Antiandrogenic activity of norgestimate in a human androgen-dependent stable-transfected cell line.

BACKGROUND: Acne occurs from an overstimulation of the sebaceous glands by high levels of androgens or because sebaceous glands are hypersensitive to normal levels of testosterone. In women with moderate acne, norgestimate (NG) in association with ethinyl estradiol (EE) is acknowledged as an effective treatment; this is related to the effect of oral contraceptives on androgen production and transport. However, the antiandrogenic properties of NG itself have been poorly studied. DESIGN: The present work was undertaken to find out whether NG and its derivative, 17-deacetylnorgestimate (dNG), present antiandrogen activity. First, we studied the effect of NG and dNG on the intracellular localization of green fluorescent protein (GFP)-labeled androgen receptor (AR). Then, we compared the AR activity of NG and dNG with that of cyproterone acetate (CPA), a gold-standard antiandrogenic compound, by investigating competitive binding, antagonist activity and transactivation level of AR. RESULTS: NG and dNG decreased GFP-AR nuclear translocation, revealing their antiandrogenic property, as for CPA. In the whole cell competition assay performed in a human cell line stably expressing an AR-responsive luminescent reporter gene (PALM cells) with (3)H-labeled R1881 as tracer, NG and dNG were slightly stronger competitors than the antiandrogen CPA. Half-maximal inhibition (K(i)) of (3)H-labeled R1881 (10(-9) M) binding occurred at 4.2 +/- 0.5 x 10(-8) M of NG, 3.4 +/- 0.4 x 10(-8) M of dNG and 6.6 +/- 0.8 x 10(-8) M of CPA. Comparison of antagonist activities of NG, dNG and CPA on AR transactivation levels showed that NG, dNG and CPA inhibited androgen-induced luciferase activity in PALM cells. We observed slight and similar inhibition with 6 x 10(-8) M respectively of NG, dNG and CPA. For the three compounds, the best inhibitory effect was found at 3 x 10(-7) M: 24% for NG and dNG vs. 47% for CPA. The antiandrogenic activity of NG and dNG was found to be 50% that of CPA. CONCLUSION: In a human androgen-dependent stable-transfected cell line, a useful tool for studying AR transcriptional activity and its subnuclear localization in the presence of androgens and antiandrogens, we demonstrated that NG and dNG have antiandrogenic properties that could partly explain the efficacy of NG in association with EE for the treatment of moderate acne in women.

Effect of multiple-dose dexloxiglumide on the pharmacokinetics of oral contraceptives in healthy women.

This study was undertaken to evaluate the effect of dexloxiglumide, a selective cholecystokinin receptor antagonist, on the pharmacokinetics of a combination oral contraceptive (OC). A single-blind, placebo-controlled, 2-period crossover study was conducted in 24 healthy young female subjects who received Ortho Tri-Cyclen containing ethinyl estradiol (EE, 0.035 mg) and norgestimate (NE, 0.180 mg/0.215 mg/0.250 mg per 7-day phase, respectively) for 5 days (days 17-21) concurrently with either 200 mg dexloxiglumide (3 times a day on days 17-20, followed by a single dose on day 21) or matching placebo during 2 consecutive 28-day OC dosing cycles. Plasma was sampled up to 24 hours for the determination of EE, NE, and 17-deactyl norgestimate (17-DNE, a rapidly formed pharmacologically active metabolite of NE). The geometric mean ratios (GMRs, dexloxiglumide/placebo) of the plasma concentration-time curve over 24 hours with corresponding 90% confidence intervals (CIs) for EE and 17-DNE were 1.21 (1.17-1.26) and 0.92 (0.89-0.95), respectively. The GMRs (90% CI) of C(max) for EE and 17-DNE were 1.15 (1.09-1.20) and 0.93 (0.90-0.96), respectively. Coadministration of OC and dexloxiglumide was well tolerated and safe. Comparable systemic exposure of EE and 17-DNE in the presence and absence of dexloxiglumide suggests that dexloxiglumide treatment is unlikely to interfere with the safety and efficacy of oral contraceptives based on the analysis of the resulting pharmacokinetic profile.

