Richard P. Blye

CDB-4124 and its putative monodemethylated metabolite, CDB-4453, are potent antiprogestins with reduced antiglucocorticoid activity: in vitro comparison to mifepristone and CDB-2914.

To obtain selective antiprogestins, we have examined the in vitro antiprogestational/antiglucocorticoid properties of two novel compounds, CDB-4124 and the putative monodemethylated metabolite, CDB-4453, in transcription and receptor binding assays and compared them to CDB-2914 and mifepristone. All four antiprogestins bound with high affinity to rabbit uterine progestin receptors (PR) and recombinant human PR-A and PR-B (rhPR-A, rhPR-B) and were potent inhibitors of R5020-induced transactivation of the PRE2-tk-luciferase (PRE2-tk-LUC) reporter plasmid and endogenous alkaline phosphatase production in T47D-CO human breast cancer cells. None of these compounds exhibited agonist activity in these cells. Induction of luciferase activity was potentiated about five-fold by 8-Br-cAMP under basal conditions and to the same extent in the presence of the PR antagonists. Mifepristone bound to rabbit thymic glucocorticoid receptors (GR) with approximately twice the avidity of the CDB antiprogestins. Inhibition of GR-mediated transcription of PRE2-tk-LUC was assessed in HepG2 human hepatoblastoma cells. Mifepristone exhibited greater antiglucocorticoid activity than CDB-2914, 4124, and 4453, about 12-, 22-, and 185-fold, respectively. Thus, while there was a good correlation between binding to PR and functional activity of these antiprogestins, GR binding was not predictive of their glucocorticoid antagonist activity. In agreement with our in vivo results, CDB-4124 and CDB-4453, as well as CDB-2914, are potent antiprogestins in vitro, but show considerably less antiglucocorticoid activity than mifepristone.

(−) Gossypol: an active male antifertility agent

The enantiomers of gossypol have been resolved by preparative HPLC of diastereomeric Schiffs base derivatives on a chiral bonded phase. Whereas (+)-gossypol has previously been reported to be inactive, (-)-gossypol is now shown to be active as a male oral antifertility agent in hamsters.

Development of the selective progesterone receptor modulator CDB-2914 for clinical indications

CDB-2914 (17 alpha-acetoxy-11 beta-[4-N,N-dimethylaminophenyl]-19-norpregna-4,9-diene-3,20-dione) is a 19-norprogesterone derivative that acts as an antagonist in progesterone-responsive tissues. It binds to progesterone receptors A and B with high affinity. After oral dosing in humans, CDB-2914 serum levels peak at 60-90 min. CDB-2914 binds to serum proteins and is cleared slowly. Doses of 1, 10 and 50 mg exhibit proportional increases in peak serum levels, but serum levels from higher doses, 100 and 200 mg, are not dose-dependent, suggesting saturation of carrier sites. The biological effects of CDB-2914 vary according to time of the menstrual cycle that the drug is given. In the mid-follicular phase, CDB-2914 (50 mg) inhibits follicular development and delays ovulation and menses. At 100 mg, in some cases the original follicle ceases development and a new follicle is recruited. Endometrial maturation is delayed at all doses tested (10, 50, 100 mg). Given at mid-luteal phase, there was a dose-dependent effect on menses, with higher doses (100-200 mg) resulting in earlier menses. On average, CDB-2914 tends to lengthen the menstrual cycle by approximately 1-2 days although the amount of delay varies with timing in the menstrual cycle and dose.

Disruption of Spermatogenesis and Sertoli Cell Structure and Function by the Indenopyridine CDB-4022 in Rats

Abstract The present studies were undertaken to determine the testicular cell type(s) affected by the antispermatogenic indenopyridine CDB-4022. At the oral threshold dose (2.5 mg/kg), CDB-4022 induced infertility in all males. CDB-4022 did not alter (P > 0.05) Leydig cell function as assessed by circulating testosterone, seminal vesicle, and ventral prostate weights or body weight gain compared to controls. Conversely, CDB-4022 reduced (P < 0.05) testicular weight, spermatid head counts, and percentage of seminiferous tubules undergoing spermatogenesis. In a second study, adult male rats received a maximally effective oral dose of CDB-4022 (12.5 mg/kg), dipentylphthalate (DPP; 2200 mg/kg; a Sertoli cell toxicant), or vehicle and were necropsied 3, 6, or 12 h after dosing to determine acute effects. Serum inhibin B levels were suppressed (P < 0.05) by 6 h after CDB-4022 or DPP treatment, but epididymal androgen-binding protein (ABP) levels were not altered (P > 0.05), compared to controls. CDB-4022 and DPP increased (P < 0.05) the percentage of tubules with apoptotic germ cells, particularly differentiating spermatogonia and spermatocytes, by 12 h after dosing. Microscopic examination of the testis indicated a greater degree of vacuolation in Sertoli cells and initial signs of apical germ cell sloughing/shedding by 3 or 12 h after CDB-4022 or DPP treatment, respectively. In a third study, prepubertal male rats were treated with vehicle, 12.5 mg/kg of CDB-4022, or 2200 mg/kg of DPP, and the efferent ducts of the right testis were ligated 23 h before necropsy. Seminiferous tubule fluid secretion (difference in weight of testes), serum inhibin B levels, and ABP levels in the unligated epididymis were reduced (P < 0.05) at 24 and 48 h after dosing in CDB-4022- and DPP-treated rats compared to controls. Collectively, these data suggest that CDB-4022 disrupts spermatogenesis by inducing apoptosis in early stage germ cells via a direct action on the Sertoli cell.

