WO2010038153A1 - Use of sox transcription factor acti\ators for stimulating chondrogenesis - Google Patents

Abstract

The invention is directed to a method for stimulating chondrogenesis comprising administering to a subject in need thereof a pharmaceutical composition comprising a compound selected from the group consisting of phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, thiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, 6,7- dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6- dehydrokawain, harringtonine, galangine, and kaempferol.

Description

METHODS FOR STIMULATING CHONDROGENESIS

[001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 61/101 ,946, filed October 1 , 2008, the entirety of which is incorporated by reference.

[002] The multistep process of cartilage formation, or chondrogenesis, plays critical roles in skeletal development and maintenance. Chondrogenesis leads to the formation of permanent cartilaginous tissue, which provides major structural support for the respiratory and auditory tracts and the articular joints. In the latter, articular or hyaline cartilage covers the ends of bones to form the smooth articular surface of joints, protecting bones from load-bearing forces and allowing for joint motion. Chondrogenesis also leads to the formation of the cartilage growth plates. These growth plates, in turn, direct longitudinal growth of the skeleton and endochondral ossification, the process by which most of the bone in the skeleton is formed.

[003] Cartilage is a highly specialized tissue, consisting of chondrocytes embedded in a network of extracellular matrix components including collagens and proteoglycans. The chondrocytes maintain cartilage architecture by performing both formation and breakdown of critical extracellular matrix components. Steinert et al., Arthritis Research & Therapy 9(3):213 (2007).

[004] Chondrogenesis begins when mesenchymal cells committed to the chondrogenic lineage aggregate together to form cell clusters, or condensations, that express extracellular matrix. In the center of the condensations, prechondrocytes emerge and turn off expression of mesenchymal and condensation markers and increase the expression of Col2a1 and other early cartilage markers. Differentiation of these prechondrocytes is accompanied by increased expression of Col2a1 and other extracellular matrix proteins, including but not limited to aggrecan and other collagens.

[005] In endochondral ossification, the chondrocytes within certain regions of the cartilage differentiate into hypertrophic chondrocytes. Once fully differentiated, hypertrophic cells become surrounded by a calcified extracellular matrix and die through apoptosis as the cartilage matrix is replaced by bone. Karsenty and Wagner, Developmental Cell, 2:389-406 (2002). Thus, the precursor cells responsible for cartilage formation also participate in bone formation. Although a few skeletal elements (including the lateral halves of the clavicles and some parts of the skull) form through an alternative osteogenic process known as intramembranous ossification, most bone in the vertebrate skeleton forms through this chondrocyte- mediated process of endochondral ossification.

[006] Nonhypertrophic chondrocytes include reserve and proliferating chondrocytes. They are considered to be the cartilage-forming cells because they express α1 (ll) collagen and aggrecan, the major components of cartilaginous extracellular matrix. The transcription factor Sox9 is essential for differentiation of mesenchymal cells into chondrocytes at the mesenchymal condensation stage. Sox5 and Sox6 are coexpressed with Sox9 in nonhypertrophic chondrocytes and are involved in the expression of various matrix genes. Increased expression and/or activity of Sox5, Sox6, and/or Sox9 is associated with the maintenance of a chondrocytic phenotype, whereas these genes are down-regulated during chondrocyte hypertrophy. Consequently, increased Sox5, Sox6, and/or Sox9 expression and/or activity is thought to be associated with inhibition of chondrocyte hypertrophy. [007] Disruptions in the chondrogenesis process can lead to severe developmental consequences. In adults, impairment of chondrocyte function can cause cartilage degradation and osteoarthritis. Also, because cartilage exists as an avascular tissue, it has little or no capacity to repair itself when damaged by trauma or disease processes. In many cases, progenitor cells that could facilitate tissue repair do not migrate to the damaged site, resulting in permanent defects.

[008] Currently, the most common methods for cartilage repair involve surgical procedures such as autologous chondrocyte transplantation and delivery of matrices seeded with chondrogenic cells or chondrogenic factors, among other procedures. While several of these surgical methods have met with short-term success, long-term clinical results are difficult to achieve, and a critical need still exists for pharmacological agents that promote articular cartilage formation, healing, and maintenance. Known mediators of chondrogenesis include members of the transforming growth factor 8 (TGF-β) superfamily such as bone morphogenic proteins (BMPs) and fibroblast growth factors (FGFs). In addition, transcription factors have been considered for use in promoting chondrogenesis. However, these may not be ideal for therapeutic applications due to stability or delivery limitations.

[009] The invention is based in part on the discovery that compounds selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, thiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6- methoxyluteolin, 6,7-dihydroxyflavone, tamahxetine, genistin, 5-methoxyflavone, pratol, 5,6-dehydrokawain, harringtonine, galangine, and kaempferol are effective to stimulate chondrogenesis. Accordingly, the invention provides methods for stimulating chondrogenesis in a subject, methods for treating a cartilage pathology or a chondrogenic disease, and methods for preventing or delaying onset of a cartilage pathology by administering a chondrogenic composition formulated for pharmaceutical administration comprising an amount of one or more of these compounds effective to stimulate chondrogenesis in a subject.

[010] It will be apparent to the person of ordinary skill in the art that many of the chondrogenesis-stimulating compounds of the invention are nutraceuticals, i.e., they are found in, or can be derived from foodstuffs and can be used to make products such as fortified foods or dietary supplements. In particular, it will also be appreciated that many of the chondrogenesis-stimulating compounds are flavonoids, including chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, tamarixetine, genistin, 5-methoxyflavone, pratol, galanganine, kaempferol, and 6,7- dihydroxyflavone.

[011] Exemplary embodiments of the invention are disclosed. Any embodiment described in connection with one aspect of the invention is intended to apply equally to other aspects of the invention. For example, each of the chondrogenic compositions disclosed may be used in each of the methods disclosed.

Brief Description of the Drawings

[012] Figure 1 shows expression of Sox9 in mouse limb bud sections. E11.5 limb buds were serially sectioned and each region was transferred to individual microfuge tubes containing 700ml of RLT lysis buffer (Qiagen RNeasy kit). After homogenizing the sections in RLT lysis buffer by repeated pipetting, RNA was isolated according to the manufacturer's protocol.

[013] Figure 2 shows the effects of treating PML cultures with phenazopyridine hydrochloride, cytochalasin B, methysticin, thiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, 6,7-dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6- dehydrokawain, harringtonine, galangine, and kaempferol. The data represent percent stimulation of pGL3(4X48) reporter activity compared to control cultures. Each compound was tested in quadruplicate in two separate experiments (4X48(1) and 4X48(2)).

[014] Figure 3 shows the effects of treating PLM cultures with various doses of rhapontin. The x-axis indicates rhapontin doses, and the y-axis represents percent stimulation of pGL3(4X48) reporter gene compared to control cultures.

[015] Figure 4 shows the activity of the pGL3(4X48) reporter in murine limb bud-derived cells in response to different treatment combinations. The x-axis indicates the different treatments and the y-axis represents percent stimulation of the pGL3(4X48) reporter construct relative to the control culture. PHCL = phenazopyridine hydrochloride, 4310 = pan RAR antagonist 4310, B4 = bone morphogenetic protein-4 (BMP-4). The reporter gene activities shown are from an experiment performed in triplicate.

[016] Figure 5 shows Mmp13 mRNA levels in treated murine limb bud- derived cell cultures, as measured by a TaqMan^® real-time PCR assay. Mmp13 expression levels are expressed as a percent of control cells' expression level on day 5. 7 = phenazopyridine hydrochloride (PHCL), 4310 = pan RAR antagonist 4310, B4 = bone morphogenetic protein-4 (BMP-4). The expression levels shown are from an experiment performed in duplicate.

