Compositions and methods for treating multiple osteochondroma and chondrogenesis-related diseases

Abstract

Compositions comprising at least one retinoid acid receptor agonist are disclosed. Methods for inhibiting, treating, and / or preventing a chondrogenesis-related disease or disorder in a subject, comprising administering to a subject at least one retinoid acid receptor (RAR) agonist are further disclosed.

Description

[0001] COMPOSITIONS AND METHODS FOR TREATING MULTIPLE OSTEOCHONDROMA AND CHONDROGENESIS-RELATED DISEASES By

[0002] Maurizio Pacifici

[0003] Christina Mundy

[0004] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 762,370, filed February 24, 2025. The foregoing application is incorporated by reference herein.

[0005] FIELD OF THE INVENTION

[0006] The present invention relates to the fields of chondrogenesis and skeletal growth. More specifically, the invention provides compositions and methods for inhibiting chondrogenesis and the treatment of related diseases.

[0007] BACKGROUND OF THE INVENTION

[0008] Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

[0009] Benign ectopic cartilaginous / bony tumors called osteochondromas or exostoses characterize the pediatric skeletal disorder multiple osteochondroma (MO) - also known as hereditary multiple osteochondromas (HMO) or hereditary multiple exostoses (HME) (Jones, K.B. (2011) J. Pediatr. Orthop., 31:577-86; Porter et al. (1999) J. Pathol., 188: 119-25). The osteochondromas are growth plate-like structures that form next to, but never within, the growth plates of long bones, vertebrae, ribs, pelvis and other skeletal elements. Because of size and location, the osteochondromas can cause a variety of health problems including skeletal growth retardation and deformities, chronic pain, compression of nerves and blood vessels, and psychological concerns (Jones, K.B. (2011) J. Pediatr. Orthop., 31:577-86; Goud et al. (2012) J. Bone Joint Surg. Am., 94:1013-20; Hosalkar et al. (2007) J. Pediatr. Orthop., 27:333-7). In most MO patients, the osteochondromas remain benign through life, but in about 2 to 5% of them the osteochondromas progress to malignancy, turn into osteosarcomas or chondrosarcomas and thus become life-threatening (Bovee et al. (1999) Am. J. Hum. Genet., 65:689-98). Most MO patients carry heterozygous loss-of-function mutations in EXT1 or EXT2 that encode glycosyltransferases responsible for heparan sulfate (HS) synthesis (Esko et al. (2002) Annu. Rev. Biochem., 71:435-71; Jennes et al. (2009) Hum. Mutat., 30:1620-7).EXT1 and EXT2 form protein complexes in the Golgi and are both required for HS synthesis (Esko et al. (2002) Annu. Rev. Biochem., 71:435-71). MO patients thus have reduced levels - but not lack - of HS in their tissues. Puzzlingly however, the cartilaginous portions of human osteochondromas display very low levels of HS (Hecht et al. (2005) Differentiation 73:212-21), indicating that osteochondroma formation may require a more severe loss of HS beyond what would be caused by a mere EXT haploinsufficiency (Reijnders et al. (2010) Am. J. Path., 177:1946-57; Stickens et al. (2005) Development. 132:5055-68). This requirement has been confirmed in transgenic mouse studies involving conditional Extl and / or Ext2 ablation (Jones et al. (2010) Proc. Natl. Acad. Sci., 107:2054-9; Matsumoto et al. (2010) Proc. Natl. Acad. Sci., 107:10932-7; Zak et al. (2011) 48:979-987). Studies have suggested mechanisms that could account for a severe drop of HS in human osteochondromas including EXT loss-of-heterozygosity, large and encompassing genomic deletions, a second hit in another gene, and background genetic traits such as modifiers (Jennes et al. (2009) Hum. Mutat., 30:1620-7; Waaijer et al. (2013) Genes Chromosomes Cancer 52:431-6; Wuyts et al. (2000) Hum. Mutat., 15:220-7). However, there are still no clear answers nor obvious genotype-phenotype correlations in MO, even though the syndrome can vary significantly in severity within affected family members and amongst individuals from different families (Pedrini et al. (2011) J. Bone Joint Surg., 93:2294-2302).

