Production of antisera against contraceptive steroids

A four step synthesis of 6-(O-carboxymethyl)oximinoethynylestradiol is reported. This compound, 6-(O-carboxymethyl)oximinomestranol, the 3-(O-carboxymethyl)oximes of norethindrone and norgestrel and the 3-hemisuccinate of ethynylestradiol were synthesized and conjugated with bovine serum albumin. Rabbits were immunized at 3 dose levels of haptene (20, 66 and 200 nmoles) and eight weeks later with a booster containing 66 nmoles of haptene. The antibody titer and association constant of responding rabbits was nearly independent of dose although most antibody production occurred after the booster injection. Antibodies to mestranol crossreacted more than 100% with ethynylestradiol and to a small extent with norethindrone and norgestrel.     

Use of diethylstilbestrol and ethynylestradiol to feminize Nile tilapia Oreochromis niloticus (L.) in an outdoor environment

Two synthetic estrogens, diethylstilbestrol (DES) and ethynylestradiol (EE), were orally administered to 8.7 mm gonadally undifferentiated Oreochromis niloticus fry for a period of 28 days in an outdoor setting. Diethylstilbestrol was administered at doses of 100 mg, 200 mg, and 400 mg per kg diet. Ethynylestradiol was administered at 50 mg, 100 mg, and 200 mg per kg diet. One group received a non-hormone-treated feed. Hormone treatments produced significantly more (P < 0.05) than 50% females indicating that genotypic male fish were sex-reversed to phenotypic females. No rate of estrogen administration resulted in a 100% female population. Ethynylestradiol (EE) treatments resulted in 58–65% females, 32–35% males, and 3–9% hermaphrodites. Diethylstilbestrol (DES) treatments resulted in 60–80% females, 13–37% males, and 1–7% hermaphrodites. The DES 400 treatment was the most effective in altering phenotypic sex: 80% females, 13% males, 7% hermaphrodites.     

Serum and placenta levels of ethynylestradiol in presence of ethynodiol diacetate after oral administration

The ethynylestradiol concentration--in the presence of ethynodiol diacetate--in serum after oral administration was measured by a rapid radioimmunoassay method developed by the authors. It was found that the peak level was reached 1 h after administration, and even after 12 h a significant amount of free ethynylestradiol was present in the serum. The transfer of ethynylestradiol into the placenta was also studied in subjects who were 10-12 weeks pregnant. Placenta/serum quotients were calculated for the ethynylestradiol, and were found to increase in parallel with the dose of the drug administered, proving that an ethynylestradiol enrichment of the placenta occurred as early as 10-12 weeks of pregnancy.     

Aromatization of norethindrone to ethynylestradiol in human adult liver

Homogenates of human adult liver are capable of aromatizing norethindrone (17 alpha-ethynyl-19-nortestosterone) to ethynylestradiol (17 alpha-ethynylestradiol). The evidence of ethynylestradiol formation was obtained using a Bio-Rad AG1-X2 column, thin layer chromatographies and co-crystallization. Neither acid nor base was used in any step in product identification.     

Development of a monoclonal antibody for use in radioimmunoassay of ethynylestradiol

Production of monoclonal antibodies (MABs) to a 17 alpha-ethynyl-1,3,5 (10)-estratriene-3,17 beta-diol (ethynylestradiol, EE2) hapten is described. Culture antibodies generated by hybridized cells tested in enzyme-linked immunosorbent assay (ELISA) bound the 6-oxoethynyl-estradiol-6-(0-carboxymethyl) oxime-bovine serum albumin conjugate (EE2-6-0 CMO-BSA) but not BSA. On radioimmunoassay (RIA) with tritiated ethynylestradiol (3H-EE2), some of the antibodies demonstrated high binding affinity (Ka = 2.8 X 10(10) M-1) to EE2. Estradiol-17 beta, estrone and estriol did not show any displacement of 3H-EE2 from the MABs even at high concentration (1 X 10(4) ng/mL). Among the widely used progestins, norethynodrel and norethisterone exhibited considerable cross-reactivity (25.7-100% and 0.3-54.1%, respectively) but not levonor-gestrel with the MABs. The high affinity demonstrated by the MABs to EE2 3-sulfate but not to EE2 17-sulfate and EE2 3,17-disulfate suggests the dominant role of the 17 beta-hydroxyl group in immunogenicity.     

