Novel reversed-phase HPLC method for simultaneous determination of ethynodiol diacetate (EDA)/ethinyl estradiol (EE) in pharmaceutical dosage form.

Ethynodiol diacetate (EDA) and ethinyl estradiol (EE) tablets are indicated to prevent pregnancy in women who use oral contraceptives as a contraception method. EDA and EE were separated by reversed-phase HPLC using Agilent ZORBAX SB-Phenyl column, 4.6 mm × 15 cm, 5 µm, using a gradient mixture of acetonitrile and Milli-Q water as mobile phase. The linearity and recovery were found in the range of 0.025-0.25 mg/mL and 0.05-0.18 mg/mL for EDA and 0.001-0.01 mg/mL and 0.002-0.007 mg/mL for EE, respectively. The method is validated according to the regulatory guidelines concerning system suitability, specificity, repeatability, recovery, linearity, robustness, and stability of the sample solution.

The use of oral contraceptives as a prevention of recurrent premenstrual psychosis.

Premenstrual psychosis is a rare and not formally recognized disorder (DSM-IVR, ICD-10). The literature mainly consists of clinical cases. There have been preliminary reports of improvement in such cases after administration of oral contraceptives. We present a case of premenstrual psychosis in which hormonal treatment was effective in preventing symptomatic relapses.

Membrane-initiated steroid signaling (MISS): genomic steroid action starts at the plasma membrane.

UNLABELLED: Plasma membrane (PM) steroid recognition sites are thought to be responsible only for rapid, non-genomic responses without any link to the nuclear receptor-mediated genomic effects of steroids. We focused on a PM "glucocorticoid-importer" (GC-importer) that imports GC into rat liver cells. This site interacts also with particular gestagens (progesterone, P; medroxyprogesterone, MP; ethynodiol, Ethy) and estrogens (ethinylestradiol, EE(2); mestranol), which do not bind to the nuclear GC receptor (GR). To elucidate the role of the GC-importer, we transfected a rat wild-type hepatocyte (CC-1) and a hepatoma cell line, unable to import GC (MH 3924), with a GC<-->GR-responsive luciferase (luc)-reporter gene. Selected steroids were tested for their ability to induce or inhibit luc expression. Corticosterone (B) and dexamethasone (Dex), but also the GC-antagonists cortexolone (Cortex), P and MP, induced luc. Even the PM-impermeable BSA-derivatives of B, Dex and Cortex did so to almost the same extent as the free steroids. MH 3924 cells respond stronger than CC-1 to luc inducing steroids. Luc expression was inhibited by RU 38 486, but also by EE(2) and Ethy. The thiol reactive mesylate-derivatives of B, Dex and Cortex induced to a considerably lesser extent than the free or BSA-steroids. The thiol reagent mersalyl blocks cellular entry of GC and inhibits luc induction in CC-1 cells. Incubation with EE(2) and B of PM-vesicles, isolated from liver cells, resulted in a decrease of the density of two 75 and 52kDa G-proteins reflecting a diminished exchange of GDP by GTP. CONCLUSION: the PM-residing GC-importer, now renamed "Steroid Hormone Recognition and Effector Complex" (SHREC) is an interdependent part of the complete GC signal propagation in which G-proteins are involved. Free SH-groups of SHREC are a prerequisite for genomic GC activity. Specific interactions between SHREC and GC-agonist/-antagonist trigger steroid-dependent signaling. However, import of the ligand into the cell terminates it. Thus, the PM-related non-genomic steroid responses are clearly linked to the GR-related genomic effects.

Estimation of impurity profiles of drugs and related materials. Part VIII: Combined application of high-performance liquid chromatography and NMR spectroscopy in the impurity profiling of drugs.

The usefulness of the joint application of HPLC and NMR spectroscopy in drug impurity profiling is demonstrated by the following examples: (1) identification of Z and E isomers of 17 alpha-ethynyl-4-oestrene-3 beta, 17-diol-3-acetate-17-(3'-acetoxy-2'-butenoate) in ethynodiol diacetate; (2) identification of the p-tolyl analogue as the impurity of enalapril maleate; (3) identification and quantification of 2'-dehydro-pipecuronium bromide in pipecuronium bromide. The possibilities of utilizing NMR spectroscopy for the identification and quantification of the impurities with and without their isolation are discussed.

Clinical experience with the progestogen-only pill.

Experience with the progestogen-only pill (POP) in a family planning clinic is presented. From the clinic records, 408 women were identified who had opted to use a POP. Of these, 50 women had used the POP during lactation and these were excluded from the analysis. The remaining 358 women used the POP for up to 150 months, giving a total of 18,125 women-months of use. Three pregnancies occurred, giving a Pearl Index of 0.2 per 100 women-years. Non-menstrual side effects were minor and were reported by 77 women. For the women who discontinued the POP, the main reason was menstrual irregularity (47.5%). However, despite the long-term use by most of the women, almost 40% maintained a mostly regular menstrual pattern. Our findings suggest that the POP provides a very acceptable method of oral contraception for many women and that it should be more actively promoted.

Investigations upon the mechanism of inhibition of spermatogenesis in the rat by a dimeric ethynodiol-testosterone ester.

