Estrogen and tibolone metabolite levels in blood and breast tissue of postmenopausal women recently diagnosed with early-stage breast cancer and treated with tibolone or placebo for 14 days.

Unlike estrogens plus progestagens, tibolone, a selective tissue estrogenic activity regulator, does not increase breast tenderness and mammographic density. To elucidate this, serum and breast levels of tibolone and estrogenic metabolites are measured. Postmenopausal women (n = 102) with early-stage, ER(+ve), primary breast cancer received tibolone or placebo for 14 days in an exploratory, double-blind, randomized trial (STEM carcinoma tissue). Baseline and presurgery sera were collected; tumor tissues were obtained at surgery. E(1) (estrone), E(2) (estradiol), E(1)S (estrone-sulfate), tibolone-its nonsulfated, monosulfated, and disulfated 3-hydroxymetabolites-and Delta(4)-tibolone were measured by validated gas chromatography and mass spectrometry and liquid chromatography with tandem mass spectrometry assays. More than 12 hours after the final dose, serum E(1), E(2), and E(1)S levels were unchanged with placebo, whereas tibolone significantly increased E(1)S and the E(1)S/(E(1) + E(2)) ratio. In tumors, E(1) and E(2) levels were higher than in serum, and E(1)S levels were lower, with placebo and tibolone administration. The percentage of E(1)S was about 90% in serum and 16% in tissue. Tibolone did not affect tissue levels of endogenous estrogens. Serum levels of estrogenic 3alpha- and 3beta-hydroxytibolone, progestagenic/androgenic Delta(4)-tibolone, and monosulfate metabolites were low. Serum 3alphaS,17betaS-tibolone and 3 betaS,17betaS-tibolone levels were 250 and 52 ng/mL, respectively. Tumor levels of 3alpha- and 3beta-hydroxytibolone and Delta(4)-tibolone were higher than in serum, but disulfate levels were lower. The percentage of sulfated tibolone metabolites was 99% in serum and 96% in tumor. Serum metabolite patterns of estradiol and tibolone are different from those in tissues and are compatible with neutral effects of tibolone on breast Ki67 expression.

High performance liquid chromatography/ion-trap mass spectrometry for separation and simultaneous determination of ethynylestradiol, gestodene, levonorgestrel, cyproterone acetate and desogestrel.

A fast and highly sensitive high performance liquid chromatographic/ion-trap mass spectrometric method (LC/MS) has been developed for simultaneous determination of ethynylestradiol (EE2), gestodene (GES), levonorgestrel (LNG), cyproterone acetate (CPA) and desogestrel (DES). Among three types of sorbents tested (C8, C18 and phenyl) from two suppliers, the best separation was achieved on reverse phase Zorbax SB-Phenyl column using aqueous methanol as a mobile phase. A linear gradient profile from 70 up to 100% (v/v) in 7th min, kept constant at 100% up to 10th min and followed by a negative gradient to 70% of methanol up to 12th min was used for elution. Applicability of electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) and influence of the mobile phase composition, its flow rate, capillary/vaporizer temperature of API source and in-source fragmentor voltage ionization are discussed. The on-column limits of quantification (10S/N) were 300 pg of EE2, 14 pg of GES and LNG, 4 pg of CPA and 960 pg of DES per injection (1 microL) using APCI with data collection in selected ion monitoring (SIM) mode. The analytical performance of the method was evaluated using the determination of EE2, GES, LNG, CPA and DES in contraceptives and river water samples.

Sulfation of tibolone and tibolone metabolites by expressed human cytosolic sulfotransferases.

Tibolone is an important therapeutic agent used in the treatment of menopausal symptoms in many countries and has beneficial effects on menopausal and postmenopausal vasomotor, bone, vaginal and mood symptoms without affecting the endometrial, breast or cardiovascular systems. The rapid metabolism of tibolone to active metabolites including 3alpha-OH-tibolone, 3beta-OH-tibolone and Delta(4)-tibolone may be important in its tissue-specific effects. Sulfation also has a major role in the metabolism and regulation of the tissue-specific activity of tibolone and its metabolites. The ability of seven major expressed human sulfotransferase (SULT) isoforms to sulfate tibolone and its three metabolites was examined. Expressed human SULT2A1 was capable of sulfating tibolone and all three metabolites with the highest affinity for 3alpha-OH-tibolone. SULT1E1 conjugated both 3-OH-tibolone metabolites and tibolone itself slightly. SULT2B1b sulfated both 3-OH metabolites but not tibolone or Delta(4)-tibolone. SULT isoforms 1A1, 1A3, 1B1 and 1C1 did not demonstrate detectable activity. Sulfation of tibolone and its metabolites by human tissue cytosols was analyzed to determine whether the pattern of tibolone sulfation corresponded to the known expression of SULT isoforms in each tissue. The tissue-specific effects of tibolone may be regulated in part by the inactivation of tibolone and its metabolites by specific human SULT isoforms.

Determination of steroid hormones in oral contraceptives by high-performance liquid chromatography.

