The potent synthetic androgens, dimethandrolone (7α,11ß-dimethyl-19-nortestosterone) and 11ß-methyl-19-nortestosterone, do not require 5α-reduction to exert their maximal androgenic effects.

Dimethandrolone (DMA: 7α,11ß-dimethyl-19-nortestosterone) and 11ß-methyl-19-nortestosterone (MNT) are potent androgens in development for hormonal therapy in men. As 5α-reduced androgens, such as 5α-dihydrotestosterone (DHT), may raise the risk of benign prostate hyperplasia, accelerate the development of prostate carcinoma, and increase male pattern baldness and acne, we investigated the role of 5α-reduction in the androgenic activity of DMA and MNT. The authentic 5α-reduced metabolites, 5α-dihydroDMA (5α-DHDMA) and 5α-dihydroMNT (5α-DHMNT), were prepared by chemical synthesis and compared in vitro and in vivo to the parent compounds. Both 5α-reduced androgens bound with high affinity to the rat androgen receptor (AR) and were potent inducers of transactivation of 3XHRE-LUC in CV-1 cells cotransfected with a human AR expression plasmid. To examine in vivo androgenic (stimulation of ventral prostate [VP] and seminal vesicle [SV] weights) and anabolic (stimulation of levator ani [LA] muscle weights) activity, 22-day-old castrate male rats were treated sc for 7 days with various doses of DMA, 5α-DHDMA, or testosterone (T) or MNT, 5α-DHMNT, or T and necropsied on day 8. 5α-DHDMA was at least threefold more potent than T in stimulating growth of the VP but only 30-40% as potent as DMA. 5α-DHMNT was four- to eightfold more potent than T, whereas MNT was approximately equipotent to T. To assess the possible role of 5α-reduction in VP and SV growth, castrate immature rats were treated with maximally effective doses of T, DHT, DMA, MNT, or the related 19-norandrogen, 7α-methyl-19-nortestosterone (MENT), or vehicle, with or without dutasteride (DUT), an inhibitor of 5α-reductases types 1 and 2. In rats treated with T+DUT, serum T was significantly higher (P<0.05) than in rats treated with T alone, and serum DHT was decreased (P<0.001) to levels observed in castrate vehicle-treated rats. DUT significantly reduced both VP and SV weights in T-treated rats, whereas there was no significant effect of DUT on weights of these accessory sex glands in rats treated with DMA, MNT, DHT, or MENT. These results indicate that inhibition of 5α-reductase activity in vivo does not affect the androgenic potency of DMA, MNT, or MENT.

Effects of synthetic androgens on liver function using the rabbit as a model.

The objective of this study was to determine whether the rabbit was a suitable model to test new synthetic androgens for potential liver toxicity within a short dosing interval. Adult male rabbits were dosed orally daily on days 0-13 with 17α-methyltestosterone (MT) as a positive control and testosterone (T) as a negative control to validate this model. Synthetic androgens tested were: 7α-methyl-19-nortestosterone (MENT), dimethandrolone-undecanoate (DMAU), and 11ß-methyl-19-nortestosterone-17ß-dodecylcarbonate (11ß-MNTDC). Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transpeptidase (GGT), and sorbitol dehydrogenase (SDH), as well as clearance of intravenous injected bromsulfonphthalein (BSP) from serum on days 0, 7, and 14, were determined. As expected, T (10 mg/kg/d) did not adversely affect BSP retention or serum liver enzymes. MT (10 mg/kg/d) increased BSP retention, and AST, ALT, GGT, and SDH levels, indicating that this model could detect androgens known to be hepatotoxic. DMAU and MENT (10 mg/kg/d) increased BSP retention and all 4 serum liver enzymes as well, but the effects were less than those observed with MT at the same dose. All parameters returned to baseline 2 weeks after cessation of dosing. 11ß-MNTDC at 10 mg/kg/d did not have an effect on BSP retention or liver enzymes, but a slight increase in serum GGT levels was observed in rabbits treated with 25 mg/kg/d. For the androgens that exhibited liver toxicity at 10 mg/kg/d (MT, DMAU, and MENT), a no-observed-effect level of 1 mg/kg/d was established. Overall ranking of the synthetic androgens from most to least hepatotoxic on the basis of percent BSP retention was: MT & DMAU > MENT > 11ß-MNTDC. Hence, the rabbit appears to be a promising model for detection of potential liver toxicity by synthetic androgens using BSP clearance and serum liver enzyme levels as early indicators of injury.

