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.

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.

Norethisterone concentration in breast milk and infant and maternal plasma during ethynodiol diacetate administration.

Twelve women with established lactation of 4-8 weeks duration were given a low-dose progestogen-only contraceptive, ethynodiol diacetate 0.5 mg (Femulen) daily. On the seventh and eight day of the study, prior to the mother's taking the pill, a blood sample was taken from her and from the infant. Further blood samples were collected from the mother 4 and 12 hours later. Breast milk samples were collected at every feed on day 7 and day 8. Ethynodiol diacetate is rapidly metabolised in humans, changing into the metabolite norethisterone which is found in both blood and milk. Hence, norethisterone concentrations were estimated. On day 7 and day 8, four hours after ingestion of the pill, the median norethisterone maternal plasma concentration was 1.60 ng/ml and it fell to a median level of 0.30 ng/ml prior to the next dose of the pill. At this time the median infant concentration was 0.10 ng/ml but the maximum observed level was 0.50 ng/ml. In the breast milk the norethisterone concentration appears to peak at around 4-8 hours following the ingestion of the pill. The maximum observed concentration in breast milk was 0.84 ng/ml. The amount of norethisterone ingested by the infant averaged 0.02% (6.65 micrograms) of the dose of ethynodiol diacetate ingested by the mother. The maximum observed on any one day was 0.07% (27.52 micrograms). The above results indicate that the amount of progestogen ingested by the infant from its mother's milk is small and is unlikely to pose a risk to the infant.

A randomized study of metabolic effects of four low-estrogen oral contraceptives: I. Results after 6 cycles.

Healthy, non-smoking, normotensive, well-motivated young women were assigned at random to one of four different, commercial, low-estrogen, oral contraceptive products. Measurements of biochemical parameters were made on blood specimens collected from fasting subjects twice during the late pretreatment cycle, then again during each late treatment cycle for six months. All women assigned to one product (0.5mg NET + 35 microgram EE) dropped out of the study before the end of the fifth cycle, but discontinuations with the other three products were few. While numbers of subjects are small, the groups are closely matched and most metabolic differences are statistically significant. Products containing EDA and NET were associated with increases in serum total cholesterol and triglycerides, but decreases in HDL-cholesterol. In contrast, the LNG-containing preparation produced significantly less effect on these tests. A similar pattern was seen with a range of blood coagulation and fibrinolytic factors, Minimal alterations were seen with the LNG preparation, while those containing NET or EDA showed marked increases in factors I. VII, VIII, X and plasminogen, associated with a decrease in antithrombin III. It is suggested that differences in the metabolic impact of the various commercially available low-estrogen preparations, combined with effects on intermenstrual bleeding, allow a choice of the progestogen component most suitable for general use.

Metabolism of ethynodiol diacetate in the rhesus monkey before and after administration of rifampicin.

1. On administration of a single oral dose of [4-(14)C]ethynodiol diacetate (0.15 mg/kg) to rhesus monkey, plasma concn. of total 14C peaked after about 4 h. About 60% of the plasma radioactivity was present as glucuronide conjugates and no unchanged drug was detected. 2. Some 67 +/- 6% (mean +/- S.D.) of the dose of 14C was excreted in 4 days, 50 +/- 6% in urine and 18 +/- 2% in faeces. Most of the urinary excretion occurred within 24 h of dosage. 3. Glucuronide conjugates accounted for 60% of the urinary 14C, and 46% of the faecal 14C was free steroids. 4. Norethisterone and its tetrahydro metabolites were identified in the free, glucuronide and sulphate fractions of plasma and urine. Keto-4,5-dihydronorethisterones and trihydroxy metabolites were identified in the conjugated fractions of urine, and a complex mixture of polar metabolites was detected in faeces. 5. Rifampicin treatment (7.5 mg/kg/day, orally) for 8 days decreased the half-life of total 14C in plasma following a single oral dose of 4-[14C]ethynodiol diacetate (0.15 mg/kg) from 44 +/- to 24 +/- 2 h. 6. Faecal elimination of total 14C was significantly increased to 29 +/- 5% of the dose following rifampicin treatment, but urinary excretion was unchanged. 7. Rifampicin treatment increased the amount of polar metabolites and decreased the amount of norethisterone in the free and conjugated fractions of plasma and urine. The amounts of sulphate and non-hydrolysed conjugates in faeces were increased after rifampicin treatment.

Bioavailability and pharmacokinetics of norethisterone in women after oral doses of ethynodiol diacetate.

Measurement by radioimmunoassay of plasma norethisterone (NE) has been used to compare the bioavailability of tablets containing ethynodiol diacetate (EDA) with that of a standard oral solution of this progestogen in 12 normal women. The tablets investigated were from three batches which showed different in vitro dissolution rates. There were no significant differences in the bioavailability of the tablet formulations, which were essentially bioequivalent to the solution. Peak blood levels of NE were reached within 4h of EDA administration in solution or tablets. After the peak, NE plasma levels declined in two phases, with a mean terminal elimination half lives of 4 to 6.9h. The pharmacokinetics of NE after EDA administration showed some similarity to those observed by other workers after oral doses of NE itself.