The combination of androgens and progestogens has been shown to be a suitable male contraceptive. Previous experiments revealed that injection of a dimeric testosterone-ethynodiol ester into rats and monkeys induces azoospermia for several weeks. In order to investigate the mechanism of action, we compared the endocrine effects of a single injection of 10 mg of the dimeric ester into intact male rats with that of 6 mg of norethisterone enanthate + 6 mg of testosterone enanthate. After the injection of the dimer there was a transitory reduction of serum FSH and a strong suppression of serum LH and testosterone, of testicular testosterone and of androgen-binding protein (ABP) in the testis and epididymis for at least 8 weeks, whereas spermatogenesis was totally depressed between the 4th and 8th week. Contrary to this, the enanthates caused only a slight suppression of spermatogenesis, although serum LH, testicular testosterone and ABP were profoundly reduced. The only conspicuous difference in the endocrine pattern of both groups during the first 4 weeks was in the serum testosterone level which remained normal in the rats treated with the enanthates. The results suggest that testicular testosterone and ABP concentrations are of minor significance for an intact spermatogenesis, and that some other factors produced by Sertoli cells might be involved and possibly maintained by normal serum testosterone levels.

Serum thymidine activity and somatomedin-C levels in children and adolescents with Turner's syndrome: effect of chronic estrogen and progestogen replacement therapy.

Turner's syndrome has been used as a model of primary hypogonadism to assess the role of estrogen-progestogen replacement therapy on serum thymidine activity (TA) and somatomedin-C (Sm-C) levels. 33 subjects with gonadal dysgenesis were studied: 10 untreated and 13 treated with estrogen-progestogen combination. In 10 untreated patients serum TA was 1.02 +/- (SEM) 0.04 U/ml and serum Sm-C value was 27.82 +/- 4.14 nmol/l, both similar to those in the age-matched normal children. A positive correlation was found between Sm-C and the bone age (r = 0.891, p less than 0.002). In the 13 treated subjects, the estrogen-progestogen combination as replacement therapy induced a significant increase in Sm-C level (40.52 +/- 4.30 nmol/l, p less than 0.05). No variation was observed for serum thymidine activity between the two groups of subjects.

Dose- and time-dependent effects of a dimeric ethynodiol-testosterone depot-preparation in female rat.

The time- and dose-dependent effects of a dimeric ethynodiol-testosterone ester upon intact and hemi-castrated female rats were compared to those of equivalent doses of norethisterone enanthate plus testosterone enanthate. In intact rats, serum LH was markedly suppressed for at least 8 weeks after a single i.m. injection of 10 or 20 mg of the dimer which exceeded the depot-effect of the combination of the enanthates. A biphasic time-and dose-dependent effect of the dimeric ester upon ovarian and uterine weight was observed. During first 2 weeks following the injection there was a pronounced enlargement of corpora lutea and a marked enhancement of progesterone secretion, probably due to a direct stimulation by the androgens. At the same time, the uteri were markedly enlarged. During the following weeks, the ovaries became progressively smaller and showed degenerative changes, the steroid levels decreased and the uteri became atrophic. When hemi-castrated rats were injected once with 1 and 5 mg of the dimer, LH became suppressed, and the compensatory hypertrophy of the ovary was inhibited. At higher doses, the weight of the ovary and uterus was increased, and the luteal function was stimulated. The results indicate that, even though the gonadotropins are suppressed, there is possibly a direct stimulatory effect of high doses of the intact dimeric molecule upon the uterus, while the stimulation of the corpus luteum function is due to an interference of the transiently elevated testosterone level with ovarian steroid biosynthesis.

Liver regeneration in oral contraceptive treated female rats--effects of moderate malnutrition.

Treatment of well-nourished female rats with a combination of 5 micrograms ethynyl estradiol and 100 micrograms ethynodiol diacetate, increased the DNA content, 3H thymidine incorporation into DNA and mitotic activity in the non-regenerating liver, but impaired liver regeneration after partial hepatectomy. In rats which were moderately malnourished by feeding 25 percent less calories and 50 percent of recommended allowance for vitamins A and B2, OC treatment had similar stimulatory effect on non-regenerating liver, but did not impair liver regeneration after partial hepatectomy. Analysis of nucleotide bases after hydrolysis of unpolymerized nucleotides and nucleosides revealed significant perturbations due to OC treatment. However, the impaired liver regeneration due to OC treatment of well-nourished rats could not be attributed to diminished availability of bases, particularly thymidine. Data on mitotic index and binucleate cell numbers suggest that besides inhibiting mitosis (DNA duplication), OC treatment of well-nourished rats may also impair partitioning of binucleate cells.

Contribution of functional groups of 19-nor-progestogens to binding to progesterone and estradiol-17beta receptors in rabbit uterus.

The structural elements of 19-norprogestogens which may be essential for binding to progesterone and estradiol-17beta(E2) receptors were investigated in the rabbit uterine cytosol. The kinetic study showed that 19-nor-progestogens are competitive inhibitors of progesterone-receptor (8S) binding and E2-receptor binding. The affinities of steroids for the progesterone receptor were as follows: norethindrone (Ki of 2.3 X 10(-9)M) greater than 5alpha-dihydronorethindrone greater than norethindrone acetate greater than lynestrenol greater than 17alpha-ethynyl-estra-4-ene-3beta, 17beta-diol greater than ethynodiol diacetate (Ki of 1.3 X 10(-7) M). The affinities of steroids for the E2 receptor were as follows: ethynodiol diacetate (Ki of 1.3 X 10(-7)M) greater than 17alpha-ethynyl-estra-4-ene-3beta, 17beta-diol greater than norethindrone acetate greater than norethindrone greater than 5alpha-dihydronorethindrone greater than lynestrenol (Ki of 8.4 X 10(-7)M). The results indicate that 3-ketone and 17beta-hydroxyl groups, and the plane of ring A/B of 19-norprogestogen are important for binding to the progesterone receptor. The affinities of 19-nor-progestogens for the E2 receptor were very weak. Their affinities for the E2 receptor increased with addition of acetate or hydroxyl groups at the 3beta and 17beta positions, and were decreased by the elimination of a 3 oxygen function or the reduction of ring A.