Dimethandrolone (7alpha,11beta-dimethyl-19-nortestosterone) and 11beta-methyl-19-nortestosterone are not converted to aromatic A-ring products in the presence of recombinant human aromatase.

Dimethandrolone undecanoate (DMAU: 7alpha,11beta-dimethyl-19-nortestosterone 17beta-undecanoate) is a potent orally active androgen in development for hormonal therapy in men. Cleavage of the 17beta-ester bond by esterases in vivo leads to liberation of the biologically active androgen, dimethandrolone (DMA), a 19-norandrogen. For hormone replacement in men, administration of C19 androgens such as testosterone (T) may lead to elevations in circulating levels of estrogens due to aromatization. As several reports have suggested that certain 19-norandrogens may serve as substrates for the aromatase enzyme and are converted to the corresponding aromatic A-ring products, it was important to investigate whether DMA, the related compound, 11beta-methyl-19-nortestosterone (11beta-MNT), also being tested for hormonal therapy in men, and other 19-norandrogens can be converted to aromatic A-ring products by human aromatase. The hypothetical aromatic A-ring product corresponding to each substrate was obtained by chemical synthesis. These estrogens bound with high affinity to purified recombinant human estrogen receptors (ER) alpha and beta in competitive binding assays (IC50's: 5-12 x 10(-9) M) and stimulated transcription of 3XERE-luciferase in T47Dco human breast cancer cells with a potency equal to or greater than that of estradiol (E2) (EC50's: 10(-12) to 10(-11) M). C19 androgens (T, 17alpha-methyltestosterone (17alpha-MT), androstenedione (AD), and 16alpha-hydroxyandrostenedione (16alpha-OHAD)), 19-norandrogens (DMA, 11beta-MNT, 19-nortestosterone (19-NT), and 7alpha-methyl-19-nortestosterone (MENT)) or the structurally similar 19-norprogestin, norethindrone (NET) were incubated at 50 microM with recombinant human aromatase for 10-180 min at 37 degrees C. The reactions were terminated by extraction with acetonitrile and centrifugation, and substrate and potential product were separated by HPLC. Retention times were monitored by UV absorption, and UV peaks were quantified using standard curves. Aromatization of the positive controls, T, AD, and 16alpha-OHAD was linear for 40-60 min, and conversion of T or AD was complete by 120 min. The nonsteroidal aromatase inhibitor, letrozole, demonstrated concentration-dependent suppression of T aromatization. Under the same conditions, there was no detectable conversion of DMA, 11beta-MNT, or NET to their respective hypothetical aromatic A-ring products during incubation times up to 180 min. Aromatization of MENT and 19-NT proceeded slowly and was limited. Collectively, these data support the notion that in the absence of the C19-methyl group, which is the site of attack by oxygen, aromatization of androgenic substrates proceeds slowly or not at all and that this reaction is impeded by the presence of a methyl group at the 11beta position.

Mechanism of action of l-CDB-4022, a potential nonhormonal male contraceptive, in the seminiferous epithelium of the rat testis.