[0010] Currently, surgery is the only treatment available to patients, but it can only be used to remove accessible tumors, with many - if not most - left in place causing lifelong health problems. Thus, a drug therapy would be highly desirable because it could inhibit tumor formation and avoid the need for invasive and dangerous surgeries in addition to alleviating long term physical problems and reduce the possibility of malignancy.

[0011] SUMMARY OF THE INVENTION

[0012] In accordance with the instant invention, methods for inhibiting, treating, and / or preventing a chondrogenesis-related disease or disorder, such as multiple osteochondroma, in a subject (e.g., female or male) are provided. Methods for inhibiting or preventing chondrogenesis and / or osteochondroma or exostosis formation and / or growth in a subject are also provided. The methods of the instant invention comprise administering to a subject at least one retinoid acid receptor (RAR) agonist. In certain embodiments, the RAR agonist is a RAR gamma agonist. In certain embodiments, the RAR gamma agonist is trifarotene.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 provides alcian blue-stained images of micromass cultures 3 days or 6 days after treatment with trifarotene (50 nM or 100 nM) or vehicle (control).

[0014] Figure 2 provides graphs showing the expression of chondrogenic genes Sox9, Acan, and Col2al in micromass cultures 3 days or 6 days after treatment with trifarotene (50 nM or 100 nM) or vehicle (control).

[0015] Figure 3 shows whole body images of male mice receiving administration of vehicle or trifarotene, with arrows pointing to skin and mouth.

[0016] Figure 4 shows whole body images of female mice receiving administration of vehicle or trifarotene, with arrows pointing to skin and mouth.

[0017] Figure 5 shows measurements of body weight in male (top) and female (bottom) mice during the duration of the treatment.

[0018] Figure 6 provides pCT images of knee from control Ext 1^ or mutant Extl^;Acan-CreER2male mice treated with vehicle (control) or trifarotene (0.75 mg / kg or 3 mg / kg). Femur (fe), tibia (ti), and fibula (fi) are shown. Most prominent osteochondromas are indicated with arrowheads.

[0019] Figure 7 provides pCT images of ribs from control Ext 1^ or mutant Extl^;Acan-CreER12male mice treated with vehicle (control) or trifarotene (0.75 mg / kg or 3 mg / kg). Location of osteochondromas is indicated by arrowheads.

[0020] Figure 8 provides pCT images of knee from control Ext 1^ or mutant Extl^;Acan-CreER12female mice treated with vehicle (control) or trifarotene (0.75 mg / kg or 3 mg / kg). Femur (fe), tibia (ti), and fibula (fi) are shown. Most prominent osteochondromas are indicated with arrowheads.

[0021] Figure 9 provides pCT images of ribs from control Ext 1^ or mutant Extl^;Acan-CreER2female mice treated with vehicle (control) or trifarotene (0.75 mg / kg or 3 mg / kg). Location of osteochondromas is indicated by arrowheads.

[0022] Figure 10A provides alcian blue and nuclear fast red stained images of femur from control Ext mice or mutant Extl^Acan-CreER12male and female mice treated with vehicle (control) or trifarotene (0.75 mg / kg or 3 mg / kg). Bottom row images represent a magnification of box areas in top row images. Figure 10B provides measurements of length and width of osteochondroma on the femur in control Extl^ mice or mutant Ext 1^; A can-CreER12mice treated with vehicle (control) or trifarotene (0.75 mg / kg or 3 mg / kg).Figure 11 A provides alcian blue and nuclear fast red stained images of tibia from control / A / / " mice or mutant Extl^Acan-CreER12male and female mice treated with vehicle (control) or trifarotene (0.75 mg / kg or 3 mg / kg). Bottom row images represent a magnification of box areas in top row images. Figure 1 IB provides measurements of length and width of osteochondroma on the femur in control Extl^ mice or mutant Ext 1^; A can-CreER12mice treated with vehicle (control) or trifarotene (0.75 mg / kg or 3 mg / kg).