Definitions

[017] The terms "stimulating chondrogenesis" and "promoting chondrogenic activity" as used herein, include increasing differentiation of mesenchymal, chondroprogenitor, dedifferentiated chondrocytes or cells with chondrogenic potential into chondrocytes, maintaining the nonhypertrophic chondrocyte phenotype by inhibiting their differentiation into hypertrophic chondrocytes, increasing cartilage formation, enhancing the production of cartilage matrix, and/or inducing cartilage repair. A "chondrogenesis stimulating agent" is a compound that stimulates one or more of these activities. A "chondrogenic composition" as used herein, refers to a composition comprising a compound selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, thiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, 6,7-dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6-dehydrokawain, harringtonine, galangine, and kaempferol together with a pharmaceutically acceptable carrier.

[018] A "chondrogenic protective agent" or "chondroprotective agent" is a compound that inhibits degeneration of cartilage. Examples of suitable chondroprotective agents include IL-1 receptor antagonists, TNF receptor antagonists, COX-2 inhibitors, viscosupplements (i.e. hyaluronic acid), and potentially glucosamine, or chondroitin sulphate. Additional chondrogenic protective agents are described in detail at columns 12 to 14 of United States Patent 7,067,144, incorporated by reference and in United States Patent 6,583,1 18, incorporated by reference in its entirety.

[019] A "cartilage defect" includes cartilage damage caused by injury, disease, or improper formation of cartilage during development. The cartilage defect may be caused by an injury to bone as well as cartilage, such as bone fractures or osteochondral defects. The cartilage defect may also result from surgical procedures.

[020] The terms "treatment," "therapeutic method," and their cognates include prophylactic/preventative measures. Thus, a subject in need of treatment may include an individual already suffering from a particular cartilage defect, damage or disorder as well as a subject that may ultimately acquire the defect, damage or disorder. The term "treatment" and its cognates do not require full resolution/repair of the cartilage defect, damage, or disorder being treated, but include full, as well as partial resolution/repair, amelioration of associated symptoms, delayed onset, or attainment of a desired biological outcome, such as increased chondrogenesis. Typically, "treatment" will include healing or repair of cartilage damage or defect in a subject by regeneration of cartilage at the site of the injury or defect.

[021] The terms "therapeutically effective dose," or "therapeutically effective amount," refer to the amount of chondrogenic compound selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, thiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6- methoxyluteolin, 6,7-dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6-dehydrokawain, harringtonine, galangine, and kaempferol in a composition that results in prevention or delay of onset or amelioration of symptoms of cartilage damage or defects in a subject or an attainment of a desired biological outcome, such as increased chondrogenesis or prevention of hypertrophy. The effective amount can be determined by methods well-known in the art and will vary with the nature and severity of the condition treated.

[022] A "subject" can be a mammal, e.g., a human, primate, ovine, bovine, porcine, equine, feline, canine, and a rodent (rat or mouse). The use of the word "a", "an" or "the" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." Chondrogenic Compositions

[023] Chondrogenic compositions are formulated for pharmaceutical use in the methods of the invention and comprise at least one compound selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, thiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6- methoxyluteolin, 6,7-dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6-dehydrokawain, harringtonine, galangine, and kaempferol in an amount effective to stimulate chondrogenesis in a subject. In certain embodiments, the compound is selected from thiabendazole, indoprofen, oxybenzone, 5,6- dehydrokawain, harringtonine, phenazopyridine, cytochalasin B, rhapontin, and methysticin. In other embodiments, the compound is a flavonoid capable of increasing differentiation of mesenchymal cells into chondrocytes and/or increasing cartilage formation, enhancing the production of cartilage matrix, and/or inducing cartilage repair. In some embodiments, the flavonoid compound is selected from 6- methoxyluteolin, tamahxetin, genistin, pratol, and galangine In other embodiments, the flavonoid compound may be chosen from chrysin, pinocembrin, 7- hydroxyflavone, 5-methoxyflavone, and 6,7-dihydroxyflavone. Suitable excipients for inclusion in the chondrogenic compositions of the invention are well known in the art. In some embodiments, the chondrogenic compound is administered in an amount sufficient to increase mRNA, protein or activity levels of Sox 5, Sox 6, or Sox 9 (human GenelDs 6660, 55553, and 6662, respectively) by at least 5, 10, 15, 20, 25, 30, 50, 70, or 90%; or 2, 4, 8, 10, 20, 40, or 80-fold. In certain embodiments, the chondrogenic compound is administered in an amount sufficient in increase expression of aggrecan (human, mouse, and rat GenelDs 176, 11595, and 58968, respectively) or Col2a1 (human, mouse, and rat GenelDs 1280, 12824, and 25412, respectively) at the mRNA or protein level by at least 5, 10, 15, 20, 25, 30, 50, 70, or 90%; or 2, 4, 8, 10, 20, 40, or 80-fold. All information associated with all GenelDs and reference protein and nucleic acid sequences referenced in this application, including references sequences and their associated annotations, is incorporated by reference.

[024] In some embodiments, the chondrogenic composition further comprises a at least one other therapeutic agent. In exemplary embodiments, the other therapeutic agent is a chondrogenesis promoter or chondrogenesis inducing agent such as, e.g., interleukin agonists, including IL-4, IL-10, IL-13 agonists, growth factors such as, e.g., TGF-61 , TGF-62, and TGF-63; bone morphogenetic protein agonists such as, e.g., BMP-2, BMP-4, BMP-6, BMP-7, BMP-8, BMP-12, BMP-13; insulin like growth factors such as, e.g. IGF-1 ; growth and differentiation factors (GDFs); WNTs; Hedgehogs; MIA; fibroblast growth factors such as, e.g., bFGF; and retinoic acid receptor (RAR) antagonists. In other embodiments, the therapeutic agent is a chondrogenic protective agent such as, e.g., IL-1 receptor antagonists, TNF receptor antagonists, COX-2 inhibitors, MAP kinase inhibitors, nitric oxide synthase inhibitors, NFkB inhibitors, and flavonoid compounds such as those described in United States Patent 6,583,118. In an exemplary embodiment, the chondrogenic composition comprises a compound selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, thiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, 6,7- dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6- dehydrokawain, harringtonine, galangine, kaempferol, together with both another chondrogenesis inducing agent and a chondroprotective agent. [025] In certain embodiments, the chondrogenic compositions of the invention may also exhibit chondroprotective activity. A composition can be evaluated for chondroprotective activity by a variety of means known in the art, including direct visualization of cells, histological grading of cartilage, or by molecular diagnostics, such as monitoring the expression level of one or more genes involved in cartilage metabolism. For example, the Mmp13 gene product (human, mouse, and rat GenelDs: 4322, 17386, 171052, respectively) has been implicated in cartilage degeneration. Neuhold et al., J. CHn. Invest. 107(1): 35-44 (2001). Accordingly, in some embodiments, a compound's ability to reduce Mmp13 activity is indicative of chondroprotective activity. In some embodiments, a composition with chondroprotective activity reduces Mmp13 activity in a target cell by at least 10, 15, 20, 30, 50, or 90%; or 2, 4, 6, 8, 10, 20, 50, or 80-fold, relative to a control cell. In more particular embodiments, a composition with chondroprotective activity reduces Mmp13 mRNA or protein levels by at least 10, 15, 20, 30, 50, 90% or 2, 4, 6, 8, 10, 20, 50, 80-fold, relative to control cells.

