, E, and / or O) in blood and synovial fluid in the subject treated with the pharmaceutical composition is at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%, higher than the subject treated with a C'-CEO composition with same dosage of C', C, E, and O but without P. In some embodiments, the level of drug components (C', C, E, and / or O) in blood and synovial fluid in the subject treated with the pharmaceutical composition is at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%, higher than the subject treated with a C'-CEO composition with a 10%-50% higher dosage of C', C, E, and O but without P. In some embodiments, the level of drug components (C', C, E, and / or O) in blood and synovial fluid in the subject treated with the pharmaceutical composition is at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%, higher than theWSGR Docket No. 53126-704.601 subject treated with a C'-CEO composition with a 50%-100% higher dosage of C', C, E, and O but without P.

[0065] In some embodiments, the subject treated with the pharmaceutical composition has reduced OARSI score indicating reduced cartilage damage. In some embodiments, the OARSI score in the subject treated with the pharmaceutical composition is at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%, lower than the subject treated with a C'-CEO composition with same dosage of C', C, E, and O but without P. In some embodiments, the OARSI score in the subject treated with the pharmaceutical composition is at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%, lower than the subject treated with a C'-CEO composition with a 10-50% higher dosage of C', C, E, and O but without P.

[0066] In some embodiments, the subject treated with the pharmaceutical composition has increased cartilage thickness and volume. In some embodiments, the cartilage thickness and volume in the subject treated with the pharmaceutical composition is at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%, larger than the subject treated with a C'-CEO composition with same dosage of C', C, E, and O but without P. In some embodiments, the cartilage thickness and volume in the subject treated with the pharmaceutical composition is at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%, larger than the subject treated with a C'-CEO composition with a 10-50% higher dosage of C', C, E, and O but without P.

[0067] In some embodiments, the subject treated with the pharmaceutical composition has reduced levels of OA biomarkers, such as C-reactive protein (CRP), type I collagen-derived cross-linked carboxy-terminal telopeptide (ICTP), and C-terminal telopeptide of collagen type II (CTX-II). The reduced levels of OA biomarkers indicate reduced progression of OA. In some embodiments, the levels of OA biomarkers in the subject treated with the pharmaceutical composition is at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%, lower than the subject treated with a C'-CEO composition with same dosage of C', C, E, and O but without P. In some embodiments, the levels of OA biomarkers in the subject treated with the pharmaceutical composition is at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%, lower than the subject treated with a C'-CEO composition with a 10-50% higher dosage of C', C, E, and O but without P.

[0068] In some embodiments, the pharmaceutical composition improves joint functionality in a subject. In some embodiments, the joint functionality is associated with improved gait, improved activity, and reduced limb lameness.WSGR Docket No. 53126-704.601

[0069] In some embodiments, the pharmaceutical composition improves gait in a subject. Gait parameters including ground reaction force are established measures of joint / limb avoidance and functional alterations with OA progression, and are associated with pain and inflammation in the joint tissues, particularly in large animals. Gait data can be generated using the force, spatial, and temporal parameters of the identified foot strikes. Force parameters including peak vertical force (PVF) as a percentage of body weight, vertical impulse (VI) as a percentage of body weight, and maximum peak pressure (kPa) are calculated for each limb. The spatiotemporal parameters include the stance time, stride time, stride length, and stride velocity. In some embodiments, the subject treated with the pharmaceutical composition has increased peak vertical forces or vertical impulses. In some embodiments, the gait improvement in the subject treated with the pharmaceutical composition is at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%, higher than the subject treated with a C'-CEO composition with same dosage of C', C, E, and O but without P. In some embodiments, the gait improvement in the subject treated with the pharmaceutical composition is at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%, higher than the subject treated with a C'-CEO composition with a 10-50% higher dosage of C', C, E, and O but without P.

[0070] In some embodiments, the present disclosure provides a method of treating a condition in a subject in need thereof or reducing a likelihood of developing the condition. In some embodiments, the present disclosure provides a method of treating a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically-effective amount of any of the pharmaceutical compositions disclosed herein. In some embodiments, the present disclosure provides a method of reducing a likelihood of developing the condition in a subject, the method comprising administering to the subject a therapeutically-effective amount of any of the pharmaceutical compositions disclosed herein. In some embodiments, the administering is oral administration. In some embodiments, the administering is systemic administration.