[0023] Figure 12A provides alcian blue and nuclear fast red stained images of ribs from control / A / / " mice or mutant Extl^Acan-CreER22male and female mice treated with vehicle (control) or trifarotene (0.75 mg / kg or 3 mg / kg). Bottom row images represent a magnification of box areas in top row images. Fig. 12B provides measurements of length and width of osteochondroma on the femur in control / A / / " mice or mutant Extl^;Acan-CreER12mice treated with vehicle (control) or trifarotene (0.75 mg / kg or 3 mg / kg).

[0024] DETAILED DESCRIPTION OF THE INVENTION Osteochondroma formation starts with cartilage development that is then partially replaced by bone, recapitulating the process by which most of the skeleton forms and grows normally. The retinoid signaling pathway negatively regulates cartilage formation via the activation of nuclear retinoid acid receptors (RARs) by ligands. The RAR gamma isoform is particularly important in this process. Herein, the RAR gamma agonist trifarotene is shown to be a preventative treatment against osteochondroma development in a mouse model of multiple osteochondroma. Trifarotene was approved for the treatment of acne in 2020 (Aklief® (Galderma); Selagmis® (Papaloisou)). Trifarotene was tested in mice with multiple osteochondroma by oral administration and it was determined to prevent osteochondroma formation in skeletal elements including long bones and ribs. In addition, trifarotene was tested in an in vitro cell micromass system and it was determined to inhibit chondrogenesis as measured by cytochemistry and gene expression. These results indicate that retinoid acid receptor agonists, particularly RAR gamma agonists such as trifarotene, can be used as a treatment for multiple osteochondroma.

[0025] The instant invention encompasses methods of inhibiting, treating, and / or preventing chondrogenesis (particularly aberrant, excessive, and / or improper chondrogenesis) and / or osteochondroma or exostosis formation. The instant invention also encompasses methods of treating, inhibiting, and / or preventing diseases or disordersassociated with aberrant, excessive, or improper chondrogenesis and / or osteochondroma or exostosis formation. Examples of such chondrogenesis related diseases and disorders associated with osteochondroma or ectopic cartilage formation include, without limitation: multiple osteochondroma (MO) (hereditary multiple osteochondromas (HMO) - also known as hereditary multiple exostoses (HME)), congenital conditions such as metachondromatosis (characterized by both osteochondromas and enchondromas), fibrodysplasia ossificans progressiva (characterized by heterotopic ossification and osteochondromas), ectopic chondrogenesis (e.g., during osteophyte formation in osteoarthritis), chondrogenic transdifferentiation (e.g., in tendons after rapture / damage), solitary or multiple enchondromas (benign tumors characterizing disorders such as Ollier’s disease), and ectopic chondrogenesis in perispinal ligaments and / or arteries such as aorta (e.g., occurring in PPi deficiency or in patients taking warfarin or other anticoagulants; see, e.g., Johnson et al. (2005) Arterioscler. Thromb. Vase. Biol., 25:686-691). In certain embodiments, the chondrogenesis related disease or disorder is MO.

[0026] The methods of the instant invention comprise administering at least one RAR agonist, particularly at least one RAR gamma agonist, to a subject in need thereof.