Therapeutic Uses

[026] The chondrogenic compositions described above may be used in methods of inducing chondrogenesis, cartilage healing, and/or cartilage regeneration; methods of treating a cartilage pathology or chondrogenic disease; and methods of preventing or delaying the onset of a cartilage pathology or chondrogenic disease. The chondrogenic compositions may also be employed in methods of inducing de novo cartilaginous tissue formation.

[027] In certain embodiments, the methods of the invention include the repair of congenital cartilage defects, trauma-induced cartilage pathology, or cartilage defects of other origins. The methods of the invention may be used in connection with surgical procedures for the attachment or repair of cartilage. In some embodiments, methods of the invention comprise the treatment and/or prophylaxis of articular cartilage tears, cartilage deformities, damage to hyaline cartilage, and other cartilage defects in humans and animals. Any of the chondrogenic compositions described above may be employed in methods to treat chondromalacia, chondrodysplasias, cartilage damage due to bone fracture, osteochondritis, congenital cartilage defects, osteochondritis dessecans, articular cartilage damage, herniated intervertebral disk, anotia, microtia cartilage defect, degenerative joint disease including arthritis (rheumatoid and osteoarthritis), and polychondritis.

[028] The chondrogenic compositions provide an environment to attract cartilage-forming cells, stimulate growth of cartilage-forming cells, induce differentiation of progenitors of cartilage-forming cells, or improve fixation of cartilage to bone or other tissues. Thus, the compositions may also be employed in tissue engineering of cartilage. The chondrogenic compositions may also be administered prophylactically to prevent damage to cartilaginous tissue.

[029] In certain embodiments, the methods of the invention comprise administration of a chondrogenic composition comprising a therapeutically effective amount of at least one compound selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, thiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, 6,7-dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6-dehydrokawain, harringtonine, galangine, and kaempferol in combination with cells that have chondrogenic potential, including stem cells, progenitor cells, mesenchymal stem cells, mesenchymal progenitor cells, chondroprogenitors, chondrocytes or dedifferentiated chondrocytes. In other embodiments, these compositions are administered in combination with a chondroprotective or joint protective agent such as a viscosupplement (i.e. hyaluronic acid), glucosamine, or chondroitin sulphate. In still other embodiments, the methods of the invention comprise administration of a therapeutically effective amount of a chondrogenic composition of the invention in combination with another chondrogenesis inducing agent and/or with a chondroprotective agent. The individual components of the combination therapy may be administered simultaneously or sequentially.

[030] Administration of a chondrogenic composition to a subject according to the methods of the invention may be carried out using methods known to those of skill in the art. In certain embodiments, a chondrogenic composition is delivered systemically by injection, irrigation, ingestion, inhalation, or topical application. Alternatively, the chondrogenic composition is administered locally by injection (including intra-articular), topical application, or as a coating or component of a device or implant. The chondrogenic compositions may also be employed to treat cells ex vivo prior to implantation pursuant to the methods of the invention.

[031] Suitable doses of the chondrogenic compounds provided by the invention will be readily determined based on the desired outcome, using routine skill in the medical arts (see, for example Physicians' Desk Reference, 58th Edition, Thompson, P D R, Montvale, N.J. 2004). For example, effective doses identified through in vitro or ex vivo assays can be calibrated for use in vivo, while doses effective in in vivo animal models can be readily interconverted between species by means known in the art (see, for example, Freienreich et al., Cancer Chemother. Rep. 50(4):219-44 (1966)). In some embodiments, suitable doses of a chondrogenic compound are at least 0.01 , 0.05, 0.1 , 0.5, 1 , 2, 5, 10, 15, 20, 30, 45, 50, 80, 100, 150, 200, 400, 600, 800, 1000, 2000, 4000, or 8000 μM. These doses may be used ex vivo (e.g., for chondrocyte transplantation) or in vivo for direct administration. Doses may also be calculated based on subject's body weight, e.g., at least 0.001 , 0.005, 0.01 , 0.05, 0.1 , 0.5, 1 , 5, 10, 20, 40, 80, 100, 200, 400, 800, 1000, 2000, 4000, 8000, 10000, 20000, 40000, or 80000 μg/kg. Dosing regimens may be carried out for a period of time sufficient to achieve the desired therapeutic outcome, e.g., at least 2, 4, 6, 12, 24, 48, 72 hours, or 4, 5, 10, 15, 20, or 30 days.

Carriers and implants

[032] Carriers may aid in forming a chondrogenic composition that possesses appropriate handling characteristics for injectable application to the site of cartilage defect or damage. Adding the chondrogenic composition to a carrier or implant allows it to remain in the site of the disease or damage for a time sufficient to allow the composition to increase the regenerative chondrogenic activity of the infiltrating mammalian progenitor or other cells, and to form a space in which new tissue can grow and allow for ingrowth of cells. The carrier may also allow the chondrogenic composition to be released from the disease or damage site over a time interval appropriate for optimally increasing the rate of regenerative chondrogenic activity of the progenitor cells.

[033] In certain embodiments, the chondrogenic compositions of the invention may be administered using an appropriate implantable matrix, carrier, or device. For instance, the implant may provide a surface for cartilaginous tissue formation and/or other tissue formation (e.g., in mediation of bone growth or repair). Some embodiments include implantable mechanical physical devices, biodegradable carriers, biodegradable synthetic carriers, prostheses, demineralized allogenic bone and demineralized xenogenic bone. The implantable matrix, carrier, or device may provide slow release of the chondrogenic compound and/or the appropriate environment for its presentation. Biodegradable materials, such as cellulose films, or surgical meshes, may also serve as matrices. Such materials could be sutured into an injury site, or wrapped around the cartilage. Some matrices include collagen- based materials, including sponges, such as Helistaf (Integra LifeSciences, Plainsboro, N. J.), or collagen in an injectable form.

[034] One family of carriers that may be used in the methods of the invention comprises collagenous materials, and can be in a form suitable for injection, such as a gel. Such gels may be crosslinked or non-crosslinked. Other forms of collagen, such as dispersions or fibrillar collagen, may also be useful in the methods of the present invention. Another family of carriers includes cellulosic materials such as alkylcellulose, including hydroxyalkylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and carboxymethylcellulose (CMC).

[035] In some embodiments using cellulosic carriers and collagen gels, the carrier may be in the form of a hydrated cellulosic viscous gel. Viscosity may be increased through mechanical means, such as high agitation for a suitable period of time, followed by autoclaving, or chemically. The active agent and cellulosic carrier may be formulated in a solution of a suitable buffer.

[036] Another class of materials for injectable carriers is resorbable hydroxyapatites, as well as minerals, ceramics and phosphates. Resorbable hydroxyapatites, for example, can be formulated at various porosities with varying resorption rates; their handling characteristics vary from hard implantable types, to gel-like consistency, to those that are injectable but harden at body temperature. Suitable hydroxyapatite and ceramic carriers are described, for example in W096/36562; and United States Patents 5,543,019; 5,306,305; 5,258,044; 5,496,399; 5,455,231 ; 5,336,264; 5,178,845; 5,053,212; 5,047,031 ; 5,129,905; 5,034,059; 4,880,610; 5,290,763; and 5,563,124; all of which are incorporated by reference for their disclosure of suitable carriers.