The present study was conducted to elucidate the possible molecular mechanisms involved in the antispermatogenic activity of l-CDB-4022, an indenopyridine. In this study 45-d-old male Sprague-Dawley rats were treated with a single oral dose of l-CDB-4022 (2.5 mg/kg) or vehicle, and blood and testes were collected at various time points. The rate of body weight gain was not affected, but a significant loss of testes weight was induced by l-CDB-4022. Serum hormones were assayed using specific RIAs or ELISAs, and testicular protein and RNA were analyzed by Western blotting and RT-PCR, respectively. There was a significant decrease in inhibin B and concomitant increase in FSH in serum from l-CDB-4022-treated rats, but serum levels of activin A, testosterone, and LH were unchanged. Western analysis of testicular lysates from l-CDB-4022-treated rats exhibited phosphorylation of ERK1/2 at 4 h and later time points. Loss of nectin/afadin complex occurred at 48 h, but there was an increase in levels of integrin-beta1, N-cadherin, alpha-catenin, and beta-catenin protein at 24 h and later time points. Increase in expression of Fas ligand and Fas receptor was detected 8 and 24 h after l-CDB-4022 treatment. The ratio of the membrane to soluble form of stem cell factor mRNA was decreased. Immunohistochemical analysis of testicular sections indicated a dramatic disruption of the Sertoli cell microtubule network in l-CDB-4022-treated rats. Collectively, these results suggest that l-CDB-4022 activates the MAPK pathway, reduces expression of prosurvival factors such as the membrane form of stem cell factor, alters expression of Sertoli-germ cell adherens junction proteins, disrupts Sertoli cell microtubule structure, and induces the proapoptotic factor, Fas, culminating in germ cell loss from the seminiferous epithelium.

Acute adverse effects of the indenopyridine CDB-4022 on the ultrastructure of sertoli cells, spermatocytes, and spermatids in rat testes: comparison to the known sertoli cell toxicant Di-n-pentylphthalate (DPP).

Acute effects of CDB-4022 on testicular ultrastructure were determined. Rats were treated orally with vehicle or a maximally effective single dose of CDB-4022 or Di-n-pentylphthalate (DPP). Preserved testes were processed for transmission electron microscopy. Sertoli and germ cells of vehicle-treated rats demonstrated normal morphological characteristics. Disruption of Sertoli cell ultrastructure was apparent in CDB-4022-treated rats by 3 hours. A decrease in the presence of nucleoli, an increase in the amount and diameter of swollen smooth endoplasmic reticulum, and decreases in cytoplasmic ground substance were observed. The severity of these degenerative effects increased at 6 and 12 hours: Vacuoles were apparent; increased cellular debris, swollen mitochondria, and phagocytic structures were observed; and membranes became more disorganized. Similar ultrastructural changes were observed in the Sertoli cells of DPP-treated rats. By 3 hours, spermatocytes and spermatids were adversely affected by CDB-4022 treatment with swelling of the nuclear envelope. The Step 8 spermatids were especially noteworthy; chromatin was more diffuse and rarefied, the nuclear envelopes were incomplete or broken, and the position of the spermatid nucleus within the cell and relative to Sertoli cell cytoplasm was unusual. Fusion of spermatids to form giant cells was observed by 12 hours. CDB-4022 acts acutely on Sertoli cells to induce marked cellular rarefaction and degeneration, but not necrosis. A rapid and direct effect of CDB-4022 on spermatocytes and spermatids was observed. The antispermatogenic activity of CDB-4022 appears to be a consequence of direct effects on Sertoli and germ cells.

Development of l-CDB-4022 as a nonsteroidal male oral contraceptive: induction and recovery from severe oligospermia in the adult male cynomolgus monkey (Macaca fascicularis).

