Drug treatment for achondroplasia

Abstract

The present disclosure relates to the treatment of achondroplasia using retinoic acid receptor (RAR) antagonists.

Description

[0001] DESCRIPTION

[0002] DRUG TREATMENT FOR ACHONDROPLASIA PRIORITY CLAIM

[0003] This application claims benefit of priority to U. S. Provisional Application Serial No.

[0004] 63 / 724,608, filed November 25, 2024, the entire contents of which are hereby incorporated by reference.

[0005] BACKGROUND

[0006] 1. Field of the Disclosure

[0007] The present disclosure relates generally to the fields medicine, genetics, pathology and skeletal biology. More particularly, the disclosure relates to the use of retinoic acid receptor antagonists in the treatment of achondroplasia.

[0008] 2. Background

[0009] Growth, structure and regeneration of the skeleton remain areas of intense research activity, due to their importance and relevance to body function and quality of life. The major engines of skeletal growth are the growth plates that are composed of a top reserve zone rich in stem cells followed by proliferating, pre-hypertrophic and hypertrophic zones of chondrocyte maturation and ossification, with cells organized in columns. Growth plate-like structures also form during skeletal repair and regeneration as observed in bone fracture healing. Growth plate activity is regulated by multiple mechanisms including signaling proteins such as hedgehog and fibroblast growth factor family members; transcription factors including retinoic acid receptors (RARs), Runx2 and Mef2c; and local and systemic hormones (Lefebvre and Bhattaram, 2010; Kozhemyakina et al., 2015). The growth plates normally remain active until the end of puberty and then undergo involution and close, establishing a fully grown skeleton. However, trauma, genetic disorders or drug treatment of certain pediatric pathologies can affect growth plates, derange proliferation and maturation rates, and cause their slowdown and involution, often with severe skeletal consequences.

[0010] For example, about 10% of adolescents with a growth plate injury in long bones develop asymmetric bone growth and require challenging surgical intervention. Drug-induced growth plate malfunction is observed in pediatric patients treated with pharmaceuticals that can have unwanted side effects on their growth plates, causing growth retardation. From a genetic standpoint, Achondroplasia (ACH) and Crouzon Syndrome are two examples of disease in 1

[0011] 4936-1856-8059, V. 1 which the growth plates are affected, with significant impact on pediatric and adult health (Unger et al., 2023). ACH is the most severe form of human dwarfism and is caused by gain-of-function (GOF) mutations in the cell surface Fibroblast Growth Factor Receptor 3 (FGFR3) (R. M. Pauli, 2019). This receptor normally regulates the overall rates of activity of the growth plates mainly by limiting chondrocyte proliferation in cooperation with FGF18 provided by perichondrium (Omitz and Marie, 2015). The GOF mutations in FGFR3 alter the normal functioning of growth plates by decreasing chondrocyte proliferation even further and inhibiting expression of Indian hedgehog, but accelerate chondrocyte hypertrophy and ossification, resulting in overall slowdown of growth plate activity and in skeletal growth retardation and dwarfism (Ornitz & Legeai-Mallet, 2017).

[0012] In sum, the growth plates are vulnerable to malfunction in several acquired and congenital pathologies, representing clinical needs in search of effective solutions.

[0013] 2

[0014] 4936-1856-8059, V. 1 SUMMARY

[0015] Thus, in accordance with the present disclosure, there is provided a method of treating a subject having or at risk of developing achondroplasia comprising administering to a subject in need thereof one or more retinoic acid receptor (RAR) antagonists. The subject may have achondroplasia or is at risk of developing achondroplasia, such as determined by genetic testing (e.g., one or two copies of mutated FGFR3 gene). Treating may comprise direct treatment of a pediatric subject or a neonatal subject. Treating may also comprise indirect treatment of a prenatal subject by administration to a pregnant female patent of said prenatal subject.

[0016] The pediatric subject may be from about 2 to about 18 years of age. The RAR antagonist may include one or more of an RARy selective antagonist, an RARa selective antagonist, an RARy / RARa selective antagonist, and / or an RARy / RARp selective antagonist. The RAR antagonist may be selected from the group consisting of AGN 193109, BMS 195814, BMS 195614, ER 50891, LE 135, LY 2955303, SR11335, LY 2813631, Ro 41-5253, AGN 193109, AGN194310, AGN194431, AGN194301, MM 11253, 7C and CD 2665.

[0017] The method may comprise multiple RAR antagonist administrations, such as 2, 3, 4 or more times. The administration is by oral, intravenous, intra-arterial, or intranasal or subcutaneous administration. The method may comprise a further achondroplasia therapy being administered to said subject. The administration may comprise an oral dose of said RAR antagonist is between 0.5 and 5.0 mg / kg daily, between 1.0 and 2.5 mg / kg daily, or about 1.5 mg / kg daily. The RAR antagonist may administered daily up to about the age of 18 years. The dosing regimen may be changed from daily to 2 or 3 times per week. The subject may exhibit inhibition of skeletal growth retardation and / or inhibition of growth plate aberration. The subject may not receive concurrent treatment with a cyclooxygenase-2 inhibitor.

[0018] Also provide is a use of one or more retinoic acid receptor (RAR) antagonists in the treatment or prevention of achondroplasia in a subject. The subject may be a pediatric subject, such as about 2 to about 18 years of age. The subject may be a prenatal or neonatal subject. The subject may have achondroplasia or is at risk of developing achondroplasia, such as determined by genetic testing (e.g., one or two copies of mutated FGFR3 gene). The RAR antagonist may include one more of an RARy selective antagonist, an RARa selective antagonist, an RARy / RARa selective antagonist, or an RARy / RARp selective antagonist, such as an RAR antagonist is selected from the group consisting of AGN 193109, BMS 195814, ER 50891, LE 135, LY 2955303, MM 11253. 7C and CD 2665.

[0019] 3

[0020] 4936-1856-8059, V. 1 The use of the word “a” or “an” 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.” The word “about” means plus or minus 5% of the stated number.

[0021] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0022] 4

[0023] 4936-1856-8059, V. 1 BRIEF DESCRIPTION OF FIGURES and DRAWINGS

[0024] The patent or application file contains figures and drawings executed in color. Copies of this patent or patent application publication with color figures and drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0025] The following figures and drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these figures and drawings in combination with the detailed description of specific embodiments presented herein.

