Targeting the retinoid signaling pathway with YCT-529 for effective and reversible oral contraception in mice and primates

Abstract

Background: The retinoic acid receptor alpha (Rarα) has been validated as a male contraceptive target by genetic knockouts resulting in male sterility. The effects on spermatogenesis in the absence of RARα resemble the loss of RAR signaling in vitamin A deficiency, and the mice are otherwise normal. The effects on spermatogenesis in animals treated orally with the dual RARα/RARγ antagonist BMS-189453 closely phenocopies the absence of RARα function. Notably, the resulting male sterility is reversible. We, therefore, wished to identify RARα-selective inhibitors for potential male non-hormonal contraception.

Methods: YCT-529 was investigated for RARα selective inhibition, physicochemical characteristics, oral bioavailability, and pharmacokinetic properties in mice and non-human primates. It was assessed in mouse mating trials to determine the most effective dosing regimen to induce infertility in male mice and in male non-human primates to reduce sperm levels.

Results: Characterization of YCT-529 shows suitable biochemical, physicochemical, and pharmacokinetic properties for in vivo testing. YCT-529 inhibits mouse fertility of male mice within 4 weeks of oral administration, correlating with disrupted spermatogenesis demonstrating specific inhibition of the RARα pathway. Within 6 weeks after cessation of dosing, mouse fertility reverses. Furthermore, YCT-529 inhibits sperm production in a non-human primate model within 2 weeks of oral dosing without adverse side effects. Within 10-15 weeks after cessation of dosing, non-human primates' sperm counts fully reverses.

Conclusions: These results lay the groundwork for evaluating YCT-529 in human clinical trials.

Fig. 1. Chemical structures of retinoic acid and RAR antagonists, and in vitro ADMET data.

a Structures of agonist all-trans-retinoic acid, antagonists BMS-189453, BMS-189532, and YCT-529. The general ligand scaffold consists of a hydrophobic ring system (blue) and a conjugated acid or a benzoic acid (green) that are connected by a linker (pink). The linker group determines receptor subtype specificity. A large substituent on the hydrophobic ring (black) is required for antagonist activity. b IC₅₀ data for YCT-529, BMS-189453, and BMS-189532, and EC₅₀ data for YCT-529 against RARα, RARβ, and RARγ using transactivation assays. ^aTransactivation assays. ^bHighest concentration tested. ^a,bAverage of at least three independent experiments. The in vitro ADMET assays were performed by Pharmaron, except for the chromosomal aberration assay and the rat micronucleus study, which were conducted by WuXi AppTec.

Fig. 2. Acute disruptive effects of modified regimens of YCT-529 on spermatid alignment and release in testicular tubules immediately post-CDT and induction of infertility as assessed by mating studies.

a–f Representative testicular and epididymal histological sections of male mice (n = 8 out of 10) treated with 10 mg/kg/day of YCT-529 for 2 weeks and terminated one day post-CDT. Cont, control sample; Pl, preleptotene spermatocytes; L, leptotene spermatocytes; P, pachytene spermatocytes, RS, round spermatids; Z, zygotene spermatocytes. Arabic numerals indicate the step of spermatid differentiation. Roman numerals indicate the stage of the tubules. Although abnormal cell associations complicate staging, an attempt was made to stage the drug-treated tubules using the acrosomal system. Drug-treated tubules are, therefore, labeled with a Roman numeral followed by an asterisk (e.g., stage IX*). Failure of spermatid translocation and sperm release in the tubules. c, d Bracket in stage IX* tubules indicates retained spermatids. Reduced numbers of sperm and germ cell sloughing in the epididymis (e). Scale bar in a-f, 50 µm. Infertility was induced more quickly in male mice treated with 10 mg/kg/day for a longer dosing regimen of YCT-529 of 4 weeks (h) with a full recovery of spermatogenesis, as compared to a higher dose of 20 mg/kg/day for 2 weeks (g). Males used for the mating studies were euthanized at 8–12 weeks post-CDT. They exhibited normal testicular weight and cauda epididymal sperm counts (53.90 × 106 for the mating± 9.34 versus control 55.5 × 106 ± 8.18, n = 10). i–l Representative testicular and epididymal histological sections of male mice treated with 20 mg/kg/day of YCT-529 for 2 weeks and terminated one day post-CDT. Morphological abnormalities included reduced numbers of epididymal sperm and germ cell sloughing, a failure of spermatid translocation (i, #1, 29.3%; #2, 7.5%), abnormal round spermatids (j, #1, 37%; #2, 60.4%), and multinucleated giant cells (k, #1, 1.4%; #2, 12.7%). Many of those abnormal round spermatids showed crescent-like chromatin condensation with juxtanuclear clear spaces (n = 2). Pl, preleptotene spermatocytes; L, leptotene spermatocytes; P, pachytene spermatocytes; B, type B spermatogonia; RS, round spermatids. Brackets in stage IX* tubules indicate retained spermatids. Arabic numerals indicate the step of spermatid differentiation. Roman numerals indicate the stage of the tubules. Drug-treated tubules are labeled with a Roman numeral followed by an asterisk (e.g., stage IX*). Scale bar in i-l, 50 µm.