Aromatization of norethindrone to ethynylestradiol in human adult liver [Poster 41]

Homogenates of human adult liver are capable of aromatizing norethindrone (17-ethynyl-19-nortestosterone) to ethynylestradiol (17-ethynylestradiol). The evidence of ethynylestradiol formation was obtained using a Bio-Rad AG1-X2 column, thin-layer chromatography and co-crystallization.     

Negative feedback effects of chlormadinone acetate and ethynylestradiol on gonadotropin secretion in patients with prostatic cancer and male rats

Serum LH and FSH levels were determined before and after LH-RH injection (100 micrograms, i.m.) in patients with prostatic cancer who were chronically treated with either chlormadinone acetate (CMA, 100 mg/day) or ethynylestradiol (EE, 1 mg/day). In patients treated with EE, the levels of serum LH and FSH before and after injection of LH-RH were significantly lower than those in controls. On the other hand in patients treated with CMA, the basal levels of serum gonadotropins did not differ from those in controls, and the increase in gonadotropin after LH-RH injection was comparable to that in controls. To examine the effects of these steroids on the hypothalamo-hypophysial axis in the regulation of gonadotropin secretion, CMA or EE was implanted in castrated male rats. CMA, EE or cholesterol (control) was implanted in the hypothalamic median eminence-arcuate nucleus region through a stainless doublecannula. EE implantation resulted in a 75% decrease in serum LH (p < 0.001) and a 38% decrease in serum FSH (p < 0.05) from the control levels on day 5 of implantation. On the other hand, CMA implantation induced a 33% decrease in serum LH (p < 0.05) from the control level on day 3 of implantation, but no significant change in serum FSH levels. The injection of 2 micrograms/kg of LH-RH on day 7 of implantation induced significant lowering of LH and FSH levels. There was no significant difference between serum levels of the hormones 20 min after LH-RH injection for these two groups and those for the control group. These studies suggest that EE has a potent negative feedback effect on both LH and FSH secretion, and that CMA has a mild negative feedback effect on LH secretion.     

Derivatives of ethynylestradiol with oxygenated 17 alpha-alkyl side chain: synthesis and biological activity

The coupling reaction of an acetylide ion with alkyl bromide or delta-valerolactone was used to synthesize twelve 17 alpha-derivatives of ethynylestradiol having various 17 alpha-side chain lengths and, except one, having mono- or di-oxygenated function on the side chain. All compounds had low (0.01-1.79%) relative binding affinity (RBA) for the rat uterine estrogen receptor. The highest RBA were obtained for compounds 26 (1.79%), 30 (1.55%) and 19 (0.42%). The length and polarity of the side chain decreases the affinity for the estrogen receptor. The in vivo estrogenic activity was measured on mouse uterine weight and was found to range from 0 to 35%, except for compound 30 (100%). The antiuterotrophic activity was measured by inhibition of estradiol-induced stimulation of uterine weight and was found to be 39% for compound 19, 25% for compound 26 and 0% for all other compounds. These two compounds (19 and 26) possess mixed agonist and antagonist activity.     