[0071] In some embodiments, the condition comprises a musculoskeletal or connective tissue condition. In some embodiments, the condition comprises tendinopathy, tendonitis / tendinitis, tenosynovitis, tendon injury, acute tendon injury, chronic tendon injury, a tendon condition, a cartilage injury, a ligament injury, a ligament condition, a soft tissue condition, a joint condition, a muscle condition, a bone condition, osteoporosis, osteoarthritis, arthritis, rheumatoid arthritis, gout, fibromyalgia syndrome, systemic lupus erythematosus, Sjogren’s syndrome, an autoimmune condition, and a non-autoimmune condition.WSGR Docket No. 53126-704.601

[0072] In some embodiments, the present disclosure provides a method of treating arthritis in a subject in need thereof, the method comprising administering to the subject a therapeutically- effective amount of any of the pharmaceutical compositions disclosed herein.

[0073] In some embodiments, the arthritis comprises osteoarthritis, rheumatoid arthritis, gout, ankylosing spondylitis, or psoriatic arthritis. In some embodiments, the arthritis is osteoarthritis. In some embodiments, the arthritis is osteoarthritis. In some embodiments, the arthritis is rheumatoid arthritis. In some embodiments, the arthritis is gout. In some embodiments, the arthritis is ankylosing spondylitis. In some embodiments, the arthritis is psoriatic arthritis. In some embodiments, the administering is oral administration. In some embodiments, the administering is systemic administration.

[0074] In some embodiments, the present disclosure provides a method of reducing a likelihood of developing arthritis in a subject in need thereof, the method comprising administering to the subject a therapeutically-effective amount of any of the pharmaceutical compositions disclosed herein. In some embodiments, the arthritis comprises osteoarthritis, rheumatoid arthritis, gout, ankylosing spondylitis, or psoriatic arthritis. In some embodiments, the arthritis is osteoarthritis. In some embodiments, the arthritis is osteoarthritis. In some embodiments, the arthritis is rheumatoid arthritis. In some embodiments, the arthritis is gout. In some embodiments, the arthritis is ankylosing spondylitis. In some embodiments, the arthritis is psoriatic arthritis. In some embodiments, the administering is oral administration. In some embodiments, the administering is systemic administration.

[0075] In some embodiments, the pharmaceutical composition can be administered for prophylactic or therapeutic treatments. In therapeutic applications, the pharmaceutical composition can be administered to a subject already suffering from a disease or condition, in an amount sufficient to cure or at least partially mitigate or arrest the symptoms of the disease or condition, or to cure, heal, improve, reduce, lessen, ameliorate, or reduce a likelihood of occurrence of the disease or condition. In some embodiments, the pharmaceutical composition can be administered to lessen or reduce a likelihood of developing, contracting, or worsening a condition. Amounts effective for this use can vary based on the severity and course of the disease or condition, previous therapy, the subject’s health status, weight, response to the drugs, and the judgment of the treating physician.

[0076] Multiple therapeutic agents can be administered in any order or simultaneously. If simultaneously, the multiple therapeutic agents can be provided in a single, unified form, or in multiple forms, for example, as multiple injections. The compounds can be packed together or separately, in a single package or in a plurality of packages. One or all of the therapeutic agentsWSGR Docket No. 53126-704.601 can be given in multiple doses. If not simultaneous, then the timing between the multiple doses can vary.

[0077] In some embodiments, the pharmaceutical composition can be administered before, during, or after the occurrence of a disease or condition, and the timing of administering the pharmaceutical composition can vary. In some embodiments, the pharmaceutical composition can be used as a prophylactic and can be administered continuously to a subject with a propensity to conditions or diseases to lessen or reduce a likelihood of the occurrence of the disease or condition. In some embodiments, the pharmaceutical composition can be administered to a subject during or as soon as possible after the onset of the symptoms. The administration of the pharmaceutical composition can be initiated within the first 48 hours of the onset of the symptoms, within the first 24 hours of the onset of the symptoms, within the first 6 hours of the onset of the symptoms, or within 3 hours of the onset of the symptoms. The initial administration can be via any route practical, such as by any route described herein using any pharmaceutical composition described herein.