Antiovulatory and postcoital antifertility activity of the antiprogestin CDB-2914 when administered as single, multiple, or continuous doses to rats

The present studies in rats were undertaken to investigate the potential of a new antiprogestin, CDB-2914, for use as an emergency postcoital contraceptive for women. When given orally at noon on the day of proestrus, both CDB-2914 and mifepristone displayed dose-dependent antiovulatory activity; however, CDB-2914 was about eight times more potent than mifepristone. Both antiprogestins were considerably less potent in blocking ovulation when injected subcutaneously. To evaluate antifertility activity during continuous low dose administration, rats were dosed orally with 0.5 mg of either CDB-2914 or mifepristone daily, commencing on the day of estrus and continuing for 24 days. Females were cohabited with proven fertile males on day 8 of treatment and were removed 1-3 days later after confirmed mating. The pregnancy rate was significantly reduced (p < 0.05) only in the CDB-2914-treated females; however, the mean number of normal implantation sites per pregnant rat was significantly reduced (p < 0.05) by mifepristone as compared with the vehicle control group. CDB-2914 was also found to prevent pregnancy when administered orally after mating from days 0-3 during tubal egg transport, or from days 4-6 during the pre- and peri-implantation periods. To determine the day of maximal sensitivity to CDB-2914, a single 2-mg dose per rat was given orally on days 0, 1, 2, 3, 4, or 5 postmating. This dose of CDB-2914 was without effect on pregnancy at days 0, 1, 2, or 3 postmating. In contrast, 2 mg CDB-2914 per rat was highly effective in blocking pregnancy when given on either day 4 or 5 postmating. Collectively, these data demonstrate that CDB-2914 is an orally active postcoital antifertility agent that is more potent than mifepristone in the rat. Hence, CDB-2914 may prove to be an effective emergency postcoital contraceptive in women.

Lupron Depot Prevention of Antispermatogenic/Antifertility Activity of the Indenopyridine, CDB-4022, in the Rat

Abstract The goals of this study were to determine the CDB-4022 dose-response relationship for induction of acute decreases in testicular weight and germ cell depopulation in rats; establish the threshold dose of CDB-4022 required to induce infertility; and investigate whether CDB-4022-induced testicular damage could be prevented by a GnRH agonist (Lupron Depot). Reduction of testis weight and germ cell depopulation were observed 7 days after a single oral dose of 1 mg CDB-4022/kg, whereas 0.5 mg/kg had no observable effect. These effects were maximal at 12.5 or 25 mg CDB-4022/kg. After a single oral dose of either 2.5 or 5 mg/kg, CDB-4022 induced infertility in five of five treated rats by Week 5, whereas only one of five males was rendered infertile at a dose of 1 mg/kg. Proven fertile male rats (6/group) were treated with vehicle, CDB-4022 alone (2.5 mg/kg on Day 0), CDB-4022 plus Lupron Depot (on Weeks −1, 2, 5, and 8), or Lupron Depot alone. Control males demonstrated normal fertility throughout a 32-wk cohabitation period. Five of six rats were rendered transiently infertile with Lupron Depot alone, but all recovered fertility. CDB-4022 treatment resulted in infertility in all six rats, and only one of six regained fertility. Combined treatment also caused infertility in all six rats, but four of six recovered fertility (P = 0.08 compared to CDB-4022 alone). Testicular weight was decreased in the three treatment groups compared to vehicle controls; testicular weights were ranked from highest to lowest as follows: vehicle > Lupron Depot > Lupron Depot + CDB-4022 > CDB-4022. The tubule differentiation index of Lupron Depot-treated rats (96 ± 4%) was not different from vehicle-treated rats (100%). CDB-4022 treatment decreased the number of differentiating tubules (15 ± 8%). Lupron Depot plus CDB-4022 treatment resulted in a greater number of differentiating tubules (53 ± 12%) than CDB-4022 alone, but this was still lower than vehicle- or Lupron Depot-treated rats. These data indicate that 2.5 mg/kg of CDB-4022 was the oral threshold dose that caused testicular damage rendering the majority of adult male rats permanently infertile within the study interval; 12.5 mg/kg of CDB-4022 induced maximal testicular damage. Suppression of gonadotropins and/or testosterone production by treatment with Lupron Depot before and after CDB-4022 prevented the CDB-4022-induced irreversible testicular damage.