[0027] Examples of RAR agonists, particularly RAR gamma agonists, include, without limitation: trifarotene (CAS 895542-09-3), palovarotene (CAS 410528-02-8), acacetin (5,7-dihydroxy-4-methoxyflavone), CD1530 (4-(6-Hydroxy-7-tricyclo[3.3.1.13,7]dec-l-yl-2-naphthalenyl)benzoic acid), CD437 (6-(4-hydroxy-3-tricyclo[3.3.1.13,7]dec-l-ylphenyl)-2-naphthalenecarboxylic acid), all-trans retinoic acid (ATRA), AM580 (CAS 102121-60-8), 9-cis-retinoic acid, TTNPB (4-[(E)-2-(5, 6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-l-propenyl]benzoic acid), AC261066 (CAS 870773-76-5), SRI 1237 (CAS 146670-40-8), BMS961 (CAS 185629-22-5), IRX4647, and pharmaceutically acceptable salts thereof. U.S. Patent No. 7,807,708 (incorporated herein by reference) also describes RAR agonists, particularly RAR gamma agonists. In certain embodiments, the RAR agonist is selective for RAR gamma. In certain embodiments, the RAR gamma agonist is trifarotene.

[0028] In a particular embodiment of the present invention, the RAR agonist (e.g., RAR gamma agonist) of the instant invention may be administered to a patient in a pharmaceutically acceptable carrier. The RAR agonist (e.g., RAR gamma agonist) of the instant invention may optionally be encapsulated into liposomes or mixed with other phospholipids or micelles to increase stability of the molecule. The RAR agonist (e.g., RAR gamma agonist) may be administered alone or in combination with other agentsknown to inhibit chondrogenesis and / or osteochondroma or exostosis formation or treat, inhibit, and / or prevent diseases or disorders associated with improper chondrogenesis and / or osteochondroma or exostosis formation (e.g., another anti-chondrogenic agent such as another RAR agonist). In certain embodiments, the other agent (anti-chondrogenesis agent is a heparanase inhibitor such as a modified heparin (e.g., roneparstat) (see, e.g., WO 2016 / 036782, incorporated herein by reference). The compounds may be contained in the same composition or may be in a separate composition. The compositions may be administered concurrently or consecutively.

[0029] The RAR agonist (e.g., RAR gamma agonist) (or composition(s) comprising the same) can be administered by any suitable route, for example, by injection (e.g., for local, direct, or systemic administration), oral, pulmonary, topical, nasal or other modes of administration. The composition may be administered by any suitable means, including parenteral, intramuscular, intravenous, intravascular, intraarterial, intraperitoneal, subcutaneous, topical, inhalatory, transdermal, intrapulmonary, intraareterial, intrarectal, intramuscular, and intranasal administration. In a particular embodiment, the composition is administered orally. In a particular embodiment, the composition is administered subcutaneously. In a particular embodiment, the composition is administered intraperitoneally. In certain embodiments, the composition is administered systemically. In certain embodiments, the composition is administered directly to the site of chondrogenesis (e.g., by injection). In general, the pharmaceutically acceptable carrier of the composition is selected from the group of diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. The compositions can include diluents of various buffer content (e.g., Tris HC1, acetate, phosphate), pH and ionic strength; and additives such as detergents and solubilizing agents (e.g., polysorbate 80), anti oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). The compositions can also be incorporated into particulate preparations of polymeric compounds such as polyesters, polyamino acids, hydrogels, polylactide / glycolide copolymers, ethylenevinylacetate copolymers, polylactic acid, polygly colic acid, etc., or into liposomes. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of components of a pharmaceutical composition of the present invention (see, e.g., Remington’s Pharmaceutical Sciences and Remington: The Science and Practice of Pharmacy). The pharmaceutical composition of the present invention can beprepared, for example, in liquid form, or can be in dried powder form (e.g., lyophilized for later reconstitution).

[0030] The therapeutic agents described herein will generally be administered to a subject as a pharmaceutical preparation. The compositions of the instant invention may be employed therapeutically or prophylactically, under the guidance of a physician.

[0031] The compositions comprising the RAR agonist (e.g., RAR gamma agonist) of the instant invention may be conveniently formulated for administration with any pharmaceutically acceptable carrier(s). The concentration of agent in the chosen medium may be varied and the medium may be chosen based on the desired route of administration of the pharmaceutical preparation. Except insofar as any conventional media or agent is incompatible with the agent to be administered, its use in the pharmaceutical preparation is contemplated.