[0078] In some embodiments, the subject can be administered with a single dose. In some embodiments, the subject can be administered with multiple doses. In some embodiments, the subject can be administered a single dose each day. In some embodiments, the subject can be administered multiple doses each day.

[0079] In some embodiments, the pharmaceutical composition can be administered as soon as is practical after the onset of a disease or condition is detected or suspected, and for a length of time necessary for the treatment of the disease, such as, for example, from about 1 month to about 3 months. In some embodiments, the length of time the pharmaceutical composition can be administered can be about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 1 month, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 3 months, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 4 months, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 5 months, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months about 23 months, about 2 years, about 2.5 years, about 3 years, about 3.5 years, about 4 years, about 4.5 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years. The length of treatment can vary for each subject.WSGR Docket No. 53126-704.601

[0080] In some embodiments, the method disclosed herein comprises assaying the subject for modulation of gene expression of CITED2, iNOS, NOS2 and / or MMP-13. In some embodiments, the assaying comprises real-time PCR. In some embodiments, the method disclosed herein comprises monitoring the subject following the administration. In some embodiments, the method disclosed herein comprises monitoring the subject for an extended period of time. In some embodiments, the method disclosed herein comprises administering an additional dose or additional doses if needed.

[0081] In some embodiments, the method disclosed herein increases expression of CITED2. In some embodiments, the method disclosed herein reduces expression of iNOS / NOS2. In some embodiments, the method disclosed herein decreases expression of MMP-13.

[0082] In some embodiments, the method disclosed herein mitigates histologic features of arthritis. In some embodiments, the method disclosed herein increases the subject’s arthritis pain threshold. In some embodiments, the method disclosed herein improves the subject’s OARSI score.

[0083] In some embodiments, the pharmaceutical composition can be packaged as a kit. In some embodiments, provided herein is a kit comprising a pharmaceutical composition disclosed herein, or pharmaceutically-acceptable salts thereof, and written instructions on use of the kit in the treatment of a condition described herein. In some embodiments, the present disclosure provides a kit comprising a pharmaceutical composition disclosed herein, or pharmaceutically- acceptable salts thereof, one or more additional pharmaceutical agents, and written instructions on use of the kit in the treatment of a condition described herein.

[0084] In some embodiments, subjects for administration of the pharmaceutical composition can be humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, and swine; domestic animals such as rabbits, dogs, and cats; and laboratory animals including rats, mice, and guinea pigs. A subject can be of any age. Subjects can be, for example, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, and infants.EXAMPLESExample 1: C'-CEO+P exerts an enhanced therapeutic effect.

[0085] Botanical compounds that exert an individual and synergistic effect on inducing CITED2, a novel chondroprotective transcriptional regulator, were identified by a drug screening strategy. C'-CEO formulation comprising curcumin [C'], carvacrol [C],WSGR Docket No. 53126-704.601 epigallocatechin gallate (EGCG) [E], and OPC [O] was identified. Initial animal studies provided direct evidence for the safety and efficacy of C'-CEO on mitigating OA pathology, and OA-related pain in rats. C'-CEO formulation, combined with piperine [P], can exert an enhanced therapeutic effect, even at a reduced dosage of C'-CEO. This example shows the enhanced therapeutic effect of C'-CEO+P over C'-CEO.

[0086] Rats (Sprague-Dawley, 4-5 months old, male) were subjected to destabilization of the medial meniscus (DMM) or sham surgery. The day following the surgery, the rats were orally administered piperine (6.95 mg / kg), C'-CEO (34.75 mg / kg, which was 50% of the effective dose for rats at 69.5 mg / kg), C'-CEO+P (C'-CEO: 34.75 mg / kg + piperine: 6.95 mg / kg), or placebo (41.7 mg / kg cellulose). FIGS. 1A-1E show histopathology (Safranin O staining) images in the articular cartilage of the rats that had been subjected to sham surgery (FIG. 1A), subjected to DMM surgery and administered placebo (FIG. IB), subjected to DMM surgery and administered piperine (FIG. 1C), subjected to DMM surgery and administered C'-CEO (FIG. ID), or subjected to DMM surgery and administered C'-CEO+P (FIG. IE). FIG. 2A shows Osteoarthritis Research Society International (OARSI) score for characterizing structural progression of OA based on cartilage damage. FIG. 2B shows Von Frey assay (assessment of OA-related pain) which measures paw withdrawal threshold to mechanical stimuli.