The use of estrogens as postcoital contraceptive agents

Tubal motility, oviduct flow, and ciliary activity are all influenced by estrogens. Tubal insufflation studies suggest that estrogens cause closure of the uterotubal junction in women. Estrogen action may reflect a release from modifications imposed by progesterone and may threaten the functional integrity of the corpus luteum under hypothalamic-hypophyseal control. Board and Bhatnagar have shown reduced progesterone levels with administration of diethylstilbestrol. Decrease of BBT following use of ethinyl estradiol and diethylstilbestrol support the theory of the luteolytic action of estrogen. After nidation, estrogens have no effect. Haspels reported on 2000 women treated with ethinyl estradiol (2-5 mg) or diethylstilbestrol (25-50 mg) administered for 5 consecutive days. Method failure was reported with 3 mg/day ethinyl estradiol and 25-30 mg/day diethylstilbestrol. No pregnancies were reported at 5 mg/day ethinyl estradiol and 50 mg/day diethylstilbestrol. Kuchera reported on 1000 cases with no pregnancies following treatment with 50 mg diethylstilbestrol administered within 72 hours of unprotected coitus (for a pregnancy rate of 2.5/1000). Other estrogens have been used successfully as postcoital contraceptive agents. Use of conjugated equine estrogens administered orally or intravenously within 72 hours of exposure prevented pregnancy. Dienestrol and dienestrol combined with ethynodiol acetate (progestogen) prevented pregnancy. Depot estradiol administered to 12 patients resulted in 2 ectopic pregnancies. Slow release of this estrogen could account for its high failure rate, although results suggest an effect on zygote transport. It is concluded that the best protection against pregnancy will be obtained by administering estrogen as soon as possible after coitus (preferably within 24 hours, no later than 72 and continued for 5 consecutive days).

Long-acting contraceptive agents: structure activity relationships in a series of norethisterone and levonorgestrel esters.

A large number of esters of norethisterone (17 alpha-ethynyl-17 beta-hydroxyestr-4-en-3-one) and levonorgestrel (D-(-)-13 beta-ethyl-17 alpha-ethynyl-17 beta-hydroxygon-4-en-3-one) were synthesized and tested for biological activity. The test employed in these studies was the duration of estrus suppression in cycling mature rats. In the norethisterone series several esters exhibited duration of activity comparable to that of norethisterone enanthate. In the levonorgestrel series the butanoic, cyclobutylcarboxylic and cyclopropylcarboxylic esters were longer acting than medroxyprogesterone acetate (17 alpha-acetoxy-6 alpha-methylpregn-4-ene-3,20-dione) when prepared as aqueous microcrystalline suspensions.

Recovery of reproductive function in rats treated with the aromatase inhibitor fadrozole

The aromatase inhibitor, fadrozole hydrochloride (CGS 16949A), was developed for the treatment of breast cancer, and has not been available for pediatric use because of the lack of information about potential reproductive toxicology. To determine the effect of fadrozole on subsequent fertility and reproductive performance in rats, peripubertal male and female Sprague-Dawley rats (10/group) were given fadrozole by oral gavage once a day for 60 consecutive days (age 21 through 80 d) at a dose of 0, 1200, or 6000 micrograms/kg/d (dose range in women with breast cancer: 60 to 240 micrograms/kg/d). Following a 30-d recovery period (days 81 through 111 of age), cohabitation with untreated rats of the opposite sex was accomplished for 30 d or until positive evidence of mating was obtained (daily vaginal smears). The nonfadrozole-treated males used for cohabitation were proven fertile breeders; the females were virgin with proven 4-d estrous cycles. The duration of pregnancy, number, sex, condition, and body weight of pups were determined. Pregnant rats were weighed on gestational days 7, 14, and 20. There was a profound decrease in the number of estrous cycles at both dose levels of fadrozole compared to the control (P < 0.001). During the 30-d recovery period, estrous cycles were reestablished within a few days in the treated rats and the number and length of estrous cycles were not statistically different between fadrozole-treated and control rats. The gestational body weights of fadrozole-treated and untreated females did not differ significantly. There were no statistically significant differences in the number of matings/number of pairings, gestational length, mean live pups/litter, % pups born alive/litter, and % male pups/litter in the three groups (vehicle-, low-, and high-dose fadrozole-treated females, cohabited with untreated males and fadrozole-treated males, cohabited with untreated females). Thus, young male and female rats treated for 60 d with large doses of fadrozole had no detectable adverse effect on subsequent reproductive function.

11β-Substituted 13β-ethyl gonane derivatives exhibit reversal of antiprogestational activity

Abstract The syntheses of three 17α-acetoxy-13β-ethyl-11β-aryl-18,19-dinorpregna-4,9-diene-3,20 diones from levonorgestrel are described. Despite their close structural similarity to the antiprogesterone CDB-2914, one of the compounds exhibits agonistic progestational activity, and the other two compounds are totally inactive.