[0032] The dose and dosage regimen of the agent according to the invention that is suitable for administration to a particular patient may be determined by a physician considering the patient’s age, sex, weight, general medical condition, and the specific condition for which the agent is being administered to be treated or prevented and the severity thereof. The physician may also take into account the route of administration, the pharmaceutical carrier, and the agent’s biological activity. Selection of a suitable pharmaceutical preparation will also depend upon the mode of administration chosen.

[0033] A pharmaceutical preparation of the invention may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment or prevention therapy. Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art.

[0034] Dosage units may be proportionately increased or decreased based on the weight of the patient. Appropriate concentrations for alleviation or prevention of a particular condition may be determined by dosage concentration curve calculations, as known in the art.

[0035] The pharmaceutical preparation comprising the agent may be administered at appropriate intervals until the pathological symptoms are reduced or alleviated, after which the dosage may be reduced to a maintenance level. The appropriate interval in a particular case would normally depend on the condition of the patient. Toxicity andefficacy (e.g., therapeutic, preventative) of the particular formulas described herein can be determined by standard pharmaceutical procedures such as, without limitation, in vitro, in cell cultures, ex vivo, or on experimental animals. The data obtained from these studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon form and route of administration. Dosage amount and interval may be adjusted individually to levels of the active ingredient which are sufficient to deliver a therapeutically or prophylactically effective amount.

[0036] Definitions

[0037] The following definitions are provided to facilitate an understanding of the present invention:

[0038] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0039] As used herein, the terms “host,” “subject,” and “patient” refer to any animal, particularly mammals including humans.

[0040] “Pharmaceutically acceptable” indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0041] A “carrier” refers to, for example, a diluent, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., polysorbate 80), emulsifier, buffer (e.g., Tris HC1, acetate, phosphate), antimicrobial, bulking substance (e.g., lactose, mannitol), excipient, auxiliary agent or vehicle with which an active agent of the present invention is administered.

[0042] Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions may be employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin (Mack Publishing Co., Easton, PA); Gennaro, A. R., Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and Kibbe, et al., Eds., Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, Washington.The term “treat” as used herein refers to any type of treatment that imparts a benefit to a patient afflicted with a disease, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc.

[0043] As used herein, the term “prevent” refers to the prophylactic treatment of a subject who is at risk of developing a condition (e.g., MO) resulting in a decrease in the probability that the subject will develop the condition.

[0044] A “therapeutically effective amount” of a compound or a pharmaceutical composition refers to an amount effective to prevent, inhibit, or treat a particular disorder or disease and / or the symptoms thereof. For example, “therapeutically effective amount” may refer to an amount sufficient to modulate chondrogenesis and / or osteochondroma or exostosis formation in a subject.

[0045] The following example is provided to illustrate various embodiments of the present invention. The example is illustrative and is not intended to limit the invention in any way.

[0046] EXAMPLE

[0047] Preparation, treatment and analysis of micromass cultures

[0048] Micromass cultures were prepared from mouse embryo limb buds as described (Sinha, et al. (2017) PLoS Genetics, 13:el006742). Briefly, limb bud mesenchyme was dissociated in 0.5% trypsin-EDTA at 37°C. The dissociated cells were suspended at a concentration of 10 x 106cells / ml in DMEM containing 3% fetal bovine serum and antibiotics. Micromass cultures were initiated by spotting 15 pl of the cell suspensions (1.5 x 105cells) onto the surface of 24-well tissue culture plates. After a 2 hour incubation at 37°C in a humidified CO2 incubator to allow for cell attachment, the cultures were given 500 pl of medium. After 24 hours, medium supplemented with trifarotene (50 nM or 100 nM) was added to the cultures. Fresh drug was given with medium change every other day. Equivalent amounts of vehicle were added to control cultures. Cultures were stained with alcian blue (pH 1.0) after 3 or 6 days to monitor chondrogenic cell differentiation (Huegel et al. (2013) Dev. Biol., 377:100-12). Images were taken with a Nikon SMZ-U microscope equipped with a SPOT insight camera (Diagnostic Instruments, Inc.) and acquired with SPOT 4.0 software. Micromass analysis was performed using ImageJ. Images were made binary under an RGB threshold and“Particle Analysis” was utilized to measure alcian blue positive area (Gutierrez, et al. (2012) Universitas Scientiarum., 17:167-78).