[0087] Using the rat model of OA induced by DMM surgery with placebo and piperine alone as treatment controls, this example shows that by combining C'-CEO with piperine (C'-CEO+P), using 50% of the effective dose of C'-CEO had a superior effect on mitigating OA cartilage damage. This mitigation led to a significant reduction of the OARSI score than by C'-CEO alone (FIG. 2A). Further, Von Frey analysis (FIG. 2B) revealed that C'-CEO+P had a significant palliative effect based on the paw withdraw threshold measured 4 weeks posttreatment, compared to the control groups and C'-CEO alone (FIG. 2B). This observation indicates that piperine can significantly enhance the therapeutic effect of C'-CEO and reduce the effective dose of C'-CEO for OA treatment. N=6-7 / group, / ?<0.05 vs. placebo (*), vs. piperine (#), and vs. C'-CEO ($) using one-way ANOVA and Tukey post-hoc test.

[0088] To gain insight into the mechanism underlying the enhancing effect of piperine on C'- CEO, blood samples were collected from the animals one hour after drug administration and the levels of curcumin were quantified by liquid chromatography. The analysis showed that no curcumin was detected in the blood samples from animals treated with placebo or piperine, while curcumin levels were elevated in rats treated with C'-CEO+P compared with those treated with C'-CEO alone. The result indicates that piperine enhanced the efficacy of C'-CEO by, atWSGR Docket No. 53126-704.601 least in part, enhancing the bioavailability of C'-CEO components, such as curcumin.

[0089] To gain insight into the molecular action and to evaluate piperine as a component of C'- CEO+P for OA treatment, an array of genes associated with OA pathogenesis and disease modification was examined by real-time PCR using human primary chondrocytes (79 year-old female donor, with a history of OA) cultured in vitro in the presence of IL- 10 and treated with piperine at 50.4 pg, C'-CEO at 252 pg, a combination of the two (C'-CEO+P), or vehicle control (DMSO) in the presence of IL-10 (10 ng / ml). Neither piperine nor C'-CEO, at the low concentration chosen for this experiment, showed a significant effect on CITED2 induction. A combination of these two (piperine and C'-CEO) in C'-CEO+P exhibited a significant effect on CITED2 induction (FIG. 3A). FIGS. 3A-3D show mRNA expression of CITED2 (FIG. 3A), matrix metalloproteinase- 13 or MMP-13 (FIG. 3B), iNOS (Inducible nitric oxide synthase) (FIG. 3C), and NOS2 (nitric oxide synthase 2) (FIG. 3D), analyzed by real-time PCR. iNOS / NOS2 can catalyze nitric oxide (NO) and lead to excessive production of NO in chondrocyte, causing cartilage destruction and cellular injury. iNOS / NOS2 has been recognized as an attractive target for OA treatment. Both piperine and C'-CEO independently suppress MMP-13 (one of the primary proteolytic enzymes involved in cartilage degradation in OA) and iNOS / NOS2, and C'-CEO+P significantly intensified the suppression of these molecules which are critical to the pathogenesis and pain in OA. n=3 / group. / ?<0.05 vs. vehicle (*), vs. piperine (#), and vs. C'-CEO ($) using one-way ANOVA and Tukey post-hoc test. FIG. 4 illustrates a working model of the pharmacologic action of C'-CEO+P. C'-CEO+P induces expression of CITED2, a molecular target for OA that plays a critical role in chrondroprotection, at least in part, by suppressing inflammatory and / or pain cytokines or mediators (e.g., iNOS / NOS2, PGE2, COX2, NF-KB, IL-10, TNF-a, IL-12, IL-17, and / or CCR-2), proteolytic enzymes such as matrix metalloproteinases (e.g., MMP-1, MMP-3, MMP-9, and / or MMP-13), and / or metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5), oxidative stress products (e.g., ROS, lipid peroxidation, and / or glutathione), and increases SOD activity and / or anti-oxidant (e.g., GPx, GR, and / or GST) activity. C'-CEO+P also suppresses chondrocyte apoptosis, for example suppressing Caspase-3 (e.g., by C and / or O). Of note, C'-CEO+P suppresses the expression of iNOS / NOS2, which catalyzes the synthesis of nitric oxide (NO), a major inflammatory mediator that promotes cartilage destruction, cellular injury and OA related pain.Example 2: Enhanced therapeutic effect of C'-CEO+P.