PIP: Bioavailability and pharmacokinetics of norethisterone (NE) were studied in 12 women, aged 21-37 years, after oral doses of ethynodiol diacetate (EDA). Plasma NE levels, measured by radioimmunoassay, were used to compare the bioavailability of EDA tablets (Ovulen 50; 1 mg EDA plus .05 mg ethinyl estradiol) with that of a standard oral solution of EDA. The 3 different batches of tablets studied showed different in vitro dissolution rates, 82.6%, 94.6%, and 99% at 3 hours. No marked differences were seen in the bioavailability of the tablet formulations, which were essentially bioequivalent to the solution. Peak plasma NE levels were reached within 4 hours of EDA administration in solution or tablets. Following the peak, NE plasma levels declined in 2 phases, with mean terminal elimination 1/2-lives of 4-6.9 hours. These results have shown that small variations in in vitro dissolution rates do not affect the bioavailability of NE from tablets containing EDA.

[Transformation of 19-norgestagens to estrogens by rumen microorganisms]. / Umwandlung von 19-Norgestagenen in Ostrogene durch Mikroorganismen des Rinderpansens.

The 19-norgestagens norethisterone acetate (17 alpha-ethinyl-4-oestren-17 beta-ol-3-on-17-acetate), ethinodiol diacetate (17 alpha-ethinyl-4-oestren-3 beta, 17 beta-diol-3, 17-diacetate), and norgestrol (17 alpha-ethinyl-18-methyl-4-oestren-17 beta-ol-3-on) are transformed to ethinyloestradiol or 18-methyl homologue by microorganisms of cattle rumen. Such transformation of steroid gestagens to oestrogens is likely to offer an explanation for the occurrence of oestrogen effects which had been observed during synchronised oestrus of cattle following oral application of 19-norgestagens.

Synthetic progestins: in vitro potency on human endometrium and specific binding to cytosol receptor.

The relative potency of six commonly used synthetic progestins has been evaluated in an organ culture system for human endometrium. The affinities of these progestins for endometrial progesterone receptor were also evaluated after removing the CBG-like protein by spheroidal hydroxylapatite chromatography. All six progestins induced an increase in tissue glycogen during culture at lower concentrations than did progesterone; only one (medroxyprogesterone acetate) had a relative affinity greater than progesterone. The relative potencies and affinities of the synthetic progestins were found to have the same relative order but to differ in relative magnitude.

Comparative metabolism of 17alpha-ethynyl steroids used in oral contraceptives.

The metabolism of mestranol, ethynylestradiol, norethynodrel, norethindrone, ethynodiol diacetate, lynestrenol, and norgestrel is reviewed. The estrogenic components of the oral contraceptives, mestranol or ethynylestradiol, have nearly identical metabolic pathways since mestranol is rapidly and almost completely converted to ethynylestradiol The major fraction of the drugs plus metabolites is excreted in the urine as conjugated materials. All of the 17beta-ethynyl progestins reviewed follow similar metabolic paths. For three of these, norethynodrel, ethynodiol diacetate and lynestrenol, a principal metabolite is norethindrone. Biotransformation to more polar metabolites and conjugation proceed rapidly for these three precursor drugs and norethindrone. Norgestrel follows metabolic paths similar to those of norethindrone. However, the ethyl moiety at the C-13 position appears to slow the metabolism of this steroid so that biotransformation to more polar metabolites and the conjugation of these steroids does not proceed as rapidly as that of the other progestins. The high progestational potency of norgestrel may be attributed to this slow rate of biotransformation. Some of the pharmacokinetic parameters derived from the research reports reviewed here are summarized. The compounds appear to be readily absorbed, and they and their metabolites are excreted to a greater extent in the urine than in the feces.

[Absorption, distribution, excretion and metabolism of SC-11800EE, a combined steroid preparation of SC-11800 (ethynodiol diacetate) and ethinyl estradiol in rats and mice (author's transl)].

Absorption, distribution, excretion and metabolism of SC-11800EE, a combined steroid preparation consisting of SC-11800(ethynodiol diacetate)as gestagen and ethinyl estradiol (EE)as estrogen in 20:1 (w:w), were studied with the use of 14C-SC-11800 and 3H-EE by radiometry in female rats and by the whole body autoradiography in female normal and pregnant mice. The gestagen orally given with EE was rapidly absorbed from digestive tracts and distributed in tissues in various levels. Gestagen levels in liver and kidney exceeded that in plasma. About 75% of dosed radioactivity was excreted in feces largely via bile and more than 20% in urine within 72 hr after administration. The gestagen was metabolized extensively to more polar products and their conjugates. The pharmacokinetic behavior of the gestagen given with EE did not alter after repeated administrations for 7 days, but was slightly different from that without EE, possibly due to the estrogen effect. The pharmacokinetic behavior of the estrogen was independent from the gestagen given simultaneously. The distribution of the gestagen given with EE revealed by the whole body autoradiography in normal mice were essentially consistent with the radiometric results in rats and that in the pregnant mice showed that the gestagen in fetus was virtually nil under the present conditions.