[0049] Gene expression analysis

[0050] Total RNA was isolated from control and trifarotene-treated micromass cultures on day 3 or 6 using TRIzol™ reagent (cat# 15596-026, Life Technologies) according to the manufacturer’s protocol. RNA quantification was determined by Nanodrop™. One microgram total RNA was reversed transcribed using the Verso™ cDNA kit (cat# AB1435 / A, Thermo Scientific). Quantitative real-time PCR was carried out using SYBR® Green PCR Master Mix in an Applied Biosystems 7500 according to manufacturer’s protocol. Gapdh was used as the endogenous control and relative expression was calculated using the AACt method.

[0051] Transgenic mouse lines, husbandry and drug treatment

[0052] Commercially available mice (JAX Labs) bearing an inducible loss-of-function allele of Extl in which exon 2 is flanked by head-to-head loxP sites (heretofore referred to as / M / " mice) have been described (Jones, et al. (2010) Proc. Natl. Acad. Sci., 107:2054-2059). All mice were housed in conventional housing (5 mice per cage) and given unlimited access to food and water. Shepard shacks were added to the cages for environmental enrichment. The mice were mated with commercially available Agr-CreERr2transgenic mice (JAX Labs) expressing tamoxifen-inducible CreER recombinase under the control of aggrecan (Acan or Agr) enhancer sequences (Henry, et al. (2009) Genesis 47:805-814) to eventually generate compound Extl^; Acan-CreER22mice. At 5 weeks of age, the Extl^; Acan-CreER22mice were given a single intraperitoneal injection of tamoxifen (1 mg / 13.3grs per body weight); stock solution was 20 mg / ml in ethanol: com oil mixture at 1 :4 ratio. Mice were euthanized at 4-, 6- or 8-weeks post tamoxifen injection to assess osteochondroma development. A total number of 3-5 mice were used per group to evaluate phenotype (Mundy, et al. (2022) J. Orthop. Res., 40:2391-2401; Sinha, et al. (2017) PLoS Genetics, 13 :el 006742).

[0053] Trifarotene was purchased from MedChemExpress (Monmouth Junction, NJ). The drug was dissolved in DMSO at 10 mg / ml stock solution. Aliquots were prepared and stored at -20°C. Fresh aliquots were used every two weeks. On the day of treatment, an aliquot was thawed and mixed with the appropriate amounts of DMSO and com oil to generate a working solution. This was used for two weeks and then replaced with a newone. Sufficient numbers of mice per experiment were placed in separate cages and randomized into control and experimental groups to minimize bias. Ext fiAcan-CreER12mice were then given Trifarotene at 0.75 or 3 mg / kg by oral gavage 5 days per week. Companion mice were given the vehicle mixture of DMSO and corn oil and served as controls. Each subgroup contained 3 to 5 mice per experiment. Treatment started one day after tamoxifen injection. Before gavage, mice were anesthetized by inhalation of 1.5% isoflurane in 98.5% oxygen to minimize discomfort as prescribed by IACUC. Oral gavage probes were 22 gauge and 25 mm long (Instech Laboratories, Plymouth Meeting, PA). After drug administration, animals were regularly monitored for signs of overt toxicity, including lethargy and dystonia, and Institutional LAS personnel provided additional daily animal monitoring and care.