[037] In other embodiments, the carrier is an injectable polymer, which may be viscous and which may optionally include a sequestering agent. Suitable polymers and sequestering agents include those described in United States Patent 5,171 ,579, incorporated by reference. Other polymers include pluronics, which are liquid (and hence syringeable) at 4⁰C and gel at body temperature. The pluronic Poloxamer 407, MW 12,500, is excreted unchanged in the urine after systemic absorption and has reportedly been shown to be non-toxic in animals. In certain embodiments, the polymer may be a polylactide and/or polyethylene glycol, including poly(lactide)/poly(ethylene glycol) gel. Polylactides may be dissolved in polyethylene glycols, such as low molecular weight (2000) PLA dissolved in PEG to produce a syringeable solution that precipitates PLA upon injection into an aqueous environment, resulting in a relatively firm gel.

[038] In some embodiments, the chondrogenic composition may include a sequestering agent such as hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer or polyvinyl alcohol), or a cellulosic material, such as hydroxycellulose or carboxymethylcellulose. These sequestering agents may themselves be useful as carriers for injection. In addition, combinations of sequestering agents may be used.

[039] The choice of a carrier material will be based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the chondrogenic compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined. Other matrices may be comprised of pure proteins or extracellular matrix components. Additional potential matrices are nonbiodegradable and chemically defined.

Chondrocyte Transplantation

[040] In certain embodiments, chondrocytes or chondrocyte precursor cells may be implanted along with any of the chondrogenic compositions described above. Some embodiments include culturing the cells with a composition comprising a chondrogenic compound ex vivo, prior to implantation at a defect site. In some embodiments, the chondrocytes or precursor cells may be harvested from the subject in need of treatment (e.g., autologous chondrocyte transplantation). The chondrocytes can be implanted along with a matrix, carrier or device. An autologous bone graft may also be implanted with the chondrocytes for certain subjects in need of bone repair.

[041] In other embodiments, genetically engineered cells may be administered along with any of the chondrogenic compositions described herein, and optionally in combination with an appropriate matrix or carrier that can provide a surface for cartilage and/or other connective tissue growth. The cells may be engineered to express proteins, growth factors, extracellular matrix materials or other chondrogenesis stimulating agents. In some embodiments, various collagenous and non-collagenous proteins are expected to be upregulated and secreted from the engineered cells. This phenomenon accelerates tissue regeneration by enhancing extracellular matrix deposition, and can enhance the engraftment and attachment of transplanted cells into the defect site. A carrier or implantable matrix may be used to provide slow release of the chondrogenic composition and/or differentiated cells. [042] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that any of the chondrogenic compounds of the invention may be used in any of the formulations described above to treat any of the conditions described herein. It is further intended that the embodiments provided in this specification are exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

[043] The following examples illustrate various embodiments of the invention and are not intended to limit the scope of the invention.

Example 1 : Establishment and Staining of Primary Limb Mesenchymal Cultures

[044] Primary limb mesenchymal (PLM) cultures were established from CD-1 murine embryonic limbs (E11.5) as previously described (Hoffman et al., J. Cell Biol., 174:101-13; Weston et al., J. Cell Biol., 158:39-51 (2002)). Limb mesenchyme was dissociated by dispase treatment and a single cell suspension was obtained by filtration through a 4OmM cell strainer (BD Biosciences). PLM cells were pelleted by centrifugation at 200 x g and resuspended to produce a stock cell suspension at a concentration of 2.0x10⁷ cells/ml. Cells were used for transfection (Example 2) or for establishment of cultures for Alcian blue staining.

[045] For the latter, 10 μL of PLM cells were spotted into the well of a 24-well plate, and allowed to adhere for 1 h. Culture medium consisting of 60% Ham's F12 nutrient mix/40% Dulbecco's modified Eagle's medium (DMEM) and supplemented with 10% FBS (Qualified, Invitrogen) was added to each well, and test compounds were added at approximately 15 micromolar concentrations to each well 24 hrs following culture establishment; this time was considered T= 1. Cultures were maintained for a period of up to 4 days, and culture media was replaced on alternate days to minimize handling.

[046] For Alcian blue staining, culture medium was aspirated and cells were washed once with PBS. Cultures were fixed in 95% ethanol at -20°C overnight. Fixative was removed by aspiration and cells were sequentially washed once with PBS, followed by 0.2M HCI. Cells were stained overnight with a 1 % Alcian blue solution prepared in 0.2M HCI.

Example 2: Primary Cell Transfections and Expression

[047] Stock plasmid DNAs were standardized to a concentration of 1mg/ml. For transfections, a ratio of 20:1 of reporter gene pGL3(4X48) to control reporter gene (phRL-SV40) was used and combined with Effectene transfection reagent according to the manufacturers instructions (Qiagen); trehalose to a final concentration of 0.4 M was added to the EC buffer. Luciferase reporter genes consist of a firefly reporter gene, pGL3(4X48), and a Renilla (Renilla reniformis) luciferase reporter phRL-SV40 to normalize for transfection efficiency. The pGL3(4X48) reporter contains four repeats of a Sox5/6/9 binding site. Reporter plasmids containing Sox5/6/9 binding sites (pGL3[4X48]) were previously described (Weston et al., J. Cell Biol., 158:39-51 (2002)).

[048] Test compounds were added to the culture medium of transfected cells 24 hours following transfection at approximately 15 micromolar concentrations. After 24 hours, cells were washed with PBS and lysed in 40ml of passive lysis buffer (Promega) per well. Plates were assayed for luciferase activity according to the manufacturer's instructions (Promega).

[049] To follow the expression of transcripts for Sox9 or other chondrogenic markers, quantitative real-time PCR was performed using the 7500 Fast Sequence Detection System (Applied Biosystems). The primer/probe set used for detection of Sox9 was as described in Weston et al., J. Cell Biol., 158:39-51 (2002). For detection of all other transcripts, TaqMan Gene Expression Assays (Applied Biosystems) were used. Total RNA was isolated from primary cultures, and an aliquot was reverse transcribed to cDNA using a High Capacity cDNA Archive kit (Applied Biosystems). Quantification was performed using ~10 ng of total RNA and the expression of all genes relative to endogenous rRNA was determined using TaqMan Ribosomal Control Reagents (Applied Biosystems).

Example 3: Chondroqenic Activity of Phenazopyridine Hydrochloride

Cl -H

[050] Phenazopyridine hydrochloride, also known as 3- phenyldiazenylpyridine-2,6-diamine hydrochloride, is most commonly known as an anesthetic compound. It is commercially available and may also be prepared according to procedures set forth in the literature. See, e.g., U.S. Patent 1 ,680,109. Phenazopyridine hydrochloride has been reported to have bacteriostatic properties. The toxicity profile for phenazopyridine hydrochloride has been described by by Becker and Swift, Toxicol. Appl. Pharmacol. 1 :42 (1959). Shang et al., Anal. Bioanal. Chem. 382:216 (2005) reports on pharmacokinetics and LC-MS determination in plasma. A review of clinical experience with phenazopyridine hydrochloride is provided by Zelenitsky and Zhanel, Ann. Pharmacother. 30:866-868 (1996).

[051] Using procedures based on those described in Example 2, phenazopyridine hydrochloride was found to significantly increase the activity of the chondrogenic responsive reporter gene pGL3(4X48), indicating that it is a chondrogenesis stimulating agent (Figure 2). These PLM transfection assays, as well as Alcian blue staining experiments, indicate that low micromolar concentrations of phenazopyridine hydrochloride stimulate chondrogenic activity. Treatment of PLM cultures with phenazopyridine hydrochloride for two days also increased aggrecan expression by more than two fold in proximal mesenchymal cells and over four fold in distal mesenchymal cells, as determined by RT-qPCR.