[0026] FIGS. 1A-G. Retinoid CD2665 drug administration rectifies body length and weight in male and female ACH mice. Schematic of experimental plan (FIG. 1A). Body length and weight accrual by P35 were decreased in ACH mutant mice receiving vehicle compared to control mice (CTL) given vehicle or drug but were ameliorated in mutants receiving CD2665 by P35 (FIG. IB, FIG.1C, FIG. ID, FIG. IF). The drug effects in mutants were all statistically significant (FIG. IE, FIG.1G). *p< 0.05; ***p<0.005; ns, not significant.

[0027] FIGS.2A-E. Retinoid CD2665 drug administration rectifies growth plate closure and mineralization in long bones. X-ray and microCT imaging show that tibia growth plate was abnormal, mineralizing and closing in P35 ACH mice receiving vehicle (red arrowheads) compared to controls (CTL, yellow arrowheads) (FIG.2A). These defects in ACH mice were all ameliorated by CD2665 administration (green arrowheads) (FIG. 2A) and in statistically significant manners (FIGS.2B-E). *p< 0.05; **p<0.01; ***p<0.005; ****p<0.0001.

[0028] FIG. 3. Retinoid CD2665 drug administration rectifies growth plate structure.

[0029] Histochemical procedures using Safranin-O / fast green staining show that tibia growth plate (gp) in control female mice given vehicle or drug (CTL) displayed typical resting, proliferating and pre-hypertrophic / hypertrophic (re, pl and ph / hy) zones followed by subchondral bone (sb) by P35 (left two sets of images). The growth plate was much shorter and interrupted by other tissues in ACH mutant mice receiving vehicle (third set from left) but appeared largely normal and preserved in mutants receiving CD2665 (far-right set of images).

[0030] FIGS. 4A-D. Retinoid CD2665 drug administration rectifies gene expression patterns in growth plate. Histochemical procedures using safranin-O / fast green (Saf-O / FG) staining and RNAscope in situ hybridization show that the growth plate in ACH mutants given vehicle was abnormal in structure (FIG.4C, top two images) but also displayed abnormal gene expression of ColX, Ihh, Ki67 and PTHrP (FIG. 4C, lower four images, red arrowheads) compared to controls (FIGS. 4A-B). These defects had all been ameliorated and largely 5

[0031] 4936-1856-8059, V. 1 rectified in mutants receiving CD2665 treatment (FIG. 4D, lower four images, green arrowheads).

[0032] FIGS.5A-R. Retinoid CD2665 drug administration rectifies hedgehog and Raldh3 gene expression patterns. Histochemical (FIG. 5A, FIG. 5G, FIG. 5M) and in situ hybridization data show that ACH mutant growth plates in vehicle-treated mice were characterized by (i) markedly decreased expression of Ihh, Glil and Gli2 (FIGS. 5H-J), (ii) excessive expression of Gli3 (FIG.5K) and (iii) upward-expanded expression of Raldh3 (FIG.

[0033] 5L) compared to control (CTL) mice (FIGS.5B-F). CD2665 administration largely prevented these changes and restored gene expression patterns similar to controls (FIGS.5N-R).

[0034] FIGS.6A-D. Retinoid CD2665 drug administration ameliorates defects in cranial base synchondroses. Analyses by micro-CT (pCT) (FIG.6A) and histochemistry (FIGS.6B-D) show that the intrasphenoidal (ISS) and sphenoccipital (SOS) synchondroses in vehicle-treated ACH mutant mice were abnormal and covered by ectopic bone (FIG.6A, FIG.6D, red arrowheads) compared to control (CTL) mice given vehicle or drug (FIG.6A, FIG.6D, yellow arrowheads). Defects in the mutants were largely prevented by CD2665 administration (FIG.

[0035] 6A, FIG. 6D, green arrowheads) and the synchondrosis growth plates were flanked by thin perichondrium (FIG.6D, green arrowheads) resembling perichondrium in controls (FIG.6D, yellow arrowheads).

[0036] FIGS.7A-G. Administration of retinoid drug 7C rectifies body length and weight in male and female ACH mice. Schematic of experimental plan (FIG.7A). Body length and weight accrual by P35 were decreased in ACH mutant mice receiving vehicle compared to controls given vehicle or drug but were ameliorated in mutants receiving the 7C drug (FIG.

[0037] 7B, FIG.7C, FIG.7D, FIG.7F). The drug effects in mutants were all statistically significant (FIG.7E, FIG.7G). *p< 0.05; ***p<0.005; ns, not significant.

[0038] FIGS. 8A-E. Administration of retinoid drug 7C prevents shrinking and mineralization of growth plate in male and female ACH mice. X-ray and µCT imaging show that tibial growth plate in vehicle-treated ACH mutantmice was closing and mineralized (FIG.8A, red arrowheads) but this was prevented by 7C drug administration (FIG.8A, green arrowheads), resembling controls (FIG.8A, yellow arrowheads). Quantification of tibia length and degree of mineralization in growth plate confirmed that drug treatment significantly ameliorated all these parameters in mutant mice (FIGS. 8B-E, red triangles), resembling control values (FIGS.8B-E, blue circles). *p< 0.05; **p<0.01; ***p<0.005; ****p<0.0001.

[0039] 6

[0040] 4936-1856-8059, V. 1 FIG.9A-E. Administration of retinoid drug 7C ameliorates defects in cranial base synchondroses. pCT imaging shows that the sphenoccipital synchondrosis in vehicle-treated ACH mutant mice was abnormal and covered by ectopic bone (red arrowheads) compared to controls given vehicle or drug (yellow arrowheads). These defects in mutant mice were largely prevented by 7C drug administration (green arrowheads).

[0041] FIG. 10. Blood cell counts and chemistry reveal no major side effects of CD2665 administration. Serum samples collected at P35 from control and ACH mutant mice receiving vehicle or CD2665 were processed for standard blood analyses by Institutional Centralized Core facilities. No major changes were observed in the several blood parameters following drug treatment in control (blue circles) or mutant (red tringle) mice.

[0042] FIG. 11. Analyses of liver function. Blood samples collected at P35 from control mice receiving vehicle or CD2665 were processed for standard serum analyses of liver function. There were no obvious changes in any parameter tested following drug administration.