[0054] Micro computed tomography (pCT) and histochemical analyses

[0055] Ribs and long bones were fixed for 72 hours in 4% paraformaldehyde, washed with IX PBS and stored in ethanol for scanning. Ribs and long bones were scanned using a pCT45 scanner (Scanco Medical AG, Switzerland) and analyzed using manufacturer’s issued software. Serial 21 pm 2D and 3D images were acquired at 55 kVp energy, 145 pA intensity and integration time of 200 msec. Raw pCT data were compiled into 2D gray scale images. Ribs and long bones were contoured, and binary images were generated using thresholds of 200 and 260, respectively. After imaging procedures, samples were decalcified and embedded in paraffin. Serial 5 pm sections were stained with alcian blue (a dye that stains cartilage by binding to acidic polysaccharides) and nuclear fast red and processed for histochemical osteochondroma analyses as described (Sinha, et al. (2017) PLoS Genetics, 13:el006742).

[0056] Osteochondroma quantification

[0057] Histological images of ribs were loaded into ImageJ software and pixel dimensions were converted to millimeters (mm). Regions of interest were drawn around the bony and cartilaginous areas of the osteochondromas from multiple ribs per animal to measure the areas. Surface areas were analyzed using Prism 9 (GraphPad Software Inc.). Student’s t-test and one-way analysis of variance (ANOVA) were used to establish statistical significance. Threshold for significance for all tests was set as p < 0.05.Results

[0058] Figure 1 provides alcian blue-stained images of micromass cultures 3 days or 6 days after treatment with trifarotene (50 nM or 100 nM) or vehicle (control). Alcian blue is a stain specific for cartilage. Comparison of treatment images with control images show that trifarotene significantly decreases cartilage nodule formation.

[0059] Figure 2 shows the results of gene expression analysis and verifies that trifarotene treatment dramatically inhibited expression of chondrogenic master gene Sox9 and cartilage matrix markers aggrecan (Acan) and collagen type II alpha- 1 chain (Col2al) at both doses used compared to vehicle control.

[0060] Figure 3 shows whole body images of male mice receiving administration of vehicle or trifarotene. The arrows point to skin affected by trifarotene treatment as depicted by some hair loss and redness.

[0061] Figure 4 shows whole body images of female mice receiving administration of vehicle or trifarotene. Arrows point to skin and mouth locations similar to those investigated in male mice but showing no obvious sign of drug effects in the female mice.

[0062] Figure 5 shows measurements of body weight in male and female mice during the duration of the treatment. The mice receiving trifarotene were slightly smaller than controls, but their body weight did not change appreciably during the entire treatment period.

[0063] Figure 6 provides pCT images of knee from male control Ext

[0064]

[0065] or mutant Ext 1^; A can-CreER12mice treated with vehicle (control) or trifarotene (0.75 mg / kg or 3 mg / kg). Femur (fe), tibia (ti), and fibula (fi) are shown. Most prominent osteochondromas in vehicle-receiving mutant mice are indicated with arrowheads and protrude away from the bone surfaces (middle left images). However, in trifarotene-treated mutant mice, the osteochodromas are markedly reduced and barely detectable (arrowheads, right side images). No osteochondromas are detected in controls (far left images).

[0066] Figure 7 provides pCT images of ribs from male control

[0067]

[0068] mutant Ext 1^; A can-CreER22mice treated with vehicle (control) or trifarotene (0.75 mg / kg or 3 mg / kg). Osteochondromas are appreciable at the location of the growth plate in vehiclereceiving mutant mice but the osteochondromas are much reduced and barely visible in trifarotene-treated mice. No osteochondromas are detected in controls (far left images).