Example 4: Chondrogenic Activity of Cytochalasin B

[052] Cytochalasin B is a cell permeable fungal toxin isolated from Helminthospohum determatioideum. It is one of the most important and biologically studied cytochalasins. It is commercially available from a number of sources. Cytochalasin B may also be isolated from Phoma exigua var. heteromorpha using the process described by Bottalico et al., Appl. Biochem. Biotech. 48(1):33-36 (1994). [053] Using procedures based on those described in Example 2, cytochalasin B was found to significantly increase the activity of the chondrogenic responsive reporter gene pGL3(4X48), indicating that it is a chondrogenesis stimulating agent (Figure 2). PLM transfection assays and Alcian blue staining experiments both indicate that low micromolar concentrations of cytochalasin B stimulate chondrogenic activity. In addition, treatment of PLM cultures with cytochalasin B for two days increased aggrecan expression by over 9 fold in proximal mesenchymal cells, and over 4 fold in distal mesenchymal cells, as determined by RT-qPCR.

Example 5: Chondrogenic Activity of Methvsticin

[054] Methysticin, also known as 6-[2-(1 ,3-benzodioxol-5-yl)ethenyl]-4- methoxy-5,6-dihydropyran-2-one, is a white, silky, crystalline substance extracted from the thick rootstock of a species of pepper (Piper methysticum) of the South Sea Islands. Synthesis of the racemate was described by Klohs et al., J. Org. Chem. 24, 1829 (1959). Structural characterizations, including absolute configuration (Snatzke et al., Tetrahedron Lett. 9:1797 (1968)) and HPLC determination (Smith et al., J. Chromatogr. 283, 303 (1984)) have also been described. Biological activities of methysticin include anticonvulsant effects (Kretzschmar, Arch. Int. Pharmacodyn. Ther. 177, 261 (1969)) and neuroprotective activity (Backhauss et al., Eur. J. Pharmacol. 215, 265 (1992)). Methysticin is commercially available from a number of sources.

[055] Using procedures based on those described in Example 2, methysticin was found to significantly increase the activity of the chondrogenic responsive reporter gene pGL3(4X48), indicating that it is a chondrogenesis stimulating agent (Figure 2). The PLM transfection assays and Alcian blue staining experiments demonstrate that low micromolar concentrations of methysticin stimulate chondrogenic activity.

Example 6: Chondrogenic Activity of Rhapontin

[056] Rhapontin, also known as 2-[3-hydroxy-5-[2-(3-hydroxy-4- methoxyphenyl) ethenyl]phenoxy]-6-(hydroxymethyl) oxane-3,4,5-triol, is isolated from rhubarb (Rheum rhaponticu) (Hesse, J. Prakt. Chem. 77, 321 (1908); Schϋrhoff et al., Arch. Pharm. 275, 281 (1937)). Rhapontin has been shown to inhibit growth and induce apoptosis in KATO III stomach cancer cells. Habasami et al., Oncol. Rep. 18, 347 (2007). Rhapontin is also commercially available from numerous sources.

[057] Using procedures based on those described in Example 2, rhapontin was found to significantly increase the activity of the chondrogenic responsive reporter gene pGL3(4X48), indicating that it is a chondrogenesis stimulating agent (Figure 2). As shown in Figure 3, a dose response study of rhapontin indicated that it was most effective at stimulating 4X48 activity at concentrations between 10OnM and 100OnM. These assays and Alcian blue staining experiments show that, ~100 nanomolar to low micromolar concentrations of rhapontin stimulate chondrogenic activity.

Example 7: Chondrogenic Activity of Oxybenzone

[058] Oxybenzone, also known as 2-hydroxy-4-methoxyphenyl)- phenylmethanone, is a derivative of benzophenone. It forms colorless crystals that are readily soluble in most organic solvents. Methods of preparing this compound are disclosed in United States Patents 2,773,903; 2,861 ,104; 2,861 ,105; and 3,073,866. Oxybenzone is commercially available.

[059] Using procedures based on those described in Example 2, oxybenzone was found to significantly increase the activity of the chondrogenic responsive reporter gene pGL3(4X48), indicating that it is a chondrogenesis stimulating agent (Figure 2). These assays and Alcian blue staining experiments indicate that low micromolar concentrations of oxybenzone stimulate chondrogenic activity. Example 8: Chondrogenic Activity of Thiabendazole

[060] Thiabendazole, also know as 2-(1 ,3-thiazol-4-yl)benzimidazole, exists as a white to off-white powder, and has low solubility in water but can readily dissolve in dilute acid and alkali solutions. The compound is used to control parasitic worm infections in humans and other animals, and as an agricultural fungicide. Gordon, Nature 191 , 1409 (1961); Dalvie et al., Drug Metab. Dispos. 34, 709 (2006). Thiabendazole is commercially available from multiple sources.

[061] Using procedures based on those described in Example 2, thiabendazole was found to significantly increase the activity of the chondrogenic responsive reporter gene pGL3(4X48), indicating that it is a chondrogenesis stimulating agent (Figure 2) and that low micromolar concentrations of thiabendazole stimulate chondrogenic activity. At the single dosage that was tested, this compound did not significantly increase Alcian blue staining relative to controls, but it is expected that experiments with varying conditions (e.g., dosage, duration) will further support the chondrogenic activity indicated in the reporter assay. Example 9: Chondrogenic Activity of Indoprofen

[062] lndoprofen, also known as 4-(1 ,3-dihydro-1-oxo-2H-isoindol-2-yl)- alpha-methyl-benzeneacetic acid, is a non-steroidal anti-inflammatory drug that may also upregulate the survival motor neuron (SMN) protein. Lunn et al., Chem. Biol. 11 , 1489 (2004). Methods of preparing indoprofen are disclosed in DE 2258088. lndoprofen is commercially available.

[063] Using procedures based on those described in Example 2, indoprofen was found to significantly increase the activity of the chondrogenic responsive reporter gene pGL3(4X48), indicating that it is a chondrogenesis stimulating agent (Figure 2). The PLM transfection assays indicate that low micromolar concentrations of indoprofen stimulate chondrogenic activity. At the single dosage that was tested, this compound did not significantly increase Alcian blue staining relative to controls, but it is expected that experiments with varying conditions (e.g., dosage, duration) will further support the chondrogenic activity indicated in the reporter assay.

Example 10: Chondrogenic Activity of Chrysin

[064] Chrysin, also known as 5,7-dihydroxy-2-phenylchromen-4-one, is a flavonoid compound extracted from multiple plant sources and tree bark. Medina et al., Biochem. Pharmacol. 40, 2227 (1990); Liu et al., J. Nat. Prod. 55, 357 (1992). The synthesis of chrysin was disclosed in Hutchins et al., J. Chem. Soc. 1 , 91 (1939). Chrysin has been used as an herbal supplement by bodybuilders, and may have anti-inflammatory effects (Woo et al., FEBS Lett. 579, 705 (2005)). The compound is commercially available from multiple sources.