[0043] FIG. 12. Analyses of kidney function. Blood samples collected at P35 from control mice receiving vehicle or CD2665 were processed for standard serum analyses of kidney function. There were no obvious changes in any parameter tested following drug administration.

[0044] FIG. 13. Chemical structures of CD 2665 (top) and 7C (bottom).

[0045] 7

[0046] 4936-1856-8059, V. 1 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0047] As discussed above, skeletal growth disorders and pathologies continue to represent therapeutic challenges in medicine, particularly in pediatrics. In previous basic science mouse studies on growth plate regulation carried out by the inventors, they focused on the nuclear retinoic acid receptors (RARs). The RARs normally act as (i) transcriptional activators when bound to physiologic ligands such as all-trans-retinoic acid (RA) or (ii) transcriptional repressors when ligand-free (Evans & Mangelsdorf, 2014). Genetic and biochemical lines of experimentation by the inventors established that the RARs exert a major balancing function in the rates of growth plate activity and do so by mainly acting as transcriptional repressors (Williams et al., 2010; 2009).

[0048] Using a standard model of growth plate defect in juvenile mice that mimics a pathology seen in pediatric patients, the inventors observed that growth plate dysfunction was caused by concurrent changes in mechanisms normally fine-tuning growth plate activity and functioning. They also showed that systemic treatment with a drug targeting one such mechanism, retinoic acid receptor signaling, was sufficient to maintain growth plate function and restore skeletal lengthening. Thus, they believe that growth plate dysfunction is amenable to this type of drugbased rescue.

[0049] These and other aspects of the disclosure are described in detail below.

[0050] I. Achondroplasia

[0051] Achondroplasia is a genetic disorder with an autosomal dominant pattern of inheritance and is the most severe form of dwarfism in humans and affects about 1 individual in 15,000-40,000 live births. In those with the condition, the arms and legs are short, while the torso is typically of normal length but may exhibit spine defects. Those affected have an average adult height of 131 cm (4 ft 4 in) for males and 123 cm (4 ft) for females. Other features can include a deformed head with prominent forehead (frontal bossing), underdevelopment of the midface (midface hypoplasia), vertebral alterations, foramen magnum stenosis and aberrant cranial base synchondroses functioning. Complications can include sleep apnea, recurrent ear infections, dental malocclusion and insufficient lengthening of the cranial base (Pauli, 2019). Achondroplasia includes the extremely rare short- limb skeletal dysplasia with severe combined immunodeficiency (Achondroplasia-SCID syndrome).

[0052] Achondroplasia is caused by a gain-of-function mutation in the fibroblast growth factor receptor 3 (FGFR3) gene - most often G380R - resulting in its encoded protein to be overactive.

[0053] 8

[0054] 4936-1856-8059, V. 1 FGFR3 is normally expressed in the upper zones of the growth plate where it mainly acts to limit the rates of chondrocyte proliferation. When overactive due to the mutation, the receptor further inhibits chondrocyte proliferation and gene expression of Indian Hedgehog, altering the normal progression of chondrocytes through maturation and hypertrophy and impairing endochondral bone growth and lengthening. The disorder has an autosomal dominant mode of inheritance, meaning only one mutated copy of the gene is sufficient for the condition to occur. About 80% of cases occur in children of parents without the disease, and result from a new (de novo, or sporadic) mutation, which most commonly originates as a spontaneous change during spermatogenesis. The remining cases are from a parent with the condition. The risk of a new mutation increases with the age of the father. In families with two affected parents, children who inherit both mutant genes typically die before birth or in early infancy from breathing difficulties and display also severe cranial base defects. The condition is generally diagnosed based on clinical features and can be confirmed by genetic testing. Other mutations in FGFR3 also cause achondroplasia related conditions including hypochondroplasia and SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans), a rare disorder of bone growth characterized by skeletal, brain, and skin abnormalities resulting in severe short-limb skeletal dysplasia with severe combined immunodeficiency.

[0055] The only approved pharmacological treatment of achondroplasia at present is Vosoritide, a C-natriuretic peptide analog that improves annualized growth velocity, although side effects such as low blood pressure were observed and its long-term effects are unknown. Because of its peptide nature and size possibly limiting its systemic distribution and availability, Vosoritide does not treat other severe complications in ACH such as foramen magnum stenosis and cranial base undergrowth and malformation (Saravirayan et al., 2020). Growth hormone therapy is used in countries such as Japan but is associated with several health complications. Efforts to treat or prevent complications such as obesity, hydrocephalus, obstructive sleep apnea, middle ear infections or spinal stenosis may be required. Advocacy groups support people with achondroplasia, including the Little People of America (LPA) and Growing Stronger. Nonprofit physician organizations also exist to disseminate information about treatment and management options, including development of patient resources.

[0056] Signs and symptoms of achondroplasia include: disproportionate dwarfism; shortening of the proximal limbs (called rhizomelic shortening); short fingers and toes, with "trident hands" (short hands with stubby fingers, and a separation between the middle and ring fingers - reminiscent of a trident on fetal ultrasound); large head with prominent forehead frontal 9

[0057] 4936-1856-8059, V. 1 bossing; small midface with a flattened nasal bridge; spinal kyphosis (convex curvature) or lordosis (concave curvature); varus (bowleg) or valgus (knock knee) deformities: frequent ear infections (due to Eustachian tube blockages); sleep apnea (which can be central or obstructive); cranial base malformations; foramen magnum stenosis; and hydrocephalus.

[0058] Children with achondroplasia often have less muscle tone. Because of this problem, it is common for them to have delayed walking and motor skills. It is also common for ACH children to have bowed legs, scoliosis, lordosis, arthritis, issues with joint flexibility, breathing problems, ear infections, and crowded teeth. These issues can be treated with surgery, braces, or physical therapy.

[0059] Hydrocephalus is a severe effect associated with achondroplasia in children. This condition occurs when cerebrospinal fluid is not able to flow in and out of the skull because of narrowing of the spinal canal associated with the cranial base defects. This fluid build-up is associated with a deformed head, vomiting, lethargy, headaches, and irritability. Shunt surgery is commonly performed to treat this condition, but an endoscopic third ventriculostomy can also be done.