[0069] Figure 8 provides pCT images of knee from female control Ext or mutant Ext 1^; A can-CreER22mice treated with vehicle (control) or trifarotene (0.75 mg / kg or 3mg / kg). Femur (fe), tibia (ti), and fibula (fi) are shown. Most prominent osteochondromas in vehicle-receiving mutant mice are indicated with arrowheads and protrude away from the bone surfaces (middle left images). However, in trifarotene-treated mutant mice, the osteochodromas are markedly reduced and barely detectable (arrowheads, right side images). No osteochondromas are detected in controls (far left images).

[0070] Figure 9 provides pCT images of ribs from female control

[0071]

[0072] mutant Ext 1^; A can-CreER12mice treated with vehicle (control) or trifarotene (0.75 mg / kg or 3 mg / kg). Osteochondromas are appreciable at the location of the growth plate in vehiclereceiving mutant mice but the osteochondromas are much reduced and barely visible in trifarotene-treated mice. No osteochondromas are detected in controls (far left images).

[0073] Figure 10A provides alcian blue and nuclear fast red stained images of femur from control / A / / " mice or mutant Extl^Acan-CreER22male and female mice treated with vehicle (control) or trifarotene (0.75 mg / kg or 3 mg / kg). Bottom row images represent a magnification of box areas in top row images. The graph in Figure 10B provides measurements of length and width of osteochondroma on the femur in control / A / / " mice or mutant Extl^Acan-CreER12mice treated with vehicle (control) or trifarotene (0.75 mg / kg or 3 mg / kg). Significant reduction in osteochondroma is readily seen with trifarotene treatment.

[0074] Figure 11 A provides alcian blue and nuclear fast red stained images of tibia from control / A / / " mice or mutant Extl^Acan-CreER12male and female mice treated with vehicle (control) or trifarotene (0.75 mg / kg or 3 mg / kg). Bottom row images represent a magnification of box areas in top row images. The graph in Figure 1 IB provides measurements of length and width of osteochondroma on the femur in control / A / / " mice or mutant Extl^Acan-CreER12mice treated with vehicle (control) or trifarotene (0.75 mg / kg or 3 mg / kg). Significant reduction in osteochondroma is readily seen with trifarotene treatment.

[0075] Figure 12A provides alcian blue and nuclear fast red stained images of ribs from control / A / / " mice or mutant Extl^Acan-CreER12male and female mice treated with vehicle (control) or trifarotene (0.75 mg / kg or 3 mg / kg). Bottom row images represent a magnification of box areas in top row images. The graph in Figure 12B provides measurements of length and width of osteochondroma on the femur in control / A / / " mice or mutant Extl^Acan-CreER12mice treated with vehicle (control) or trifarotene (0.75mg / kg or 3 mg / kg). Significant reduction in osteochondroma is readily seen with trifarotene treatment.

[0076] While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

WHAT IS CLAIMED IS1. A method for inhibiting, treating, and / or preventing a chondrogenesis-related disease or disorder in a subject, said method comprising administering to a subject at least one retinoid acid receptor (RAR) agonist.

2. The method of claim 1, wherein said chondrogenesis-related disease or disorder is multiple osteochondroma.

3. A method for inhibiting or preventing chondrogenesis and / or osteochondroma or exostosis formation and / or growth in a subject, said method comprising administering to a subject at least one retinoid acid receptor (RAR) agonist.

4. The method of any one of claims 1-3, wherein said RAR agonist is a RAR gamma agonist.

5. The method of claim 4, wherein said RAR gamma agonist is trifarotene.

6. The method of any one of claims 1-5, wherein said RAR agonist is administered systemically.

7. The method of any one of claims 1-5, wherein said RAR agonist is administered directly to the site of chondrogenesis.

8. The method of any one of claims 1-5, wherein said RAR agonist is administered orally.

9. The method of any one of claims 1-5, wherein said RAR agonist is administered by injection.

10. The method of any one of claims 1-9, wherein said RAR agonist is contained within a composition further comprising a pharmaceutically acceptable carrier.

11. The method of any one of claims 1-10, further comprising at least one other anti-chondrogenesis agent to the subject.

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