[065] It has been reported in US 6,583,118 that chrysin may reduce loss of glycosaminoglycan content in chondrocyte matrix in the presence of a proteoglycan depleting agent, thus identifying chrysin as a potential chondroprotective agent. Using procedures based on those described in Example 2, chrysin has now been found to significantly increase the activity of the chondrogenic responsive reporter gene pGL3(4X48), indicating that it is also a chondrogenesis stimulating agent (Figure 2) that it is effective at low micromolar concentrations. At the single dosage that was tested, this compound did not significantly increase Alcian blue staining relative to controls, but it is expected that experiments with varying conditions (e.g., dosage, duration) will further support the chondrogenic activity indicated in the reporter assay. Example 11 : Chondrogenic Activity of Pinocembrin

[066] Pinocembrin, also known as 5,7-dihydroxyflavone, is a flavonoid compound that comes from European eucalyptus honeys. Martos et al., J. Agric. Food Chem. 48, 4744 (2000). It was isolated from propolis and identified as a bacteriostatic substance. Villaneuva et al., Ann. Inst. Pasteur 118, 84 (1970). Pinocembrin is also available from multiple commercial sources.

[067] It has been reported in US 6,583,118 that pinocembrin, like chrysin, may reduce loss of glycosaminoglycan content in chondrocyte matrix in the presence of a proteoglycan depleting agent and that consequently, pinocembrin may function as a chondroprotective agent. Using procedures based on those described in Example 2, pinocembrin has now been found to significantly increase the activity of the chondrogenic responsive reporter gene pGL3(4X48), indicating that it is also a chondrogenesis stimulating agent (Figure 2) that it is effective at low micromolar concentrations. At the single dosage that was tested, this compound did not significantly increase Alcian blue staining relative to controls, but it is expected that experiments with varying conditions (e.g., dosage, duration) will further support the chondrogenic activity indicated in the reporter assay. Example 12: Chondrogenic Activity of 7-hydroxyflavone

[068] 7-hydroxyflavone, also known as 7-hydroxy-2-phenylchromen-4-one, is present in many edible vegetables. It is a well characterized flavonoid compound, shown to act as an antimetastatic agent (Yang et al., Arch Oral Biol 53(3) :287- 94.(2008)), an aromatase inhibitor (Ta and WaIIe, Steroid Biochem MoI Biol 107(1- 2):127-9.(2007)), and a potent competitive inhibitor of xanthine oxidase, i.e, it is an antioxident (Mei-Ling et al., Free Radical Biology and Medicine 20(1):35-43 (1996)). 7-hydroxyflavone is commercially available.

[069] While it has not been specifically reported in US 6,583,118 that 7- hydroxy flavone will reduce loss of glycosaminoglycan content in chondrocyte matrix in the presence of a proteoglycan depleting agent, it is possible that this flavonoid may function as a chondroprotective agent. However, it has now been confirmed, using procedures based on those described in Example 2, that 7-hydroxyflavone significantly increases the activity of the chondrogenic responsive reporter gene pGL3(4X48), indicating that it is also a chondrogenesis stimulating agent (Figure 2) that it is effective at low micromolar concentrations. At the single dosage that was tested, this compound did not significantly increase Alcian blue staining relative to controls, but it is expected that experiments with varying conditions (e.g., dosage, duration) will further support the chondrogenic activity indicated in the reporter assay. Example 13: Chondrogenic Activity of 6-Methoxyluteolin

[070] 6-Methoxyluteolin, also known as nepetin, 3',4',5,7-tetrahydroxy-6- methoxyflavone, and 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-6-methoxychromen-4- one, has been isolated from Eupatorium arnottianum Griseb and Eupatorium ballotaefolium. 6-Methoxyluteolin has been shown to inhibit tumor cells. Militao et al., Pharmazie 59(12):965-6 (2004). It has also been shown to possess antiinflammatory properties. Clavin et al., Ethnopharmacol 112(3):585-9 (2007). 6- Methoxyluteolin is commercially available.

[071] Using procedures based on those described in Example 2, 6- methoxyluteolin was found to significantly increase the activity of the chondrogenic responsive reporter gene pGL3(4X48), indicating that it is a chondrogenesis stimulating agent (Figure 2). The PLM transfection assays show that low micromolar concentrations of 6-methoxyluteolin stimulate chondrogenic activity. At the single dosage that was tested, this compound did not significantly increase Alcian blue staining relative to controls, but it is expected that experiments with varying conditions (e.g., dosage, duration) will further support the chondrogenic activity indicated in the reporter assay. Example 14: Chondrogenic Activity of 6,7-dihydroxyflavone

[072] 6,7-Dihydroxyflavone, also known as 6,7-dihydroxy-2-phenylchromen- 4-one, is commercially available. It is reported to have a weak antibacterial effect on methicillin-resistant Staphylococcus aureus by itself, but has been shown to dramatically intensify the susceptibility of methicillin-resistant or -sensitive Staphylococcus aureus to beta-lactams. Sato et al., Antimicorb Agents Chemother 48(4): 1357-60 (2004). Using procedures based on those described in Example 2, 6,7-dihydroxyflavone was found to significantly increase the activity of the chondrogenic responsive reporter gene pGL3(4X48), indicating that it is a chondrogenesis stimulating agent (Figure 2). PLM transfection assays and Alcian blue staining assays indicate that low micromolar concentrations of 6,7- dihydroxyflavone stimulate chondrogenic activity.

Example 15: Chondrogenic Activity of Tamarixetine

[073] Tamarixetine, also known as 3,5,7-trihydroxy-2-(3-hydroxy-4- methoxyphenyl)chromen-4-one, is commercially available. Tamarixetine may be isolated from plants or synthesized as described by, e.g., Peng et al. Carbohydr Res 340(10): 1682-8 (2005). It is believed to possess antioxidant properties. Using procedures based on those described in Example 2, tamarixetine has now been found to significantly increase the activity of the chondrogenic responsive reporter gene pGL3(4X48), indicating that it is capable of stimulating chondrogenesis at low micromolar concentrations. At the single dosage that was tested, this compound did not significantly increase Alcian blue staining relative to controls, but it is expected that experiments with varying conditions (e.g., dosage, duration) will further support the chondrogenic activity indicated in the reporter assay.

Example 16: Chondrogenic Activity of Genistin

[074] Genistin, also known as 5-hydroxy-3-(4-hydroxyphenyl)-7- [(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one, is a flavonoid derivative. Genistin has been shown to promote the proliferation of primary mouse bone MSC and osteoblasts. Li et al., Acta Pharmacol Sin 26(9): 1081- 6 (2005). Genistin is also believed to have anticancer properties. Choi et al. Life Sci 80(15):1403-8 (2007).

[075] Using procedures based on those described in Example 2, genistin was found to significantly increase the activity of the chondrogenic responsive reporter gene pGL3(4X48), indicating that it is a chondrogenesis stimulating agent (Figure 2). The PLM transfection assays demonstrate that low micromolar concentrations of genistin stimulate chondrogenic activity. At the single dosage that was tested, this compound did not significantly increase Alcian blue staining relative to controls, but it is expected that experiments with varying conditions (e.g., dosage, duration) will further support the chondrogenic activity indicated in the reporter assay.

Example 17: Chondrogenic activity of 5-methoxyflavone

[076] 5-Methoxyflavone, also known as 5-methoxy-2-phenylchromen-4-one, is widely available from commercial sources. Large-scale synthesis of 5- methoxyflavone has been described by Ares et al., J. Org. Chem. 58(27):7903-05 (1993). Using procedures based on those described in Example 2, 5- methoxyflavone was found to significantly increase the activity of the chondrogenic responsive reporter gene pGL3(4X48), indicating that it is capable of stimulating chondrogenesis at low micromolar concentrations (Figure 2). At the single dosage that was tested, this compound did not significantly increase Alcian blue staining relative to controls, but it is expected that experiments with varying conditions (e.g., dosage, duration) will further support the chondrogenic activity indicated in the reporter assay.