[0060] Adults with achondroplasia often face issues with obesity and sleep apnea. It is also typical for adults to experience numbness or tingling in their legs because of nerve compression. Some research has found that adults with achondroplasia may also experience psychosocial complications, usually associated with short stature.

[0061] Pregnancy in women with achondroplasia is considered higher risk. Women with achondroplasia generally have their babies delivered through C-section to prevent complications that could occur with a natural birth. Intelligence and life span are usually near normal, although craniocervical junction compression increases the risk of death in infancy.

[0062] As indicated above, achondroplasia is caused by a gain-of-function mutation in fibroblast growth factor receptor 3 (FGFR3) gene. In normal development, FGFR3 has a negative regulatory role on chondrocyte proliferation in the upper zones of growth plate. In achondroplasia, the mutated form of the receptor is constitutively active, and this leads to a much stronger inhibition of chondrocyte proliferation and a concurrent decrease in expression of the signaling protein Indian hedgehog, causing an overall slowdown of growth plate activity and endochondral ossification and ultimately causing severely shortened bones. The effect is genetically dominant, with one variant of the FGFR3 gene being sufficient to cause achondroplasia. People with achondroplasia are often born to parents who do not have the condition due to spontaneous mutation. However, patients bom from achondroplasia parents

[0063] 10

[0064] 4936-1856-8059, V. 1 can inherit two copies of the mutant gene. This is invariably fatal and patients die before or shortly after birth due to respiratory failure from an underdeveloped ribcage.

[0065] In couples where one partner has achondroplasia, there is a 50% chance of passing the disorder on to their child every pregnancy. In situations where both parents have achondroplasia there is a 50% chance the child will have achondroplasia, 25% chance the child will not, and a 25% chance that the child will inherit the mutant gene from both parents resulting in double dominance and leading to death before or shortly after birth.

[0066] Studies have demonstrated that new gene mutations for achondroplasia are most often inherited from the father and occur during spermatogenesis; it has been theorized that sperm carrying the mutation in FGFR3 have a selective advantage over sperm with normal FGFR3. The frequency of mutations in sperm leading to achondroplasia increases in proportion to paternal age, as well as in proportion to exposure to ionizing radiation. The occurrence rate of achondroplasia in the children of fathers over 50 years of age is 1 in 1,875, compared to 1 in 15,000-40,000 in the general population. Research by urologist Harry Fisch of the Male Reproductive Center at Columbia Presbyterian Hospital in 2013 indicated that in humans this defect may be exclusively inherited from the father and becomes increasingly probable with paternal age, specifically males reproducing after 35.

[0067] There are other syndromes with a genetic basis similar to that of achondroplasia, including hypochondroplasia and thanatophoric dysplasia.

[0068] Achondroplasia can be detected before birth by prenatal ultrasound, although signs are often subtle and not apparent before the 24th week of pregnancy. A DNA test can be performed before birth to detect gene sequence mutations. Postnatal diagnosis of achondroplasia is typically uncomplicated, involving an assessment of physical and radiographic features. Clinical features include megalocephaly, short limbs, prominent forehead, thoracolumbar kyphosis and mid- face hypoplasia. Complications like dental malocclusion, hydrocephalus and repeated otitis media can be observed. The risk of death in infancy is increased due to the likelihood of compression of the spinal cord with or without upper airway obstruction.

[0069] A skeletal survey is useful to confirm the diagnosis of achondroplasia. The skull is large, with a narrow foramen magnum, and relatively small skull base. The vertebral bodies are short and flattened with relatively large intervertebral disk height, and there is congenitally narrowed spinal canal. The iliac wings are small and squared, with a narrow sciatic notch and horizontal acetabular roof. The tubular bones are short and thick with metaphyseal cupping and flaring and irregular growth plates. Fibular overgrowth is present. The hand is broad with short metacarpals and phalanges, and a trident configuration. The ribs are short with cupped 11

[0070] 4936-1856-8059, V. 1 anterior ends. If the radiographic features are not classic, a search for a different diagnosis should be entertained. Because of the extremely deformed bone structure, people with achondroplasia are often "double jointed". The diagnosis can be made by fetal ultrasound by progressive discordance between the short femur length and biparietal diameter by age. The trident hand configuration can be seen if the fingers are fully extended. Another common characteristic of the syndrome is thoracolumbar gibbus in infancy.

[0071] There is no known cure for achondroplasia even though the causative mutation in FGFR3 has been known for years. Although used by those without achondroplasia to aid in growth, human growth hormone does not help people with achondroplasia, which involve more complex hormonal and protein signaling defects. Usually, the best results appear within the first and second year of therapy. After the second year of growth hormone therapy, beneficial bone growth decreases, so the therapy is not a satisfactory long-term treatment. As of December 2020, the treatment of achondroplasia with human growth hormone was approved only in Japan.

[0072] The peptide drug Vosoritide was approved recently and is used to improve growth velocity in children with achondroplasia, although its long-term effects are unknown and it is unable to treat severe ACH complications, including foramen magnum stenosis and cranial base undergrowth and malformation (Saravirayan et al., 2020). Vosoritide indirectly inhibits the activity of FGFR3. It has been gradually made available in different countries starting from 2021. Surgical limb-lengthening can be used to lengthen the legs and arms of someone with achondroplasia, but little medical consensus exists regarding this practice. The age of surgery can vary from early childhood to adulthood.

[0073] Research has also shown that introducing parents of children with achondroplasia to support and advocacy groups at the time of diagnosis can improve outcomes. Several patient advocacy groups exist to support people with achondroplasia and their families. Resources are available to support patients and their caregivers with information that they can distribute to their physicians, who may not be familiar with the unique medical requirements of managing achondroplasia. Physician-oriented best practice guidelines are also available to guide physicians managing the spinal disorders, foramen magnum stenosis, craniofacial implications, pregnancy, and peri -operative needs of people with achondroplasia.