The intrinsic transcriptional estrogenic activity of a non-phenolic derivative of levonorgestrel is mediated via the estrogen receptor-alpha.

Levonorgestrel (LNG), a 19-nor-testosterone derivative, is widely used in contraceptive formulations. This compound does not bind to the estrogen receptor (ER), but it shows estrogen-like effects under in vivo and in vitro conditions. The estrogenicity of LNG may be attributed to its bio-transformation into non-phenolic metabolites. In this study, the ability of A-ring reduced LNG metabolites to activate transcription via an estrogenic mechanism of action, including differences between ER alpha and ER beta subtypes, were investigated. Transactivation assays were performed in HeLa cells transfected with expression vectors for ER alpha and ER beta and an estrogen-responsive reporter gene. Cells were also transfected with expression vectors for both progesterone receptor (PR) isoforms (A or B). As expected, the tetrahydro derivatives of LNG (3 alpha,5 alpha- and 3 beta,5 alpha-LNG) showed significantly lower PR-mediated transcriptional activities through both isoforms when compared with progesterone (P(4)) and LNG. In contrast, the 3 beta,5 alpha-tetrahydro derivative resulted in a significant activation of estrogen-dependent gene transcription. This effect was selectively confined to the ER alpha, since little if any activity could be observed with the ER beta and no antagonistic activities were demonstrated. This study provides structural and molecular clues for the well documented in vitro and in vivo intrinsic estrogenicity of 19-nor-testosterone-derived progestins and ligand requirements for ER alpha recognition.

Efficacy and safety of a transdermal contraceptive system.

OBJECTIVES: To evaluate the efficacy, cycle control, compliance, and safety of a transdermal contraceptive system that delivers norelgestromin 150 microg and ethinyl estradiol 20 microg daily. METHODS: In this open-label, 73-center study, 1672 healthy, ovulatory, sexually active women received ORTHO EVRA/EVRA for six (n = 1171) or 13 cycles (n = 501). The treatment regimen for each cycle was three consecutive 7-day patches (21 days) followed by 1 patch-free week. RESULTS: The overall and method-failure probabilities of pregnancy through 13 cycles were 0.7% and 0.4%, respectively. The incidence of breakthrough bleeding was low throughout the study. Perfect compliance (21 consecutive days of dosing, followed by a 7-day drug-free interval; no patch could be worn for more than 7 days) was achieved in 90% of subject cycles; only 1.9% of patches detached completely. Adverse events were typical of hormonal contraception, and most were mild-to-moderate in severity and not treatment limiting. The most common adverse events resulting in discontinuation were application site reactions (1.9%), nausea (1.8%), emotional lability (1.5%), headache (1.1%), and breast discomfort (1.0%). CONCLUSIONS: The transdermal contraceptive patch provides effective contraception and cycle control, and is well tolerated. The weekly change schedule for the contraceptive patch is associated with excellent compliance and wearability characteristics.

Assessment of the oestrogenic activity of the contraceptive progestin levonorgestrel and its non-phenolic metabolites.

Levonorgestrel (13beta-ethyl-17alpha-ethynyl-17beta-hydroxy-4-gonen-3-one), a potent contraceptive progestin stimulates growth and proliferation of cultured breast cancer cells through a receptor-mediated mechanism, even though levonorgestrel does not bind to the oestrogen receptor (ER). To assess whether the oestrogen-like effects induced by this synthetic progestin are exerted via its metabolic conversion products, we studied the binding affinity of three A-ring levonorgestrel derivatives to the ER and their capability to transactivate an oestrogen-dependent yeast system co-transfected with the human ER gene and oestrogen responsive elements fused to a beta-galactosidase reporter vector. The results demonstrated that the 3beta,5alpha reduced levonorgestrel derivative and to a lesser extent its 3alpha isomer interact with the oestrogen receptor, with a significantly lower relative binding affinity (2.4% and 0.4%, respectively) than that of oestradiol (100%), while levonorgestrel does not. Both levonorgestrel metabolites were able to activate, in a dose-dependent manner, the beta-galactosidase reporter gene in the yeast expression system, an effect that was precluded by a steroidal antioestrogen. The oestrogenic potency of levonorgestrel metabolites was significantly lower (750-fold) than that of oestradiol. Furthermore, high doses of 3beta,5alpha levonorgestrel (2.5 mg/day/6 days) induced an increase of oestrogen-dependent progestin receptor in the anterior pituitary of castrated rats. The overall data offer a plausible explanation for the weak oestrogenic effects induced by high, non-pharmacological doses of levonorgestrel.

