Development of the retinoic acid receptor alpha-specific antagonist YCT-529 for male contraception: A brief review

Abstract

Genetic studies in mice have demonstrated that retinoic acid receptor alpha (RARα) deficiency leads to male infertility without affecting overall viability, suggesting that pharmacological inhibition of this receptor could be a viable contraceptive strategy. This review describes the use of experimental approaches to develop RARα-selective antagonists for male contraception. Initial studies with BMS-189453, a pan-RAR antagonist, showed significant testicular degeneration and reversible infertility in mice. The search for RARα-specific antagonists led to the development of YCT-529, a potent and selective RARα antagonist with favorable pharmacokinetics. YCT-529 demonstrated excellent in vivo efficacy in inhibiting spermatogenesis and inducing infertility in mice, with fertility recovery following drug discontinuation. YCT-529 is now in clinical development as a candidate for male contraception.

1. Introduction

Vitamin A has long been recognized as an essential micronutrient (1), with its dietary deficiency (VAD) linked to serious health issues, including heightened infection risk, growth retardation, ocular conditions such as night blindness and xerophthalmia, and of particular relevance to the present discussion, reproductive health. Indeed, in VAD rodents, male sterility due to disrupted spermatogenesis was established as early as the 1920s (2).

Dietary vitamin A (retinol) undergoes sequential metabolic processes as part of the extensively studied vitamin A metabolite or retinoid signaling pathway. Initially, cytosolic alcohol dehydrogenases (ADHs) and microsomal retinol dehydrogenases (RDHs) convert retinol to retinaldehyde, which is further oxidized by three retinaldehyde dehydrogenases (RALDH1, RALDH2, and RALDH3) to produce all-trans (ATRA) or 9-cis retinoic acid (3), which function intra-cellularly by binding to the nuclear factors, retinoic acid receptor (RARs) or retinoid-X receptors (RXRs). RARs and RXRs are families of isoforms designated as α, β, and γ.

2. Focus on retinoic acid receptor-alpha

Genetic ablation approaches have shown that retinoic acid receptor alpha (RARα) is essential for spermatogenesis. Mice deficient in RARα are viable, but the males are sterile (4). RARα-deficient testes exhibit distinct abnormalities in spermatogenesis, including a temporary arrest at step 8-9 spermatids in the first wave, delayed development of preleptotene/leptotene (PL/L) spermatocytes in the second wave, and an accumulation of PL/L spermatocytes in the third wave (5). Additional issues include abnormal cellular associations due to asynchronous spermatogenic progression, reduced germ cell proliferation, increased apoptosis in elongating spermatids during their early transformation phase, improper orientation of step 8-9 spermatids relative to the lumen, entrenchment in Sertoli cells, and defects in spermiation at stage VIII.(4–8) These abnormalities occur primarily at stages VIII-IX of the seminiferous epithelium cycle (Fig. 1), (9) which coincides with the peak of expression of RARα.

Figure 1.

Diagrammatic representation of the spermatogenic cycle illustrating the profound abnormalities in RARα-deficient mice clustered in stage VIII–IX tubules. Details of the symbols used in the staging map shown can be found in Russell et al.(29). The green bar line indicates the particular stage, stage VIII, that showed the highest frequency of cellular abnormalities. Red arrows point to the specific cell type at stage VIII, where various abnormalities were found. Reprinted with permission from Wolgemuth and Chung (9).

While these gene ablation analyses provide unequivocal evidence for the essential and specific role of RARα in male fertility and hence its consideration as an attractive target for inducing male sterility, the irreversible nature of genetic disruption of RARα makes such approaches unsuitable for male contraception. We, therefore, turned to pharmacologic approaches using retinoid receptor antagonists to reversibly target its function during spermatogenesis.

BMS-189453 (Fig. 2), a pan-RAR antagonist that targets RARα and also RARβ and RARγ, was initially characterized as a “testicular toxin,” causing significant testicular degeneration in adult rats (10). We explored the effects of RAR antagonism utilizing BMS-189453, which is orally bioactive, on inhibiting spermatogenesis (11). Oral administration of BMS-189453 at a dose of 5 mg/kg for seven days in mice led to immediate disruptions in spermiogenesis, notably the failure of spermatid translocation and release (11). Several stages of germ cell differentiation were also adversely affected, with effects observable within one month or less (11). Importantly, these effects were specific to the testes, with no observed side effects, including no changes in testosterone levels (11). Furthermore, fertility inhibition was reversible, as demonstrated by the healthy and fertile progeny (22 males and 22 females) produced by two recovered males (11).

Figure 2.

Structures of BMS-189453 and YCT-529 and associated transactivation assay results (26, 28).

Further studies on the dosing period and dosage requirements revealed that infertility could be induced with doses as low as 1 mg/kg, achieving 100% sterility by the end of the treatment period, followed by a rapid recovery of normal spermatogenesis (12). Importantly, the observed abnormalities in the testes were similar to those seen in RARα-deficient mice (Fig. 1) (11, 12). Even with long-term dosing, no changes in testosterone levels were observed, and the progeny from the recovered males exhibited no abnormalities (12). Interestingly, a more rapid recovery was noted upon cessation of long-term administration (16 weeks versus 4 or 8 weeks) at a 1 mg/kg/day dose (12).