The present study was undertaken to examine the antispermatogenic effect of l-CDB-4022 in the adult male cynomolgus monkey. Monkeys (four per group) were dosed via nasogastric tube for 7 d with l-CDB-4022 at 12.5 mg/kg.d or vehicle (d 0=first day of dosing). Plasma levels of l-CDB-4022 and its deesterified metabolite were nondetectable prior to treatment and in all vehicle-treated monkeys. Peak levels of l-CDB-4022 and its metabolite were observed at 4 h after dosing with steady-state levels apparent around d 4. Sperm concentration and total sperm per ejaculate were decreased to levels below 1x10(6) sperm/ml or sperm/ejaculate in l-CDB-4022-treated monkeys by d 17 and remained suppressed through wk 6. Sperm motility also declined to 0% for 6 wk. Testicular volume was reduced in l-CDB-4022-treated monkeys through d 21. The left testis and epididymis were removed from all monkeys on d 24. At this time, the most mature germ cells in the seminiferous tubules of testes from l-CDB-4022-treated monkeys were either spermatocytes or round spermatids. Immature germ cells, but not mature sperm, were found in the efferent ducts and collapsed epididymal lumen of l-CDB-4022-treated monkeys. A steady recovery in sperm motility, concentration, and total sperm per ejaculate was observed in l-CDB-4022-treated monkeys such that these parameters were not different from those of vehicle-treated monkeys by wk 16. Volume of the remaining testis increased in vehicle- and l-CDB-4022-treated monkeys after hemicastration; however, the increase in l-CDB-4022-treated monkeys was delayed compared with that observed in the vehicle-treated monkeys. The morphology of the remaining testis and epididymis, which were removed on wk 17, was normal. Serum inhibin B levels were increased in l-CDB-4022-treated monkeys during the dosing interval; thereafter serum inhibin B levels declined such that there was no difference between the groups by wk 3. l-CDB-4022 treatment did not affect circulating levels of testosterone, LH, FSH, or estradiol. In conclusion, these data indicate that in the cynomolgus monkey, a representative higher primate, l-CDB-4022 exerts a selective antispermatogenic action, which was reversible under the conditions of this study and thus has potential as a nonhormonal oral male contraceptive.

Dimethandrolone undecanoate: a new potent orally active androgen with progestational activity.

Dimethandrolone (DMA), the 17beta-undecanoic acid ester of dimethandrolone (DMAU; 7alpha,11beta-dimethyl-19-nortestosterone) is a potent androgen currently in development for therapeutic uses in men. Cleavage of the 17beta-ester bond liberates the biologically active DMA. In this study we investigated the activity of DMAU and DMA both in vivo and in vitro. DMAU was active orally in castrate rat bioassays, and when administered sc, a single dose produced prolonged androgenic activity and suppression of LH with sustained circulating levels of DMA. DMA, other 19-norandrogens, and C-19 androgens bound to recombinant rat androgen receptor with high affinity and were equipotent in stimulating luciferase activity (EC50, 10(-10) -10(-9) M) in CV-1 cells cotransfected with a human androgen receptor expression vector and a luciferase reporter plasmid with three hormone response elements. Because various 19-norandrogens are also known to bind to progestin receptors (PR) and to possess progestational activity in vivo, we evaluated the binding affinity of DMA for rabbit PR and recombinant human PR-A and PR-B and its ability to induce PR-mediated transcription and endogenous alkaline phosphatase activity in T47DCO human breast cancer cells. DMA and related 19-norandrogens bound with high affinity to both rabbit and human PR, whereas the less active 11alpha-methyl stereoisomer of DMA and C-19 androgens showed low or negligible binding to PR. In T47DCO cells, 10(-8) M DMA and other 19-norandrogens stimulated transcription of a progestin/glucocorticoid/androgen response element-thymidine kinase-luciferase reporter plasmid to the same extent as R5020, the potent progestin promegestone (EC50, approximately 10(-9) M), but C-19 androgens had no effect. Antiprogestins were potent inhibitors of transactivation and alkaline phosphatase activity induced by DMA and other 19-norandrogens in T47DCO cells, whereas antiandrogens were weak inhibitors. DMA and DMAU also exhibited dose-dependent progestational activity in the estrogen-primed immature female rabbit, as assessed by induction of endometrial gland arborization. The dual androgenic and progestational activities of DMA make it a potential candidate for a single-agent male contraceptive as well as for androgen therapy in men, pending a successful outcome of pharmacokinetic and toxicity studies currently in progress.

Steroid hormonal regulation of growth, prostate specific antigen secretion, and transcription mediated by the mutated androgen receptor in CWR22Rv1 human prostate carcinoma cells.