[0090] Botanical compounds that exert an individual and synergistic effect on inducing CITED2, a novel chondroprotective transcriptional regulator, were identified by a drugWSGR Docket No. 53126-704.601 screening strategy. C'-CEO formulation comprising curcumin [C'], carvacrol [C], epigallocatechin gallate (EGCG) [E], and OPC [O] was identified. Initial animal studies provided direct evidence for the safety and efficacy of C'-CEO on mitigating OA pathology, and OA-related pain in rats. C'-CEO formulation, combined with piperine [P], can exert an enhanced therapeutic effect, even at a reduced dosage of C'-CEO. This example shows piperine enhanced the potency of C'-CEO in OA treatment.

[0091] Rats (Sprague-Dawley, 4-5 months old, male) were subjected to destabilization of the medial meniscus (DMM) or sham surgery. The day following surgery, the rats were orally administered piperine (6.95 mg / kg), C'-CEO50%(34.75 mg / kg, which was 50% or half the effective dosage for rats at 69.5 mg / kg), C'-CEO50° / o+P (C'-CEO: 34.75 mg / kg + pipeline: 6.95 mg / kg), C'-CEO10°o / o(69.5 mg / kg, which was 100% or the full effective dosage for rats), or placebo (41.7 mg / kg cellulose).

[0092] FIGS. 5A-5F show histopathology (Safranin O staining) images in the articular cartilage of the rats that had been subjected to sham surgery (FIG. 5 A), subjected to DMM surgery and administered placebo (FIG. 5B), subjected to DMM surgery and administered piperine (FIG. 5C), subjected to DMM surgery and administered C'-CEO50° / o(FIG. 5D), subjected to DMM surgery and administered C'-CEO50° / o+P (FIG. 5E), or subjected to DMM surgery and administered C'-CEO100%(FIG. 5F).

[0093] FIG. 6A shows Osteoarthritis Research Society International (OARSI) score for characterizing structural progression of OA based on cartilage damage. Scale bar = 200 pm. FIG. 6B shows Von Frey assay (assessment of OA-related pain) which measures paw withdrawal threshold to mechanical stimuli. N=5-7 / group. <0.05 vs. placebo (*) using oneway ANOVA and Tukey post-hoc test. Human primary chondrocytes were treated with piperine at 50.4 pg, C'-CEO at 252 pg, a combination of the two (C'-CEO+P), or vehicle control (DMSO) in the presence of IL-ip (10 ng / ml). FIGS.7A-7D show mRNA expression of CITED2 (FIG. 7A), MMP-13 (FIG. 7B), iNOS (FIG. 7C), and NOS2 (FIG. 7D), analyzed by real-time PCR. n=3 / group. / ?<0.05 vs. vehicle (*), vs. piperine (#), and vs. C'-CEO (J) using one-way ANOVA and Tukey post-hoc test.

[0094] The results showed that neither C'-CEO50° / o(50% or “half-the-dose” of the effective dosage of C'-CEO as determined for rat model), nor piperine alone exerts a significant effect on OA cartilage damage based on OARSI score (FIGS. 5A-5F and 6A). C'-CEO50%+P, a formulation that combines C'-CEO50%with piperine, demonstrated an effect on mitigating OA cartilage damage comparable to C'-CEO10°o / o(100% or “full-dose” of the effective dosage of C'-WSGR Docket No. 53126-704.601CEO as determined for rat model) based on OARSI score (FIGS. 5A-5F and 6A). Further, von Frey analysis revealed that C'-CEO50° / o+P exerted a palliative effect in that the paw withdraw threshold (PWT) significantly increased in animals treated with C'-CEO50° / o+P and C'-CEO10°o / ocompared to that treated with placebo, but the PWT was not significantly different in animals treated with C'-CEO50%or piperine alone (FIG. 6B). The data indicate that while piperine alone neither exerted an effect on OA histopathology, nor improved PWT, piperine significantly enhanced the potency of C'-CEO. C'-CEO+P slows OA progression and improves functional outcomes with minimal adverse effects.