Controlled drug release from polymeric delivery devices. III: In vitro-in vivo correlation for intravaginal release of ethynodiol diacetate from silicone devices in rabbits.

Forty female rabbits were implanted with silicone vaginal devices containing ethynodiol diacetate for up to 8 weeks. As predicted from in vitro studies, a Q - t1/2 (matrix-controlled) release profile was observed in vivo. The in vivo drug release profile was compared with in vitro data measured at three hydrodynamic conditions, and the diffusional resistance across the vaginal wall was estimated. Drug released from silicone devices yielded a prolonged plasma level when compared with data following intravaginal or intravenous administration of a solution dose. The rate constant for elimination was unchanged. The plasma concentration of the drug was related to the intravaginal drug release profile both theoretically and experimentally and was above the concentration required to inhibit fertilization.

Metabolic fate of ethynodiol diacetate in the baboon.

The enterohepatic circulation and metabolism of ethynodiol diacetate (3beta,17beta-diacetoxy-17alpha-ethynyl-estr-4-ene) in baboons were studied following the intravenous injection of this contraceptive steroid labeled with 14C (4-position) and with 3H (in either the 3- or 17-acetoxy moieties). Bile and urine from four baboons with biliary fistulas and urine from four intact baboons were collected for 7 hours. On the average, 40% and 44% of the injected dose were excreted in the bile and urine, respectively. Only 48% was recovered in the urine of intact baboons. Analysis of these excretion rates indicates an insignificant enterohepatic circulation of this compound. The steroid was excreted mostly (over 80%) as a glucosiduronate in urine and bile. Very little excretion of the 3-acetoxy compound was detected in the urine or bile at any time interval. 17-Monoacetoxy compounds, however, were detected both in urine and bile, suggesting a difference in the rate of in vivo hydrolysis of the 17beta- vs. the 3beta-acetate.

Interaction of contraceptive progestins and related compounds with the oestrogen receptor. Part I: Effect on (3H)oestradiol distribution pattern in the ovariectomized rat.

The distribution pattern of oestradiol in ovariectomized rats as a function of time has been studied following intravenous adminstration of the tritiated hormone. Oestrogen specific binding with limited capacity was observed in the uterus, vagina, anterior pituitary, adrenals, preoptic area, hypothalamus, amygdala, septum and tractus diagonalis. Maximal uptake of oestradiol in the pituitary occurred within 5 min, in the uterus 60 min after injection, and remained almost unchanged at this level for more than two hours. The binding capacity per mg tissue decreased in the order pituitary, uterus, vagina, preoptic area, adrenals, hypothalamus, amygdala, spetum and tractus diagonalis. The hormone concentration in these tissues one hour after (3H)oestradiol injection was lowered by previous administration of ethinodiol, norethinodrel, lynestrenol and norethindrone, whereas medroxyprogesterone, chlormadinone, megestrol and methyllynestrenol had no effect. The same results were obtained, when instead of the steroid alcohols the corresponding acetate esters were administered. For norgestrel, oestrenol and nortestosterone the effect in the dose range studied was limited to the pituitary and preoptic area. For lynestrenol the inhibition of oestradiol binding in the target tissues was almost the same when the progestin was given 60 and 5 min before oestradiol, whereas in the case of administration 30 min after oestradiol no inhibition was observed. The reduction of oestrogen binding appeared to be dose-dependent, but the dose required to obtain a certain effect for the uterus was four times as high as for the pituitary. Discrepancies between previous studies and the implications of the present findings for the mechanism of action of ovulation inhibition by these progestins are discussed.

Interaction of contraceptive progestins and related compounds with the oestrogen receptor. Part II: Effect on (3H)oestradiol binding to the rat uterine receptor in vitro.

Incubation of the 105 000 times g supernatant of rat uterus homogenate with (3H)oestradiol resulted in an oestrogen specific binding of limited capacity to a macromolecule sedimenting in the 8-9S region after density gradient centrifugation. The contraceptive progestins of the 19-nortestosterone series were able to interfere with oestradiol binding in contrast to the hydroxyprogesterone derivatives chlormadinone, medroxyprogesterone and megestrol. The interaction appeared to be competitive. The strongest inhibition of oestradiol binding was observed in the presence of ethinodiol, followed by northinodrel, lynestrenol and norethindrone respectively. Norgestrel was almost inactive. Of the related structures tested oestrenol displayed more activity than norethindrone, nortestosterone and ethisterone were less active and 6alpha-methyllynestrenol showed only border line activity. In comparison with norethinodrel and norethindrone, lynestrenol and oestrenol appeared in vitro to be stronger competitors for oestradiol than in vivo (Part I; Van Kordelaar et al. 1975). This may be due to the great difference in lipophilic character, which is reflected in the RM values of these compounds. From the results obtained it may be concluded, that the presence of a 17alpha-ethynyl substituent promotes receptor binding, whereas the introduction of methyl substituents in the position 6, 10 and 18 causes the opposite effect. The relationship between the various ring A structures and the affinity to the receptor is discussed.