[0074] 12

[0075] 4936-1856-8059, V. 1 IL Retinoic Acid Receptors (RARs) and Antagonists Thereof

[0076] A. RARs

[0077] The RARs are nuclear receptors which act as transcriptional activators when associated with an endogenous physiologic ligand and as transcriptional repressors when ligand-free. Physiologic ligands include all-trans retinoic acid and 9-cis retinoic acid that derive from the cytoplasmic metabolism of circulating biologically inactive Vitamin A and other precursors. There are three retinoic acid receptors (RARs), RAR-alpha, RAR-beta, and RAR-gamma, encoded by the RARa, RAR / 3, A71 / ? / genes, respectively. Within each RAR subtype there are various isoforms that differ in their N-terminal region A. Multiple splice variants have been identified in human RARs: four for RARa, five for RAR / 3, and two for RARy. As observed with other type II nuclear receptors, the RARs heterodimerize with RXRs and the RAR / RXR dimers bind to DNA hormone response elements -known as retinoic acid response elements (RAREs)- present in target genes and in complex with corepressor proteins (Evans et al., 2014). Binding of retinoid agonist ligands to the RARs results in dissociation of corepressors and recruitment of coactivator proteins that promote transcription of target genes into mRNAs and eventually proteins. In addition, the expression of RAR genes is under epigenetic regulation by promoter methylation. Length and magnitude of retinoid response by cells and tissues depend on several parameters including availability and metabolism of active ligands and degradation of RARs and RXRs through the ubiquitin-proteasome. RARs and RXRs are also known to play other retinoid-independent roles as they bind to and regulate other nuclear receptor pathways, such as the estrogen receptor.

[0078] RARs play a crucial role in embryonic development. Mice studies have revealed that knocking out RARs can replicate the spectrum of defects associated with fetal vitamin A deficiency syndrome, unveiling additional abnormalities beyond previously known vitamin A functions. Notably, double RAR mutants exhibited the most severe defects, including ocular, cardiovascular and skeletal defects, indicating some level of redundancy among RARs. RXR / RAR heterodimers transmit retinoid signals in diverse ways to control the expression of networks of retinoic acid (RA) target genes. This process plays a crucial role in shaping both the axial and limb patterning during early embryo development, as well as influencing various aspects of organ formation in later stages of development.

[0079] B. RAR Antagonists

[0080] The compounds include AGN 193109, BMS 195814, BMS 195614, ER 50891, LE 135, LY 2955303, SR11335, LY 2813631, Ro 41-5253, AGN 193109, AGN194310, AGN194431,

[0081] 13

[0082] 4936-1856-8059, V. 1 AGN194301, MM 11253, 7C and CD 2665. CD 2665 (see FIG. 3) is a selective RARPy antagonist (KD values are 110, 306 and > 1000 nM for RARy, RAR[3 and RARa respectively). It can block retinoic acid-induced apoptosis ex vivo. 7C is a highly selective RAR-gamma antagonist (IC50 (nM): RAR-alpha = > 50,000; RAR-beta = > 100,000; RAR-gamma = 2).

[0083] CD 2665 (1 pM) did not antagonize the luciferase activity induced by 9-cis- or all-trans-RA in HiB5 cells, but partially blocked the luciferase activity in 3XbRARE-Luc-transfected cells induced by CD 666 (a RARy-selective agonist, 100 nM). CD 2665 at 1 pM reduced the viable cell number to about 60% of the level of vehicle-treated cells. CD 2665 (1 pM) clearly antagonized the increase in cell number resulted from the treatment with CD 666 (100 nM) (Chung etal., 2000).

[0084] The biosynthesis of retinoic acid can also be disordered by chronic ethanol consumption. CD 2665 administration did not modify blood alcohol levels in alcohol-treated mice, but totally reversed the impairment of spontaneous alternation rates resulted from alcohol consumption. Administration with CD2665 for 22 days in alcohol-treated mice significantly decreased both mRNA expression and enzymatic activity of tTG (a retinoic acid-target gene) and normalized the expression levels to the level in control mice (Alfos et al., 2001).

[0085] III. Formulation and Administration

[0086] The present disclosure provides pharmaceutical compositions. Such compositions comprise a prophylactically or therapeutically effective amount of an agent, and a pharmaceutically acceptable earner. In a specific embodiment, the term “pharmaceutically acceptable” means approved 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. The term “carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid earners, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol

[0087] 14

[0088] 4936-1856-8059, V. 1 monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0089] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington's Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, intra-rectal, vaginal, topical or delivered by mechanical ventilation.

[0090] Pharmaceutically acceptable salts include the acid salts and those which arc formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0091] Generally, the ingredients of compositions according to the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0092] IV. Combination Therapy

[0093] Combination dmg treatment including two or more RAR antagonists, such as with distinct affinities for RARy, RAR and RARa, is contemplated. The combination therapy would permit lower doses of each retinoid, thus possibly reducing side effects.

[0094] 15

[0095] 4936-1856-8059, V. 1 Another combination therapy would be to combine a retinoid antagonist as described herein with the recently approved drug Vosoritide, likely requiring lower doses of each for effectiveness and thus reducing unwanted side effects such as low blood pressure.

[0096] Combinations may be achieved by contacting subjects with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with multiple distinct compositions or formulations, at the same lime, wherein one composition includes a first RAR antagonist and the other includes a second RAR antagonist or other therapy / agent. Alternatively, the RAR antagonist factors may precede or follow administration of the other RAR antagonist / other agent / therapy by intervals ranging from minutes to weeks to months. In embodiments where the RAR antagonist and other agent / therapy are applied separately to the subject, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the RAR antagonist and other agent / therapy would still be able to exert an advantageously combined effect on the subject. In such instances, it is contemplated that one would typically contact the cell with both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other, with a delay time of only about 12 hours being most preferred. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

[0097] It also is conceivable that more than one administration of either the first RAR antagonist, or the other RAR antagonist / other agent or therapy will be desired. In this regard, various combinations may be employed. By way of illustration, where the first RAR antagonist is “A” and the other RAR antagonist or other agent / therapy is “B,” the following permutations based on 3 and 4 total administrations are exemplary:

[0098] A / B / A B / A / B B / B / A A / A / B B / A / A A / B / B B / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / A B / A / B / A B / A / A / B B / B / B / A A / A / A / B B / A / A / A A / B / A / A A / A / B / A A / B / B / B B / A / B / B B / B / A / B Other combinations are likewise contemplated.

[0099] V. Examples

[0100] The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice.