CWR22Rv1 (22Rv1) is an androgen-responsive human prostate carcinoma cell line derived from a primary prostate tumor that expresses mutant (H874Y) androgen receptors (AR) and secretes low levels of prostate specific antigen (PSA). In this study, we examined the effects of various androgens and other steroid hormones on proliferation of 22Rv1 cells, PSA secretion, and transactivation. Incubation of 22Rv1 cells with various concentrations of testosterone resulted in a dose-dependent 50-80% increase in growth over 72 h. PSA release and transactivation of PRE2-tk-LUC in 22Rv1 cells were stimulated by low concentrations of natural and synthetic androgens (EC(50)s = 10(-10) to 10(-9)M) and a broad range of other classes of steroid hormones, albeit with lower potency. Uniform positive immunocytochemical staining was observed in 22Rv1 cell nuclei with mouse monoclonal antibodies to human AR. Competitive binding assays indicated that the mutant AR in 22Rv1 cytosol is more promiscuous than a wild-type AR (ARLBD: rat AR ligand binding domain). Testosterone (10(-8)M)-induced PSA release and transactivation were blocked by both antiandrogens and antiprogestins with IC(50)s of 10(-7) to 10(-6)M. At high concentration (10(-6)M), these antagonists showed partial agonist activity in terms of PSA secretion but not transactivation. In conclusion, the mutant AR in 22Rv1 cells binds and responds to low levels of androgens and a wide spectrum of other natural and synthetic steroid hormones, mechanisms proposed to contribute to tumor progression following androgen ablation.

In vitro antiprogestational/antiglucocorticoid activity and progestin and glucocorticoid receptor binding of the putative metabolites and synthetic derivatives of CDB-2914, CDB-4124, and mifepristone.

In determining the biological profiles of various antiprogestins, it is important to assess the hormonal and antihormonal activity, selectivity, and potency of their proximal metabolites. The early metabolism of mifepristone is characterized by rapid demethylation and hydroxylation. Similar initial metabolic pathways have been proposed for CDB-2914 (CDB: Contraceptive Development Branch of NICHD) and CDB-4124, and their putative metabolites have been synthesized. We have examined the functional activities and potencies, in various cell-based assays, and relative binding affinities (RBAs) for progesterone receptors (PR) and glucocorticoid receptors (GR) of the putative mono- and didemethylated metabolites of CDB-2914, CDB-4124, and mifepristone and of the 17alpha-hydroxy and aromatic A-ring derivatives of CDB-2914 and CDB-4124. The binding affinities of the monodemethylated metabolites for rabbit uterine PR and human PR-A and PR-B were similar to those of the parent compounds. Monodemethylated mifepristone bound to rabbit thymic GR with higher affinity than monodemethylated CDB-2914 or CDB-4124. T47D-CO cells were used to assess inhibition of R5020-stimulated endogenous alkaline phosphatase activity and transactivation of the PRE(2)-thymidine kinase (tk)-luciferase (LUC) reporter plasmid in transient transfections. The antiprogestational potency was as follows: mifepristone/CDB-2914/CDB-4124/monodemethylated metabolites (IC(50)'s approximately 10(-9)M) > aromatic A-ring derivatives (IC(50)'s approximately 10(-8)M) > didemethylated/17alpha-hydroxy derivatives (IC(50)'s approximately 10(-7)M). Antiglucocorticoid activity was determined by inhibition of dexamethasone-stimulated transcriptional activity in HepG2 cells. The mono- and didemethylated metabolites of CDB-2914 and CDB-4124 had less antiglucocorticoid activity (IC(50)'s approximately 10(-6)M) than monodemethylated mifepristone (IC(50) approximately 10(-8)M) or the other test compounds. At 10(-6)M in transcription assays, none of these compounds showed progestin agonist activity, whereas mifepristone and its monodemethylated metabolite manifested slight glucocorticoid agonist activity. The reduced antiglucocorticoid activity of monodemethylated CDB-2914 and CDB-4124 was confirmed in vivo by the thymus involution assay in adrenalectomized male rats. The aromatic A-ring derivatives-stimulated transcription of an estrogen-responsive reporter plasmid in MCF-7 and T47D-CO human breast cancer cells but were much less potent than estradiol. Taken together, these data suggest that the proximal metabolites of mifepristone, CDB-2914, and CDB-4124 contribute significantly to the antiprogestational activity of the parent compounds in vivo. Furthermore, the reduced antiglucocorticoid activity of CDB-2914 and CDB-4124 compared to mifepristone in vivo may be due in part to decreased activity of their putative proximal metabolites.