Example 18: Chondrogenic Activity of Pratol

[077] Pratol, also known as 7-hydroxy-4-methoxyflavone and 7-hydroxy-2-(4- methoxyphenyl)chromen-4-one, is commercially available through a variety of sources. It has been described in WO/2001/003681 as useful to treat viral and parasitic infections. Using procedures based on those described in Example 2, pratol was found to significantly increase the activity of the chondrogenic responsive reporter gene pGL3(4X48), indicating that it is a chondrogenesis stimulating agent (Figure 2). The PLM transfection assays show that low micromolar concentrations of pratol stimulate chondrogenic activity. At the single dosage that was tested, this compound did not significantly increase Alcian blue staining relative to controls, but it is expected that experiments with varying conditions (e.g., dosage, duration) will further support the chondrogenic activity indicated in the reporter assay.

Example 19: Chondrogenic Activity of 5,6-Dehvdrokawain

[078] 5,6-Dehydrokawain, also known as 4-methoxy-6-[(E)-2-phenylethenyl] pyran-2-one, is commercially available. It may be isolated from Alpinia speciosa rhizoma. Teng et al., Chin J Physiol 33(1 ):41-8 (1990). It is believed to possess antiplatelet properties. Following procedures outlined in Example 2, 5,6- dehydrokawain was found to significantly increase the activity of the chondrogenic responsive reporter gene pGL3(4X48), indicating that it is capable of stimulating chondrogenesis at low micromolar concentrations. At the single dosage that was tested, this compound did not significantly increase Alcian blue staining relative to controls, but it is expected that experiments with varying conditions (e.g., dosage, duration) will further support the chondrogenic activity indicated in the reporter assay.

Example 20: Chondrogenic Activity of Harrinqtonine

[079] Harringtonine, also know as 4-methyl-, 2-hydroxy-2-(3-hydroxy-3- methylbutyl) butanedioate-cephalotaxine is a commercially available natural product that has been shown to possess anti-cancer properties. It has now been shown, using the procedures outlined in Example 2, that harringtonine significantly increases the activity of the chondrogenic responsive reporter gene pGL3(4X48), indicating that it is a chondrogenesis stimulating agent (Figure 2). The PLM transfection assays show that low micromolar concentrations of harringtonine stimulate chondrogenic activity. At the single dosage that was tested, this compound did not significantly increase Alcian blue staining relative to controls, but it is expected that experiments with varying conditions (e.g., dosage, duration) will further support the chondrogenic activity indicated in the reporter assay.

Example 21 : Chondrogenic Activity of Galanqine

[080] Galangine, also known as 3,5,7-trihydroxyflavone and 3,5,7-trihydroxy- 2-phenylchromen-4-one, is a mutagen believed to posses both anti-cancer and antibacterial properties. Using procedures based on those described in Example 2, galangine was found to significantly increase the activity of the chondrogenic responsive reporter gene pGL3(4X48), indicating that it is capable of stimulating chondrogenesis at low micromolar concentrations (Figure 2). At the single dosage that was tested, this compound did not significantly increase Alcian blue staining relative to controls, but it is expected that experiments with varying conditions (e.g., dosage, duration) will further support the chondrogenic activity indicated in the reporter assay.

Example 22: Chondrogenic Activity of Kaempferol

[081] Kaempferol, also known as 3,5,7-trihydroxy-2-(4- hydroxyphenyl)chromen-4-one, is commercially available and may be isolated from a variety of plants. Kaempferol is believed to possess a number of biological activities, including antioxidant, antimicrobial, anti-inflammatory, and anti-cancer activities. Using procedures based on those described in Example 2, kaempferol was found to significantly increase the activity of the chondrogenic responsive reporter gene pGL3(4X48), indicating that it is a chondrogenesis stimulating agent (Figure 2). These PLM transfection assays, as well as Alcian blue staining experiments, indicate that low micromolar concentrations of kaempferol stimulate chondrogenic activity. Example 23: Combinations of Phenazopyridine Hydrochloride and Pro-chondrogenic Compounds Increase Pro-chondrogenic Activity

[082] High-density murine limb bud-derived cultures were established as previously described (Hoffman 2006). Media was replaced every two days. Cells were transfected on day 0 with pGL3(4X48) (reporter) and phRL-SV40 (to normalize transfection efficiency) plasmids using Effectene transfection reagent (Qiagen). Twenty-four hours after transfection, cells were treated with the following final concentrations of compounds, either individually or in combination: BMP4 (20 ng/ml), phenazopyridine hydrochloride (PHCL) (10 μM), and the pan-RAR antagonist 4310 (100 nM). Lysates were collected at 24h and 72h after treatment. Luciferase activity was measured using the Dual-Luciferase^® kit according to the manufacturer's instructions (Promega). Reporter activity was normalized to untreated controls (-) on the same day. Results of an experiment performed in triplicate are shown in Figure 4.

[083] PHCL, 4310, and BMP4 all increased reporter gene activity individually. Reporter gene activity was further increased by combinations of PHCL with 4310 or BMP4. These results demonstrate that PHCL, either alone or in combination with BMP4 or 4310, significantly increases pGL3(4X48) reporter activity, indicating its effectiveness as a pro-chondrogenic compound. Example 24: Phenazopyridine Hydrochloride and Pan-RAR Antagonist 4310 Reduce Mmp13 Expression in Limb Mesenchymal Cultures

[084] MMP13 is a major contributor to cartilage degradation in osteoarthritis, and inhibiting its activity is of therapeutic interest. RAR antagonists promote chondrocyte differentiation and inhibit expression of a hypertrophic phenotype. BMPs stimulate both chondrogenesis and chondrocyte hypertrophy. Accordingly, in this study, 4310 and BMP4 were used as negative and positive controls for chondrocyte hypertropertrophy-inducing activity, respectively.

[085] Murine limb bud-derived high-density cultures were established as previously described (Hoffman 2006). Media was replaced every three days. After 24 hours of culture, compounds were added at the following final concentrations: BMP4 (20 ng/ml), phenazopyridine hydrochloride (PHCL, #7) (10 μM), and 4310 (100 nM). After 72 hours of culture, ascorbic acid and beta-glycerol phosphate were added to the culture at final concentrations of 126 μM and 1 mM, respectively. RNA was isolated from cultures at five and ten days. Gene expression was assessed using real-time PCR with TaqMan^® probe and primer sets (Applied Biosystems,Taqman Gene Expression Assay Mm00439491_m1). All gene expression levels were normalized to the 5-day control (set at 100%). Results from an experiment performed in duplicate are shown in Figure 5.

[086] Control cells exhibited an approximately three-fold increase in Mmp13 expression between 5 and 10 days. Consistent with previous reports, BMP4 increased the expression of Mmp13. Phenazopyridine hydrochloride and 4310 both markedly decreased the expression of Mmp13, indicating that they have chondroprotective activity. Thus, these compounds are attractive alternatives to decrease MMP13 activity, without the side-effects associated with MMP inhibitors.

Claims

WHAT IS CLAIMED IS:

1. A method for stimulating chondrogenesis comprising administering to a subject in need thereof a pharmaceutical composition comprising an amount of at least one compound selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, tiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, 6,7-dihydroxyflavone, tamarixetine, genistin, 5- methoxyflavone, pratol, 5,6-dehydrokawain, harringtonine, galangine, and kaempferol effective to stimulate chondrogenesis.