[0101] 16

[0102] 4936-1856-8059, V. 1 However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0103] Example 1

[0104] The inventors used a transgenic model that carries a Cre-inducible knock-in Fgfr3G380Rmutation representing a most common ACH human mutation (Wang et al., 1999) and focused first on the defects caused hy ACH on the appendicular skeleton and treatments thereof. In these transgenic mice, allele expression initiates after Cre-mediated removal of a neo cassette. Accordingly, double hetero Fgfr3G380R / +lAgr-CreERA1' juvenile male and female mice (heretofore referred to as mutant mice or ACH) and companion control Agr-CreER+l~ juvenile male and female mice (heretofore referred to as control mice or CTN) were pre-administered the retinoid antagonist drag CD2665 (1.6 mg / kg) or vehicle (DMSO-com oil) by gavage on postnatal day 9 (P9) (Koyama et al., 2021; Szondy et al., 1997). On PIO, all mice were given a single tamoxifen injection (25 mg / kg) to activate Fgfr3G380Rexpression and continued to receive a daily dose of retinoid drag or vehicle until P35 (FIG. 1A). As expected based on previous studies (6), the Fgfr3G380Rl Agr-CreER mutant mice receiving vehicle exhibited growth retardation and an appreciably smaller body by P35 (FIGS. IB and 1C, second mouse from right side) compared to controls (FIGS. IB and 1C, mice on the left). In addition, the mutant mice displayed a noticeable degree of thoracic kyphosis when viewed laterally (not shown) as reported previously (Wang et al., 1999), a pathology affecting ACH patients as well (Engberts et al., 2011). In contrast, mutant mice receiving CD2665 displayed an improvement of body growth, resembling control mice (FIGS. IB and 1C, mouse on the right). To verify visual observations, the Inventors monitored and measured body weight from the start of the experiments until collection of the mice (FIGS. ID to 1G). There was an obvious decrease in body weight accrual over time in mutant male and female mice receiving vehicle (FIGS. ID and IF, red circles) compared to controls given vehicle or drag (FIGS. ID and IF, blue circles and triangles). However, body weight had been accrued in mutants receiving CD2665 (FIGS. ID and IF, red triangles). These drug effects were statistically significant as shown in FIGS. IE and 1G.

[0105] As indicated by x-ray imaging, the growth plates in mutant male and female mice receiving vehicle were closing by P35 (FIG. 2A, red arrowheads) compared to those in controls that were open (FIG. 2A, yellow arrowheads). Such trend toward premature growth plate closure was clearly rectified in mutant mice receiving CD2665 (FIG. 2A, green arrowheads).

[0106] 17

[0107] 4936-1856-8059, V. 1 Measurement of long bones showed that compared to controls (FIG. 2B and 2C, blue circles), average tibia length was significantly decreased in mutants receiving vehicle but was improved by CD2665 treatment in both male and female mice (FIG. 2B and 2C, red triangles). Quantification of radiographic mineralization showed that control growth plates displayed minimal mineral content, reflecting the fact that they were open and fully cartilaginous as expected (FIGS. 2D and 2E, blue circle histograms). In comparison, the growth plate area in mutants receiving vehicle was highly mineralized (reflecting its ongoing pathogenic closure and ossification) (FIGS. 2D and 2D, red triangle histograms). Such abnormality was greatly corrected in mutants receiving CD2665 (FIGS. 2D and 2E, red triangle histograms).

[0108] Histochemistry was used to evaluate the structural organization of the growth plates. In controls (receiving vehicle or drug), the growth plates (gp) were open, stained strongly with safranin O, displayed typical zones of resting, proliferating and hypertrophic chondrocytes (re, pl and ph / hy), and were followed by normal subchondral bone (sb) by P35 (FIGS. 3A and 3B). However, the growth plates in mutants receiving vehicle were markedly reduced in height, stained poorly with Safranin O, and displayed structural interruptions indicative of encroachment by fibrotic tissue or bone (FIG. 3C). These obvious defects had been largely prevented in mutants receiving CD2665 (FIG. 3D). Though the growth plates had not regained their full height, they were well organized, displayed strong Safranin O staining and lacked interruption or tissue encroachment (FIG. 3D).

[0109] To further examine the character of the growth plates, the Inventors carried out a combination of histiochemistry and gene expression analysis by in situ hybridization. In controls (receiving vehicle or drug), the normal organization and structure of the growth plates were reflected by normal gene expression patterns of such typical cartilage genes as collagen X (Col X) and Indian hedgehog (Ihh) in the lower zones by P35 (FIGS. 4A and 4B). The upper zones displayed normal expression patterns of the mitotic markers Ki67 and PTHrP (FIGS. 4A and 4B). In mutants receiving vehicle, the shrinking of the growth plates was accompanied by obvious reductions in expression of Col X, Ihh, Ki67 and PTHrP (FIG. 4C, red arrowheads). All these phenotypic parameters had been ameliorated in mutants receiving CD2665 (FIG. 4D, green arrowheads), resembling controls.

[0110] IHH gene expression is downregulated in the growth plate of ACH patients and the same occurs in the ACH mutant mice (FIGS. 4C and 5G-5H). This correlated well with the fact that expression of the hedgehog signaling transducers GUI and Gli2 was markedly decreased as well in mutants receiving vehicle by P31 (FIGS. 5I-5J, brackets) compared to controls (FIGS.

[0111] 5C-5D). Indeed, the expression of the hedgehog signaling repressor GU3 was much higher in 18

[0112] 4936-1856-8059, V. 1 the mutants receiving vehicle (FIG. 5K) compared to controls (FIG. 5E). The Gli expression patterns had largely been renormalized in mutants receiving CD2665 (FIGS. 50, 5P and 5Q) along with restoration of Ihh expression (FIG. 5N) and growth plate organization (FIG. 5M). Endogenous cellular levels of retinoids are regulated by the cytoplasmic enzyme retinaldehyde dehydrogenase (RALDH) that converts retinal into biologically active retinoic acid (Evans et al., 2014). In control growth plates, Raldh3 gene expression was confined to the hypertrophic zone as expected (Williams et al., 2010) (FIG. 5F) but its expression had been increased and shifted toward the upper zones of the growth plate of mutants receiving vehicle (FIG. 5E). This shift likely deranged the normal endogenous basal retinoid gradients occurring in growth plate and likely caused an overall increase and derangement of retinoid levels. These changes also were prevented in mutant mice receiving CD2665 (FIG. 5R).