Fig. 3. Assessment of spermatogenesis in testes 4 weeks after treatment with higher dose.

a–f Histological sections of testes and epididymides from mice treated with 20 mg/kg/day of YCT-529 for 2 weeks and terminated 4 weeks post-CDT. Several males still exhibited a failure of spermatid release (a, #1,16.7%; #2, 14.3% and #3, 8.5%), an excess of elongated spermatids (b, 5%, n = 1) aligned at the tubular lumen, along with tubules almost completely lacking entire layers of specific germ cells (c–e, #1, 36%; #2, 19.4%; #3, 25.8%) and reduced numbers of epididymal sperm. Pl, preleptotene spermatocytes; L, leptotene spermatocytes; P, pachytene spermatocytes; Z, zygotene spermatocytes; RS, round spermatids; ES, elongated spermatids; MI/MII, meiosis I and II; 2^o, secondary spermatocytes. #?, the reduced number of specific cell types in a particular layer is indicated with pound symbol and question mark; RS, 3?, round spermatids, three only. Brackets in stage IX* tubules indicate retained spermatids. Arabic numerals indicate the step of spermatid differentiation. Roman numerals indicate the stage of the tubules. The drug-treated tubules are labeled with a Roman numeral followed by an asterisk (e.g., stage IX*). Scale bar in (a–f), 50 µm.

Fig. 4. The temporal and cell-specific disruption of spermatogenesis was more severe in testes at one day post-CDT with 10 mg/kg for 4 weeks vs 20 mg/kg for 2 weeks.

a–e Histological testicular and epididymal sections of male mice treated with 10 mg/kg/day of YCT-529 for 4 weeks and terminated one day post-CDT. Histological evaluation revealed more severely disrupted testicular morphology than at the same time point in the higher-dose regimen, with tubules displaying either pachytene spermatocytes or round spermatids as the most advanced germ cells (a, #1: 89.1%; #2: 96.3%; #3: 76.4%, n = 3). Pl, preleptotene spermatocytes; L, leptotene spermatocytes; P, pachytene spermatocytes; Z, zygotene spermatocytes; RS, round spermatids; SC, Sertoli cells. Arabic numerals indicate the step of spermatid differentiation. Roman numerals indicate the stage of the tubules. The drug-treated tubules are labeled with a Roman numeral followed by an asterisk (e.g. stage IX*). Scale bar in (a–e), 50 µm.

Fig. 5. A fast recovery of spermatogenesis in testes of males treated with longer dosing period and examined 4 weeks post-CDT.

a–d Histological testicular and epididymal sections of male mice treated with 10 mg/kg/day of YCT-529 for 4 weeks and terminated 4 weeks post-CDT. A recovery of spermatogenesis was observed in the majority of tubules (a #1: 79.6%; #2: 92.5%; #3: 97.9%; #4: 85.8%; #5: 82.3%, n = 5) with some remaining residual characteristic abnormalities, in particular failure of sperm release (b #1: 5%; #2: 5.2%; #3: 2.1%; #4: 2.3%; #5: 1.8%, n = 5). Other characteristic residual abnormalities included tubules with either pachytene spermatocytes or round spermatids as the most advanced germ cells (, #1: 9.5%; #2: 0.9%; #3: 0%; #4: 11.9%; #5: 16.4%, n = 5) and reduced numbers of epididymal sperm and some tubules almost completely lacking entire layers of specific germ cells were observed (#1: 5.9%; #1: 2.3%, n = 2). Pl, preleptotene spermatocytes; L, leptotene spermatocytes; P, pachytene spermatocytes; Z, zygotene spermatocytes; D, diplotene spermatocytes; RS, round spermatids; ES, elongated spermatids; B, type B spermatogonia; SC, Sertoli cells; SG, spermatogonia. MI/MII, meiosis I and II. Bracket in stage IX* tubules indicates retained spermatids. Arabic numerals indicate the step of spermatid differentiation. Roman numerals indicate the stage of the tubules. The drug-treated tubules are labeled with a Roman numeral followed by an asterisk (e.g., stage IX*). Scale bar in a–d, 50 µm.

Fig. 6. Decrease and recovery of sperm counts and unchanged hormone levels in cynomolgus monkeys.

Three sexually mature male animals were dosed per cohort. Fresh semen samples were obtained via electroejaculation at indicated time points. Cohort 1 (n = 3) was dosed with YCT-529 at 0.5 mg/kg/day (Day 1-46) followed by 1.5 mg/kg/day (Day 47-51), 2.5 mg/kg/day (Day 52-101) and 5 mg/kg/day (Day 102-108); the recovery phase was from Day 109 to Day 257. Cohort 2 (n = 3) was dosed with YCT-529 at 5 mg/kg/day (Day 1–30), followed by 7.5 mg/kg/day (Day 31-37); the recovery phase was from Day 38 to Day 145. The blue dashed line in panels (a) and (b) indicates the upper limit of reported sperm counts of non-breeders. Shown are the individual sperm counts per ejaculate (light grey, dark grey, and black lines) of Cohort 1 (a) and Cohort 2 (b) animals. Blood from all Cohort 1 and Cohort 2 animals was collected at pre-dose, 24 h post-CDT (end of the dosing period [EoD]), as well as 78 and 148 days (Cohort 1), and 93 and 107 days (Cohort 2) post-CDT (end of the recovery period [EoR]) to determine serum hormone levels with ELISA. Each sample was measured in duplicate. Shown are free serum testosterone (c), FSH (d) and inhibin-B (e) levels as individual historical control values (white circles) of 150 control animals and individual values of all Cohort 1 animals (light grey circles) and Cohort 2 animals (dark grey circles) at pre-dose, EoD and EoR. White striped, light grey, and dark grey bars represent mean values from all 150 historical control animals, all 3 Cohort 1 animals, and all 3 Cohort 2 animals, respectively, ± S.D. (in blue). No significant differences were noted as per one-way ANOVA analyses.