2. A method for preventing chondrocyte hypertrophy or maturation comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a compound selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, thiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, 6,7- dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6- dehydrokawain, harringtonine, galangine, and kaempferol.

3. A method for treating a chondrogenic disease comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of a compound selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, thiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, 6,7-dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6-dehydrokawain, harringtonine, galangine, and kaempferol.

4. The method of claim 3, wherein the chondrogenic disease is selected from the group consisting of chondromalacia, chondrodysplasias, osteochondritis, congenital cartilage disease, osteochondritis dessecans, degenerative or fibrotic joint disease, rheumatoid arthritis, osteoarthritis, and polychondritis.

5. A method for treating or repairing a cartilage defect comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a compound selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, thiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, 6,7-dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6-dehydrokawain, harringtonine, galangine, and kaempferol.

6. The method of claim 5, wherein the cartilage defect is selected from a group consisting of articular cartilage tears, congenital cartilage defects, and cartilage damage induced by bone fractures.

7. A method of treating, ameliorating or repairing a skeletal defect, a large segmental skeletal gap or a non-union fracture arising from trauma or surgery comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a compound selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, thiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, 6,7- dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6- dehydrokawain, harringtonine, galangine, and kaempferol.

8. The method of claim 7, wherein the composition is provided at the site of surgery, and wherein the composition mediates the formation of new bone tissue.

9. The method of claim 7, wherein the composition is delivered at the site of the segmental skeletal gap or non-union fracture, and wherein the composition mediates the formation of new bone tissue.

10. A method for the ex vivo engineering of chondrocytes comprising:

(a) culturing a population of precursor cells of chondrocyte lineage with a composition comprising a compound selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, thiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, 6,7-dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6-dehydrokawain, harringtonine, galangine, and kaempferol for a time sufficient to stimulate chondrogenesis; and

(b) implanting the cells from (a) into a desired site in a subject.

11. The method of claim 10, wherein the cells from step (a) are applied to an implantable device selected from the group consisting of a mechanical physical device, biodegradable carrier; biodegradable synthetic carrier, prostheses, demineralized allogenic bone and demineralized xenogenic bone, and implanted into the desired site.

12. The method of any one of claims 1 to 11 , wherein the subject is a mammal.

13. The method of any one of claims 1 to 7, wherein the composition is administered systemically.

14. The method of any one of claims 1 to 7, wherein the composition is administered locally.

15. The method of any one of claims 1 to 9, wherein the composition is administered by injection.

16. The method of any one of claims 1 to 9, wherein the composition is administered as a coating on a device or implant.

17. The method of any one of claims 1 to 9, wherein said composition is used in conjunction with an implantable device selected from the group consisting of a mechanical physical device, biodegradable carrier, biodegradable synthetic carrier, prostheses, demineralized allogenic bone, and demineralized xenogenic bone.

18. The method of any one of claims 1 to 17, wherein the composition further comprises at least one other therapeutic agent.

19. The method of claim 18, wherein at least one other therapeutic agent is a chondrogenesis inducing agent.

20. The method of claim 19, wherein the chondrogenesis inducing agent is selected from BMPs, GDFs, FGFs, WNTs, Hedgehog, MIA or RAR antagonists.

21. The method of claim 18, wherein at least one other therapeutic agent is a chondroprotective agent.

22. The method of claim 21 , wherein the chondroprotective agent is selected from IL-1 receptor antagonists, TNF receptor antagonists, COX-2 inhibitors, MAP kinase inhibitors, nitric oxide synthase inhibitors, and NFkB inhibitors.

23. The method of any one of claims 1 to 16, wherein the composition is administered simultaneously or sequentially with a chondrogenesis inducing agent and/or a chondroprotective agent.

24. The method according to any one of claims 1 to 23, wherein the compound is selected from thiabendazole, indoprofen, oxybenzone, 5,6- dehydrokawain, harringtonine, phenazopyridine, cytochalasin B, rhapontin, and methysticin.

25. A method for stimulating chondrogenesis comprising administering to a subject in need thereof a pharmaceutical composition comprising an amount of at least one flavonoid effective to stimulate chondrogenesis.

26. The method of claim 25, wherein the flavonoid is chrysin, pinocembrin, 7- hydroxyflavone, 6-methoxyluteolin, tamarixetine, genistin, 5-methoxyflavone, pratol, galanganine, kaempferol, and 6,7-dihydroxyflavone.

27. Use of at least one compound selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, tiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, 6,7- dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6- dehydrokawain, harringtonine, galangine, and kaempferol in the preparation of a medicament for stimulating chondrogenesis in a subject in need thereof.

28. At least one compound selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, tiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, 6,7-dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6-dehydrokawain, harringtonine, galangine, and kaempferol for stimulating chondrogenesis in a subject in need thereof.

29. Use of at least one compound selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, tiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, 6,7- dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6- dehydrokawain, harringtonine, galangine, and kaempferol in the preparation of a medicament for preventing chondrocyte hypertrophy or maturation in a subject in need thereof.

30. At least one compound selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, tiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, 6,7-dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6-dehydrokawain, harringtonine, galangine, and kaempferol for preventing chondrocyte hypertrophy or maturation in a subject in need thereof.

31. Use of at least one compound selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, tiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, 6,7- dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6- dehydrokawain, harringtonine, galangine, and kaempferol in the preparation of a medicament for treating a chondrogenic disease in a subject in need thereof.

32. At least one compound selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, tiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, 6,7-dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6-dehydrokawain, harringtonine, galangine, and kaempferol for treating a chondrogenic disease in a subject in need thereof.

33. Use of at least one compound selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, tiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, 6,7- dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6- dehydrokawain, harringtonine, galangine, and kaempferol in the preparation of a medicament for treating or repairing a cartilage defect in a subject in need thereof.

34. At least one compound selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, tiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, 6,7-dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6-dehydrokawain, harringtonine, galangine, and kaempferol for treating or repairing a cartilage defect in a subject in need thereof.

35. Use of at least one compound selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, tiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, 6,7- dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6- dehydrokawain, harringtonine, galangine, and kaempferol in the preparation of a medicament for treating, ameliorating or repairing a skeletal defect, a large segmental skeletal gap or a non-union fracture arising from trauma or surgery in a subject in need thereof.

36. At least one compound selected from phenazopyridine hydrochloride, cytochalasin B, methysticin, rhapontin, tiabendazole, indoprofen, oxybenzone, chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, 6,7-dihydroxyflavone, tamarixetine, genistin, 5-methoxyflavone, pratol, 5,6-dehydrokawain, harringtonine, galangine, and kaempferol for treating, ameliorating or repairing a skeletal defect, a large segmental skeletal gap or a non-union fracture arising from trauma or surgery in a subject in need thereof.

37. The use of any one of claims 29, 31 , 33, or 35, or the compound of any one of claims 30, 32, 34, or 36, wherein the compound is selected from thiabendazole, indoprofen, oxybenzone, 5,6-dehydrokawain, harringtonine, phenazopyridine, cytochalasin B, rhapontin, and methysticin.

38. Use of a flavonoid in the preparation of a medicament for stimulating chondrogenesis in a subject in need thereof.

39. A flavonoid for stimulating chondrogenesis in a subject in need thereof.

40. The use of claim 38 or the flavenoid of claim 39, wherein the flavonoid is chrysin, pinocembrin, 7-hydroxyflavone, 6-methoxyluteolin, tamarixetine, genistin, 5- methoxyflavone, pratol, galanganine, kaempferol, and 6,7-dihydroxyflavone.