Given the genetic data showing that RARα was the essential retinoid receptor for regulating spermatogenesis, we examined the effects of available RARα-selective antagonists, instead of pan-RAR antagonists, on spermatogenesis in male mice. However, oral administration of the RARα-selective antagonists BMS-189532 and BMS-195614 at doses of 2 mg/kg or 10 mg/kg for seven days resulted in minimal or no inhibition of overall spermatogenesis, likely due to poor bioavailability (13).

Consequently, we wished to develop a new, orally active RARα-selective antagonist specifically for male contraception. RARs exert their function as nuclear proteins crucial in regulating gene expression by binding to coactivators (14). When the in vivo ligand ATRA or its synthetic analog agonists bind to the ligand-binding domain of RARs, they induce a conformational change in helix 12 (H12) that facilitates coactivator recruitment (14). However, introducing a bulky substituent to the agonist creates steric hindrance by moving H12 into an inactive conformation and inhibiting coactivator binding (14–17). This strategy has successfully led to the development of several RAR antagonists (18–21).

Despite the structural similarities among the three RAR subtypes, the unique presence of serine 232 in RARα, compared to alanine residues at the corresponding position in RARβ and RARγ, allows for the selective targeting of RARα (22–25). Synthetic retinoids designed with H-bond donors can interact specifically with serine 232, achieving RARα selectivity (22, 26). The design of the new RARα-selective antagonist builds on this principle, incorporating a hydrophobic ring and terminal benzoic acid to mimic all-trans-retinoic acid, a bulky substituent to induce antagonism, and an H-bonding donor linker to recognize RARα specifically (27).

3. Identification of YCT-529

Through structure-activity relationship (SAR) analysis, we identified YCT-529 (Fig. 2), which features a chromene hydrophobic ring, a pyrrole linker, a benzoic acid moiety, and a toluene substituent. YCT-529 demonstrates strong selectivity for RARα, with an IC₅₀ of 6.8 nM, while showing no activity at concentrations as high as 3300 nM against RARβ and RARγ and, unlike BMS-189453, no agonist activity (28). Modifications to the chromene, toluene, and benzoic acid components of the molecule resulted in reduced activity, confirming the importance of these structural elements.

YCT-529 has a Log D of 3.5 and is soluble at physiological pH (79 μM). Further evaluations of metabolic stability, hERG activity, Mini Ames, HepG2 and human lung fibroblast cytotoxicity, chromosomal aberration studies, a rat micronucleus study, and a Pharmaron 47 off-target panel indicate high metabolic stability, no toxicity, no mutagenicity, and no significant off-target effects. Pharmacokinetic studies in mice revealed peak plasma levels of 2.1 μM at 1 hour post-dose, with a half-life of 11 hours and significant testicular exposure (28). At a single oral dosage of 1000 mg/kg, no necropsy alerts were observed, and a dosage of 500 mg/kg showed no adverse effects on blood urea nitrogen, creatinine kinase, creatinine, aspartate aminotransferase, white blood cells, lymphocytes, platelets, or monocytes (28).

4. In vivo effects of YCT-529 on male fertility

To determine if YCT-529 could inhibit spermatogenesis in vivo, a pilot study was performed in which adult male mice were treated with oral administration of the drug at 10 mg/kg/day for 2 weeks (n=10) (28). One day after the cessation of drug treatment (CDT), there was a drop in the cauda epididymal sperm counts, although overall testicular weights did not decrease significantly (28). Histological analysis revealed reduced numbers of sperm and germ cell sloughing in the epididymis and a failure of spermatid translocation and sperm release in stage VIII*-IX* tubules (28). Four weeks post-CDT, sperm counts were still reduced in half of the males, consistent with the histological observation of proper spermatid translocation but a failure of sperm release in most of the testes (n=8 out of 10). A limited mating assessment in this pilot study revealed that 3 of the 10 males failed to yield pregnancies after mating for 2 weeks (3-4 weeks post-CDT), suggesting that YCT-529 can affect male fertility.

A follow-up full-scale study revealed that the patterns of changes in testis weight and sperm counts and the morphological abnormalities observed in the pilot study were again observed in males one day and 4 weeks post-CDT (28). Half of the males (n=5 of 10) failed to yield pregnancies after mating for 2 weeks (3-4 weeks post-CDT) (28). Importantly, the morphological abnormalities in the testes occurred in the same stages shown to be affected in the RARα-deficient mice (Fig. 1).

To further assess the effectiveness of YCT-529 in inducing sterility, two dosing regimens were utilized: 20 mg/kg/day orally for two weeks and 10 mg/kg/day orally for four weeks.(28). Both regimens were highly effective in inhibiting spermatogenesis and reducing sperm counts and testicular weight, with histological abnormalities resembling those seen in RARα-deficient testes ((28); Fig.1). Mating studies confirmed the high efficacy of YCT-529 in inducing infertility, in particular with the 10 mg/kg/day for 4 weeks dosage, reducing the number of embryos by almost 100%. Importantly, fertility began to recover within four weeks of discontinuing YCT-529 administration.

5. Conclusions and future directions

Thus, we suggest that YCT-529 represents an orally bioavailable, effective, and reversible RARα-selective antagonist and, therefore, a promising candidate for male contraception. YCT-529 was licensed to YourChoice Therapeutics, which conducted additional animal studies in non-human primates and preclinical safety studies for the first-in-human Phase I clinical trial of a male non-hormonal contraceptive that commenced in December 2023 (NCT06094283) and was completed in June 2024. A Phase 1a/2a clinical trial (NCT06542237) started in September 2024.