The ability of a gonadotropin-releasing hormone antagonist, acyline, to prevent irreversible infertility induced by the indenopyridine, CDB-4022, in adult male rats: the role of testosterone.

Intratesticular testosterone (ITT) is known to play a critical role in the maintenance of spermatogenesis. We have used acyline, a GnRH antagonist, to suppress testosterone (T) production, and acyline and T implants to study the prevention of irreversible infertility induced by CDB-4022. Vehicle or acyline was administered to proven fertile male rats (n = 5/group) at a dose (210 microg/day) that completely suppressed (P < 0.05) T production, as measured by serum T, and testicular function, either before, concurrent with, or after vehicle or a single oral dose of 2.5 mg CDB-4022/kg (Week 0). Vehicle-treated males remained fertile, whereas acyline-treated males exhibited transitory infertility. CDB-4022 alone caused irreversible infertility in all males. Importantly, CDB-4022-treated males recovered fertility when acyline was started before CDB-4022 (Weeks -4 to 0; Weeks -4-9), but not when acyline was administered concurrently with or after CDB-4022 (Weeks 0-9; Weeks 10-19). At the end of this study (Week 34), testes weights, spermatid head counts (SHC), and tubule differentiation indices (TDI) were suppressed (P < 0.05) in infertile CDB-4022-treated males, but in rats that recovered fertility, these parameters were similar (P > 0.05) to those in vehicle-treated males. In addition, serum inhibin B and epididymal androgen-binding protein levels were nondetectable in infertile CDB-4022-treated rats. To test whether suppression of ITT was critical for prevention of CDB-4022-induced infertility, proven fertile rats (n = 7-8/group) received vehicle, acyline alone, or acyline and a T implant for 4 wk before CDB-4022 (Week 0). The T implant increased ITT in acyline-treated rats. Although ITT was lower (P < 0.05) in the T-implanted males than in untreated rats, it was sufficient to sustain spermiogenesis. Serum rFSH levels were also elevated in rats treated with acyline + T as compared with acyline alone during the treatment interval, but rFSH was still lower than in vehicle-treated rats. Rats in all treatment groups were rendered infertile initially, but the acyline + CDB-4022-treated rats recovered fertility by Week 10. In contrast, rats treated with CDB-4022 alone or acyline + T + CDB-4022 remained infertile until at least Week 16. Testes weights, SHC, and TDI were within normal ranges for acyline + CDB-4022-treated rats, but were decreased (P < 0.05) in CDB-4022- or acyline + T + CDB-4022-treated rats. Serum inhibin B levels were nondetectable by Week 1 in males rendered irreversibly infertile by CDB-4022; levels increased transiently and returned to baseline in rats protected by acyline pretreatment. These data indicate that pretreatment with acyline was able to prevent irreversible infertility in CDB-4022-treated rats, whereas posttreatment with acyline did not promote spermatogonial differentiation, as has been observed by others in rats that received GnRH analogs and various other testicular toxicants. Suppression of ITT and possibly rFSH by acyline appeared to be crucial in preventing irreversible infertility induced by CDB-4022. In this regard, our results are similar to those of investigators who have studied other testicular toxicants. Continued development of CDB-4022 as a potential male contraceptive will depend largely on its safety profile and whether its antispermatogenic activity is reversible in primates.

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.