The aim of this research was to standardize a high-performance liquid chromatographic method for quantitative determination of steroid hormones, like ethinylestradiol (ETE), levonorgestrel (LEVO), and gestodene (GEST), in commercially available oral contraceptives (OCs). The combination ETE-LEVO was analyzed using a LiChrospher 100 RP-8 column (5 microns, 125 x 4 mm) in LiChroCART, with a mobile phase constituted of acetonitrile: water (60:40 v/v). Using the same column, ETE-GEST was analyzed with a mobile phase constituted of acetonitrile:water (50:50 v/v) at pH 7.5 adjusted with 0.02 M ammonium hydroxide. For both methods, a flow rate of 0.8 mL/min was utilized and detection was carried out at 215 nm. All analyses were performed at room temperature (24 +/- 2 degrees C). Calibration curves for ETE-LEVO were obtained using solutions with concentration ranges from 2.40 to 60.0 micrograms/mL (ETE), and from 12.0 to 300.0 micrograms/mL (LEVO). Calibration curves for ETE-GEST were obtained using solutions with concentration ranges from 2.40 to 60.0 micrograms/mL (ETE), and from 9.0 to 160.0 micrograms/mL (GEST). Correlation coefficients obtained were from 0.9999 to 0.9990. Coefficients of variation for samples containing ETE-LEVO were 0.47% and 0.38%, respectively. For samples with ETE-GEST they were 0.39% and 0.44%, respectively. The average recovery for samples with ETE-LEVO was 103.46% and 100.78%, respectively. For samples containing ETE-GEST it was 100.89% and 101.03%, respectively.

Different methods for the determination of gestodene, and cyproterone acetate in raw material and dosage forms.

Four new precise accurate and selective methods have been developed for the determination of gestodene (I) and cyproterone acetate (II). The first method (A) depends on reaction of (I) and (II) with isoniazide in an acid medium and the colored products were measured at 378 and 400 nm, respectively. The second method (B) depends on the reaction of (I) and (II) with tetrazolium blue in an alkaline medium and the colored products were measured quantitatively at 515 and 520 nm, respectively. The optimum conditions for the analysis were studied. Both methods determined gestodene (I) in concentration range from 4 to 24 microg ml(-1) with mean percentage recoveries 99.54%+/-1.20 and 99.63%+/-1.89 for method A and B, respectively. For cyproterone acetate, the concentration ranges were 4-36 and 8-40 microg ml(-1) with mean percentage recoveries 99.94%+/-1.19 and 99.23%+/-2.00 for methods A and B, respectively. The third method (C) depends on the quantitative evaluation of (I) and (II) densitometrically using dichloroethane:methanol:water (95:5:0.2) as mobile phase and the chromatogram were scanned at 247 and 281 nm, respectively. Method (C) determines (I) and (II) in concentration ranges from 0.2 to 1.6 and 0.1-0.7 microg microl(-1) using Hamilton syringe 10 microl, with mean percentage recoveries 99.94%+/-1.19, and 99.82%+/-1.75, respectively. The fourth method (D) is a first derivative one depends on measuring the D(1) value at 303 nm for (II) only in concentration range 10-20 microg ml(-1) with mean percentage recoveries 99.95%+/-1.49.

Classification and comparison of oral contraceptives containing new generation progestogens.

New generation-oral contraceptives containing desogestrel or gestodene, and possibly also norgestimate, are more or less similar with respect to contraceptive efficacy, cycle control and acceptability. They also show a more favourable metabolic profile in comparison with older preparations. The desogestrel-containing preparations Gracial and Marvelon, and possibly also the gestodene-containing preparation Gynera, have demonstrated a good efficacy in well-controlled studies in the treatment of mild to moderate acne and/or hirsutism. There may be differences between new generation oral contraceptives with respect to their effects on metabolic variables like high-density lipoprotein cholesterol and sex hormone-binding globulin. These differences are most probably modulated by variations in both the pharmacokinetics and selectivity of the progestogenic components. Of particular relevance here may be the recent finding that approximately 20% of administered norgestimate is metabolized into levonorgestrel. For use in clinical practice, it is of considerable help to have different preparations containing a range of oestrogen doses with the same progestogen. They allow the clinician to 'tailor make' the choice of oral contraceptives for those starting pill use or those switching to another combination due to symptomatology or changed circumstances, e.g. advancing age, smoking, etc. In this respect, desogestrel-containing oral contraceptives allow the most flexible approach.

Characterization of the novel progestin gestodene by receptor binding studies and transactivation assays.

Gestodene is a novel progestin used in oral contraceptives with an increased separation of progestogenic versus androgenic activity and a distinct antimineralocorticoid activity. This specific pharmacological profile of gestodene is defined by its pattern of binding affinities to a variety of steroid hormone receptors. In the present study the affinity of gestodene to the progesterone receptor (PR), the androgen receptor (AR), the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR) and the estrogen receptor (ER) was re-evaluated by steroid binding assays and compared to those obtained for 3-keto-desogestrel and progesterone. The two synthetic progestins displayed identical high affinity to rabbit PR and similar marked binding to rat AR and GR, while progesterone showed high affinity to PR but only low binding to AR and GR. Furthermore, 3-keto-desogestrel exhibited almost no binding to MR, whereas gestodene, similar to progesterone, showed marked affinity to this receptor. In addition to receptor binding studies, transactivation assays were carried out to investigate the effects of gestodene on AR-, GR- and MR-mediated induction of transcription. In contrast to progesterone, which showed antiandrogenic activity, gestodene and 3-keto-desogestrel both exhibited androgenic activity. Furthermore, all three progestins exhibited weak GR-mediated antagonistic activity. In contrast to progesterone, which showed almost no glucocorticoid activity, gestodene and 3-keto-desogestrel showed weak glucocorticoid action. In addition, gestodene inhibited the aldosterone-induced reporter gene transcription, similar to progesterone, whereas unlike progesterone, gestodene did not induce reporter gene transcription. 3-Keto-desogestrel showed neither antimineralocorticoid nor mineralocorticoid action.

[Structure elucidation of gestodene]. / Strukturaufklärung von Gestoden.

The structure of 17 alpha-ethinyl-17 beta-hydroxy-18-methyl-4,15-estradien-3-one (gestodene) was elucidated by the spectrometric methods UV, IR, NMR, MS.