[0095] To gain insight into the mechanism of action (MO A) and to evaluate piperine as a component to combine with C'-CEO for OA treatment, an array of genes associated with OA pathogenesis and disease modification was examined by real-time PCR using human primary chondrocytes (79 year-old female donor, with a history of OA) cultured in vitro in the presence of IL-1 and treated with piperine, C'-CEO, C'-CEO combined with piperine, or vehicle. Neither piperine nor C'-CEO, at a low concentration chosen for this experiment, showed a significant effect on CITED2 induction. FIG. 7A shows that a combination of piperine and C'- CEO achieved a significant effect on CITED2 induction. FIGS. 7B-7D show that both piperine and C'-CEO independently suppressed MMP-13, iNOS, and NOS2, and C'-CEO combined with piperine significantly intensified the suppression of these molecules which are critical to the pathogenesis and pain in OA. The suppression by C'-CEO+P was higher than the combined suppression by C'-CEO and suppression by P, illustrating the synergistic effect of C'-CEO and P. The results demonstrate the novel molecular action and the specific value of piperine as the potency enhancer of C'-CEO on OA treatment.

[0096] To gain insight into the mechanism underlying the enhancing effect of piperine on C'- CEO, blood samples from the rats one hour after drug administration were collected for quantifying the levels of curcumin by liquid chromatography-mass spectrometry (LC-MS). The analysis showed that curcumin levels were elevated in rats treated with C'-CEO50%+P compared with those treated with C'-CEO without piperine, indicating that piperine enhanced the efficacy of C'-CEO by, at least in part, enhancing the bioavailability of C'-CEO components, such as curcumin.

[0097] FIG. 8 illustrates a working model of the pharmacologic action of C'-CEO+P. C'- CEO+P induces expression of CITED2, a molecular target for OA that plays a critical role in chrondroprotection, at least in part, by suppressing proteolytic enzymes such as matrix metalloproteinases (e.g., MMP-1, MMP-3, MMP-9, and / or MMP-13) (e.g., by C and / or E), andWSGR Docket No. 53126-704.601 metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5) (e.g., by E). C'-CEO+P suppresses inflammatory and / or pain cytokines or mediators, for example, iNOS / NOS2 (e.g., by C', C, E, O, and / or P); PGE2 and C0X2 (e.g., by C', C, and / or E); NF-KB, IL-ip, TNF-a, IL-12, and IL-17 (e.g., by C and / or E); and CCR-2. C'-CEO+P suppresses oxidative stress products, for example ROS (e.g., by C, E, and / or O), lipid peroxidation (e.g., by C'), and glutathione (e.g., by C). C'-CEO+P increases SOD activity (e.g., by E and / or O) and anti-oxidant (e.g., GPx, GR, and / or GST) activity (e.g., by O). C'-CEO+P suppresses chondrocyte apoptosis, for example suppressing Caspase-3 (e.g., by C and / or O). The working model indicates that C'-CEO+P mitigates OA progression, at least in part, by suppressing pathways that contribute to the development of OA.

Claims

WSGR Docket No. 53126-704.601CLAIMSWhat is claimed is:

1. A pharmaceutical composition, comprising: a therapeutically-effective amount of curcumin; a therapeutically-effective amount of carvacrol; a therapeutically-effective amount of epigallocatechin-3 -gallate; a therapeutically-effective amount of oligomeric proanthocyanidins; a therapeutically-effective amount of piperine; and a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is in an oral dosage form.

3. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is in a capsule form.

4. The pharmaceutical composition of claim 1, wherein curcumin is present in the pharmaceutical composition at a level from about 10% (w / w) to about 30% (w / w).

5. The pharmaceutical composition of claim 1, wherein carvacrol is present in the pharmaceutical composition at a level from about 20% (w / w) to about 40% (w / w).