Multiple-dose pharmacokinetics of a contraceptive patch in healthy women participants.

This open-label, randomized study evaluated the pharmacokinetics of norelgestromin (NGMN) and ethinyl estradiol (EE) following the application of a contraceptive patch (1/week) for three cycles (3 weeks/cycle). Healthy women (n = 24) wore a 20-cm(2) patch (ORTHO EVRA/EVRA) on either their abdomen or buttock during blood sampling weeks and on any of four approved sites at other times. Serum was analyzed for NGMN and EE from samples taken during Week 1 of Cycle 1 and Weeks 1-3 of Cycle 3. Steady-state conditions were achieved during the three-cycle study. The patch delivered NGMN and EE at steady-state concentrations within their reference ranges throughout three cycles of treatment; reference ranges are based on studies with ORTHO-CYCLEN/Cilest. Steady-state serum concentrations and area under the curve from 0 to 168 h increased only slightly from Cycle 1, Week 1 to Cycle 3, Week 3 for NGMN and EE, indicating minimal accumulation. Treatment was well tolerated, and patch adhesion was excellent.

Determination of norgestimate and its metabolites in human serum using high-performance liquid chromatography with tandem mass spectrometric detection.

A rapid and reliable analytical method is described for the simultaneous determination of a synthetic progestin norgestimate (NGM), and its metabolites, 17-deacetylnorgestimate (17-DA-NGM), 3-ketonorgestimate (3-keto-NGM) and norgestrel (NGL) in human serum using reversed phase high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS-MS) detection. The assay was linear over the concentration ranges of 0.1-5.0 ng/ml for 17-DA-NGM and NGL and 0.5-5.0 ng/ml for NGM and 3-keto-NGM. The inter-assay reproducibility was consistently less than 10%. The overall recovery of the analytes ranged from 72 to 92%. Serum profiles following oral administration of norgestimate to female volunteers are presented.

11 beta-substituted 13 beta-ethyl gonane derivatives exhibit reversal of antiprogestational activity.

The syntheses of three 17 alpha-acetoxy-13 beta-ethyl-11 beta-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.

Comparative progestational activity of norgestimate, levonorgestrel-oxime and levonorgestrel in the rat and binding of these compounds to the progesterone receptor.

The progestational activity of norgestimate (NORG), levonorgestrel-oxime (LNG-oxime) and levonorgestrel (LNG) were compared in a pregnancy maintenance study in rats. The compounds were administered subcutaneously to pregnant rats at several doses, blood samples were collected repeatedly, and the concentration of LNG was measured in these samples. It could be demonstrated that following the administration of NORG and LNG-oxime, LNG was a major metabolite present in the serum. The pharmacological response in rats treated with NORG and LNG-oxime could be related to the systemic exposure of these animals to metabolically derived LNG. Thus, both NORG and LNG-oxime can be regarded as pro-drugs of LNG, the latter being almost exclusively responsible for the pharmacological activity of both pro-drugs. This notion was further supported by studies on the comparative binding affinity of these compounds to rabbit and human progesterone receptor (PR). LNG exhibited the highest binding affinity of the compounds studied. Relative binding affinity (RBA) values of LNG using progesterone as reference (100%) were found to be 125% for rabbit PR (rPR), 143% for human uterine PR (hPR) and 125% for recombinant hPR, respectively. In contrast to LNG, NORG exhibited only a low affinity to the PR, which is documented by RBA values of 1.2% for rPR, 3.2% for uterine hPR and 9% for recombinant hPR. The corresponding values of LNG-oxime were 30% (rPR), 20% (uterine hPR) and 18% (recombinant hPR), respectively. Thus, the combined experimental evidence of the present study does not support the view of NORG being a progestogen on its own as has been suggested by others.