[0113] Example 2

[0114] As underlined above, the current FDA-approved treatment for ACH - Vosoritide - is not able to treat and rectify the defects in cranial base and associated structures usually seen in ACH patients, thus failing to address some of the major pathological complications of the disease. This is likely because Vosoritide is a protein and may not be able to diffuse into the cranial area and exert action. Thus, it was important to establish whether retinoid drugs such as CD2665 would in fact be able to address and treat cranial base defects in ACH. Like the axial and appendicular skeleton in growing children, the cranial base contains growth plates that are organized in tandem and are called synchondroses. Micro CT imaging of control mice at P35 (receiving vehicle or drug) showed that the intrasphenoidal synchondrosis (ISS) and the sphcnoccipital synchondrosis (SOS) were normal and open (FIGS. 6A, yellow arrowheads) and displayed typical histological growth plate zonal organization (FIGS. 6B and 6C). In mutants receiving vehicle however, the synchondroses appeared closed by micro CT analysis and their anatomical site was covered by bone (FIG. 6A, red arrowheads). In mutants receiving CD2665 however, it was clear that the synchondroses appeared largely open (Fig. 6A, green arrowheads). Histochemistry showed that compared to controls, the synchondrosis growth plates in mutants receiving vehicle were reduced in length and their zonal organization was less apparent, with a reduced hypertrophic zone (FIGS. 6B and 6C). In addition, the mutant synchondroses were flanked by a thick ectopic bone tissue (FIGS. 6B to 6D, red arrow) rather than perichondrium as seen in controls (FIGS. 6B to 6D, yellow arrows). These defects had been largely prevented in mutants receiving CD2665 and the synchondroses were flanked by a seemingly normal perichondrium (FIGS. 6B to 6D, green arrows).

[0115] 19

[0116] 4936-1856-8059, V. 1 Example 3

[0117] To verify that the beneficial effects on the appendicular and craniofacial skeleton in ACH mice can be exerted by other retinoid antagonists and are not limited to CD2665, the inventors tested the retinoid antagonist 7C. As above, control and ACH mice were administered vehicle or 7C (1.6 mg / kg) by gavage at P9 and were then all given a single injection of tamoxifen at PIO. Thereafter, mice continued to receive vehicle or 7C daily by gavage until P35. As seen above, body length was shorter in male and female mutant mice receiving vehicle by P35 (FIGS. 7B and 7C, second mouse from right) compared to controls, and this defect was ameliorated by administration of retinoid 7C (mouse on the right). Body weight accrual from PIO to P35 was also lower in ACH mutants receiving vehicle compared to controls (FIGS. 7D to 7G) and this deficiency also was ameliorated by treatment with 7C in a statistically significant manner (FIGS. 7E and 7G, blue circles versus red triangles).

[0118] These results were sustained by x-ray and micro CT analyses of long bones at P35. The tibia growth plates were normal and open in control male and female mice given vehicle or drug (FIG. 8A, panels on the left, yellow arrowheads). However, the growth plates were abnormal, interrupted and partially ossified in ACH mutants receiving vehicle (Fig. 8A, middle panels on the right, red arrowheads). These defects had been largely prevented by administration of retinoid 7C (FIG. 8 A, panels on the right, green arrowheads).

[0119] Quantification of skeletal element length and degree of mineralization verified that the shortening of tibia length in vehicle-treated mutants was ameliorated in mutants receiving 7C treatment (FIGS. 8B and 8C, red triangles) and resembled the controls (FIGS. 8B and 8C, blue circles). The same trends were observed with regard to growth mineralization that was excessive in mutants receiving vehicle but was ameliorated by 7C treatment (FIGS. 8D and 8E, red triangles), mimicking controls (FIGS. 8D and 8B, blue circles). These phenotypic changes and rectifications of were statistically significant (FIGS. 8B to 8E).

[0120] Next, the Inventors determined the effects of 7C treatment on cranial base synchondroses. Micro CT imaging showed that in male and female control mice at P35 given vehicle or 7C drug, the sphenoccipital synchondrosis (SOS) was evident and open (FIG. 9, yellow arrowheads) but was covered by bone and seemingly closed in mutants receiving vehicle (FIG. 9, red arrowheads). However, this defect had been clearly ameliorated in mutants receiving administration of 7C drug (FIG. 9, green arrowheads).

[0121] 20

[0122] 4936-1856-8059, V. 1 Example 4

[0123] Drug treatment can be associated with side effects that may elicit unwanted consequences and could introduce complications in therapies (Loke et al., 2011). A common test to assess side effects is general blood cell count and chemistry analysis. Peripheral blood was collected from WT and ACH mice receiving vehicle or CD2665 from PIO to P35. The resulting serum samples were analyzed by centralized facilities at the Inventors’ institution. Standard parameters evaluated included red blood cell (RBC) number, whole blood cell (WBC) number, hemoglobin levels, hematocrit, platelet number, mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH). For the most part, there were no major changes in these parameters after administration of vehicle or CD2665. One general observation was that these parameters appeared to be slightly decreased in mutants versus controls (regardless of treatment), a condition that may resemble pancytopenia.

[0124] Conclusion

[0125] The data obtained so far demonstrate that the retinoid antagonists CD2665 and 7C are able to counteract to a major and statistically significant extent the deleterious effects of Fgfr3G880Rmutant receptor action on skeletal growth, limb skeletal element length, body size, spine features and curvature, and cranial base synchondroses. In addition, drug administration can correct changes in expression of key growth plate regulatory genes including Ihh, Glil, Gli2 and Gli3. It can also restore the loss of Ki67- and PTHrP-expressing progenitor cells in the reserve zone that are essential for continuous and healthy growth plate function through the end of puberty (Mizuhashi et al., 2018). It is also important to note that the normal gradients of endogenous physiologic retinoids in growth plate are maintained by expression of Raldh3 restricted to the hypertrophic zone (Williams et al., 2010; Koyama et al., 2021). The Fgfr3G880Rmutant receptor alters such mechanisms by provoking an increase in Raldh3 expression and a shift toward the upper zones (FIG. 5L), another change rectified by CD2665 administration (FIG. 5R). In sum, the retinoid antagonist drags appear to exert comprehensive and concerted beneficial effects on ACH mutant growth plates, bringing several key phenotypic traits and regulatory mechanisms back to normal ranges and restoring normal skeletal growth, morphogenesis and mineralization patterns. These two drags as well as drags with similar chemical, biological and pharmacological characteristics are thus likely to provide a novel and effective treatment for human ACH.