6. The pharmaceutical composition of claim 1, wherein epigallocatechin-3 -gallate is present in the pharmaceutical composition at a level from about 2% (w / w) to about 20% (w / w).

7. The pharmaceutical composition of claim 1, wherein oligomeric proanthocyanidins is present in the pharmaceutical composition at a level from about 30% (w / w) to about 60% (w / w).

8. The pharmaceutical composition of claim 1, wherein piperine is present in the pharmaceutical composition at a level from about 10% (w / w) to about 30% (w / w).

9. The pharmaceutical composition of claim 1, wherein a weight ratio of curcumin / carvacrol / epigallocatechin-3 -gallate / oligomeric proanthocyanidins / piperine is about 0.18:0.29:0.07:0.46:0.2.WSGR Docket No. 53126-704.60110. A method of treating a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically-effective amount of the pharmaceutical composition of any one of claims 1-9.

11. The method of claim 10, wherein the condition is tendinopathy, tendonitis / tendinitis, tenosynovitis, tendon injury, acute tendon injury, chronic tendon injury, a tendon condition, a cartilage injury, a ligament injury, a ligament condition, a soft tissue condition, or a joint condition.

12. The method of claim 11, wherein the condition is arthritis.

13. The method of claim 12, wherein the arthritis is osteoarthritis.

14. The method of claim 12, wherein the arthritis is rheumatoid arthritis.

15. The method of claim 12, wherein the arthritis is gout.

16. The method of claim 12, wherein the arthritis is ankylosing spondylitis.

17. The method of claim 12, wherein the arthritis is psoriatic arthritis.

18. The method of claim 10, wherein the administering is oral administration.

19. The method of claim 10, wherein the administering comprises administering multiple doses of the therapeutically-effective amount of the pharmaceutical composition.

20. The method of claim 10, wherein the administering comprises administering a single dose of the therapeutically-effective amount of the pharmaceutical composition a day for a period of one week.

21. The method of claim 10, wherein the administering comprises administering a single dose of the therapeutically-effective amount of the pharmaceutical composition a day for a period of one to four weeks.

22. The method of claim 10, wherein the method increases expression of CITED2, reduces expression of iNOS / NOS2, and decreases expression of MMP-13.

23. The method of any one of claims 10-22, further comprising assaying the subject for modulation of gene expression of CITED2, iNOS, NOS2 and / or MMP-13.WSGR Docket No. 53126-704.60124. The method of any one of claims 12-23, wherein the treating comprises mitigating histologic features of arthritis.

25. The method of any one of claims 12-24, wherein the treating comprises increasing the subject’s arthritis pain threshold and / or improving the subject’s OARSI score.

26. A method of reducing a likelihood of developing arthritis in a subject in need thereof, the method comprising administering to the subject a therapeutically-effective amount of the pharmaceutical composition of any one of claims 1-9.

27. The method of claim 26, wherein the arthritis is osteoarthritis.

28. The method of claim 26, wherein the arthritis is rheumatoid arthritis.

29. The method of claim 26, wherein the arthritis is gout.

30. The method of claim 26, wherein the arthritis is ankylosing spondylitis.

31. The method of claim 26, wherein the arthritis is psoriatic arthritis.

32. The method of claim 26, wherein the administering is oral administration.

33. The method of claim 26, wherein the administering comprises administering multiple doses of the therapeutically-effective amount of the pharmaceutical composition.

34. The method of claim 26, wherein the administering comprises administering a single dose of the therapeutically-effective amount of the pharmaceutical composition a day for a period of one week.

35. The method of claim 26, wherein the administering comprises administering a single dose of the therapeutically-effective amount of the pharmaceutical composition a day for a period of one to four weeks.

36. The method of claim 26, wherein the method increases expression of CITED2, reduces expression of iNOS / NOS2, and decreases expression of MMP-13.

37. The method of any one of claims 26-36, further comprising assaying the subject for modulation of gene expression of CITED2, iNOS, NOS2 and / or MMP-13.WSGR Docket No. 53126-704.60138. The method of any one of claims 26-37, wherein the treating comprises mitigating histologic features of arthritis.

39. The method of any one of claims 26-38, wherein the treating comprises increasing the subject’s arthritis pain threshold and / or improving the subject’s OARSI score.

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