[0126] 21

[0127] 4936-1856-8059, V. 1 As indicated above, human ACH is caused by activating mutations in FGFR3 which impair growth plate function, causing a steep decrease in chondrocyte proliferation and IHH gene expression. Notably, there are other human pediatric genetic diseases that are caused by activating mutations in FGFR2 such as Crouzon Syndrome or are caused by deficiencies in IHH such as Brachydactyly with Short Stature and Acrocapitofemoral Dysplasia (Unger et al., 2023). Our data so far indicate that the retinoid drugs can rectify defects caused by mutant FGFR3, including restoring Ihh expression in mouse growth plates. Thus, it is possible that the retinoid drugs may be able to serve also as treatments for pediatric conditions caused by mutations in FGFR2 and deficiencies in IHH.

[0128] * * * * * * * * * * * * * * * * *

[0129] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

[0130] 22

[0131] 4936-1856-8059, V. 1 VI. References

[0132] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0133] Alfos etal. (2001) Alcohol Clin, Exp. Res. 25, 1506-1514.

[0134] Chung et al. (2000) Mol. Brain Res. 83, 52-62.

[0135] Engberts et al. (2011) J. Child. Orthop. 6, 69-73.

[0136] Evans and Mangelsdorf (2014) Cell 157, 255-266.

[0137] Koyama <? / al. (2021) J. Bone Min. Res. 36, 1387-1402.

[0138] Kozhemyakina et al. (2015) Development 142, 817-831

[0139] Lefebvre and Bhattaram (2010) Curr. Op. Dev. Biol. 90, 291-317

[0140] Loke et al. (2011) Ther. Adv. Drug. Saf. 2, 59-68.

[0141] Maeda et al. (2010) Bone 46, 472-478.

[0142] Mizuhashi et al. (2018) Nature 563, 254-258.

[0143] Ornitz and Legeai-Mallei (2017) Dev. Dyn. 246, 291-309.

[0144] Ornitz and Marie (2015) Genes Dev. 29, 1463-1486.

[0145] Pauli (2019) Orphanet J. Rare Dis. 14, 1.

[0146] Savarirayan et al., (2020) Lancet 396, 684-692.

[0147] Szondy et al. (1997) Mol. Pharmacol. 51, 972-982.

[0148] Unger et al. (2023) Am. J. Med. Genet. 191, 1164-1209

[0149] Voitkamp et al. (1996) Science 273, 613-622.

[0150] Wang etal. (1999) Proc. Nat. Acad. Sci. USA 96, 4455-4460.

[0151] Williams et al., (2009) Dev. Biol. 328, 315-327.

[0152] Williams et al., (2010) J. Biol. Chem. 285, 36674-36681.

[0153] 23

[0154] 4936-1856-8059, V. 1

Claims

WHAT IS CLAIMED:

1. A method of treating a subject having or at risk of developing achondroplasia comprising administering to a subject in need thereof one or more retinoic acid receptor (RAR) antagonists.

2. The method of claim 1, wherein said subject has achondroplasia or is at risk of developing achondroplasia, such as determined by genetic testing (e.g., one or two copies of mutated FGFR3 gene).

3. The method of claim 1 or claim 2, wherein treating comprises direct treatment of a pediatric subject or a neonatal subject.

4. The method of any one of claims 1-3, wherein treating comprises indirect treatment of a prenatal subject by administration to a pregnant female patent of said prenatal subject.

5. The method of claim 4, wherein said pediatric subject is from about 2 to about 18 years of age.

6. The method of any one of claims 1-5, wherein the RAR antagonist includes one or more an RARy selective antagonist, an RARoc selective antagonist, an RARy / RARa selective antagonist, and / or an RARy / RARp selective antagonist.

7. The method of any one of claims 1-5, wherein the RAR antagonist is selected from the group consisting of AGN 193109, BMS 195814, BMS 195614, ER 50891, LE 135, LY 2955303, SR11335, LY 2813631, Ro 41-5253, AGN 193109, AGN194310, AGN194431, AGN19430L MM 11253, 7C and CD 2665.

8. The method of any one of claims 1-7, wherein more than one RAR antagonist is administered, such as 2, 3, 4 or more times.

9. The method of any one of claims 1-8, wherein a further achondroplasia therapy is administered to said subject.

10. The method of any one of claims 1-9, wherein administration is by oral, intravenous, intra-arterial, or intranasal or subcutaneous administration.244936-1856-8059, V.

111. The method of any one of claims 1-10, wherein an oral dose of said RAR antagonist is between 0.5 and 5.0 mg / kg daily, between 1.0 and 2.5 mg / kg daily, or about 1.5 mg / kg daily.

12. The method of any one of claims 1-11, wherein said RAR antagonist is administered daily up to about the age of 18 years.

13. The method of claim 12, wherein the regimen changes from daily to 2 or 3 times per week.

14. The method of any one of claims 1-13, wherein said subject exhibits inhibition of skeletal growth retardation and / or inhibition of growth plate aberration.

15. The method of any one of claims 1-14, wherein said subject does not receive concurrent treatment with a cyclooxygenase-2 inhibitor.

16. Use of one or more retinoic acid receptor (RAR) antagonists in the treatment or prevention of achondroplasia in a subject.

17. The use of claim 16, wherein the subject is a pediatric subject, such as about 2 to about 18 years of age.

18. The use of claim 16, wherein the subject is a prenatal or neonatal subject.

19. The use of claim 16, wherein said subject has achondroplasia or is at risk of developing achondroplasia, such as determined by genetic testing (e.g., one or two copies of mutated FGFR3 gene).

20. The use of any one of claims 16-19, wherein the RAR antagonist includes an RARy selective antagonist, an RARa selective antagonist, an RARy / RARa selective antagonist, or an RARy / RARp selective antagonist, such as an RAR antagonist is selected from the group consisting of AGN 193109, BMS 195814, ER 50891, LE 135, LY 2955303, MM 11253, 7C and CD 2665.254936-1856-8059, V. 1

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