Irreversible protein binding of metabolites of ethynylestradiol in vivo and in vitro

During incubation of 6,7-³H-ethynylestradiol with rat liver microsomes up to 20 % of the radioactivity was bound irreversibly to the microsomal proteins. Incubations in presence of albumin resulted in a further radioactive labelling of the albumin. The irreversible nature of the steroid-protein bond was established by solvent extraction and charcoal treatment. Further evidence was obtained after hydrolyzing the microsomal protein with trypsin and submitting the labelled tryptic peptides to ion exchange chromatography and electrophoresis. The labelled albumin was applied to sephadex gel filtration which showed the association of the ethynylestradiol radioactivity to the albumin peak.The binding reaction required supply of NADPH, could be stimulated by pretreatment of the animals with phenobarbital and was inhibited by CO and SKF 525 A. On these characteristics the concept was based that, in analogy to the well known binding of estradiol and estrone, 2hydroxylation is also an essential prerequisite for the binding of ethynylestradiol. The concept was confirmed by trapping off the 2-hydroxy-ethynylestradiol with glutathione, which led to a decrease of the ethynylestradiol-protein binding.Further evidence resulted from experiments $\text{in vivo}$, dosing rats with 6,7-3H-ethynylestradiol and 6,7-3H-estradiol 48 hrs prior to sacrifice and examining the amount of radioactivity irreversibly bound to the liver endoplasmic reticulum. 3H-ethynylestradiol caused a radioactive labelling of microsomes twice as much as that after ³H-estradiol.     

Refractoriness of ovarian adenylate cyclase to continued hormonal stimulation.

Culture of preovulatory rat follicles with luteinizing hormone, follicle-stimulating hormone or prostaglandin E2 for 24 h reduced the subsequent response of adenylate cyclase to the homologous by 80, 50 and 90%, respectively; yet follicles refractory to luteinizing hormone fully responded to follicle-stimulating hormone responded to luteinizing hormone and prostaglandin E2, and those refractory to prostaglandin E2 could be stimulated by either gonadotropin. Desensitization of the adenylate cyclase system by luteinizing hormone was achieved by hormone concentrations of 0.8--2.0 mug/ml in the medium; a lower dose of luteinizing hormone (0.4 mug/ml), though effective in stimulating adenylate cyclase, did not induce refractoriness. Prostaglandin E2 caused partial refractoriness at dose levels of 0.1--0.25 mug/ml; higher dose levels were more effective. These findings suggest that continued exposure to the preovulatory follicle to elevated levels of hormones may cause perturbations in either the interaction between the hormone and its specific receptor or in a subsequent step essential for activation of adenylate cyclase.     

Antigens from synthetic and natural estrogens: formation by conjugation with protein through a thiopropionic acid link

Formation of estra-1,3,5(10)-trienes substituted in the 7-position by a 3-thiopropionic acid side chain occurs by light-catalyzed reaction of β-mercaptopropionic acid with 1,3,5(10),6-estratetraenes. The reaction yields a mixture of α and β-epimers, which can be distinguished by their nmr spectra in pyridine. Both estrone and estradiol analogs were prepared. Addition of acetylene to estrone derivatives led to mestranol and ethynylestradiol analogs. The carboxylic acids were coupled with bovine serum albumin (with an incorporation of 25–30 steroid residues per albumin molecule) to yield antigens for the synthetic and natural estrogens which caused formation of specific antibodies.     

The effect of oral contraceptive steroid therapy on the urinary metabolites of radioactive estrone and estradiol-17beta

Eight urinary metabolites of radioactive estrone and estradiol-17β (estrone, estradiol-17β, 2-hydroxyestrone, 2-methoxyestrone, 2-hydroxyestrone 3-methyl ether, 16α-hydroxyestrone, 2-hydroxyestradiol and estriol) have been measured by reverse isotope dilution from young women on oral contraceptive therapy. There was a decrease in the sum of the 16α-hydroxy1ated metabolites in the Ketodase liberated fraction from the subjects taking ethynylestradiol containing preparations as compared to those taking preparations containing mestranol and those subjects who were taking no oral contraceptives. This report is also the first to document and measure 2-hydroxyestradiol as a urinary metabolite of radioactive estrone and estradiol.     

A kinetic study on the lactoperoxidase catalyzed oxidation of estrogens

Lactoperoxidase, which is produced in mammary glands, is proposed to be involved in carcinogenesis, because of its ability to react with estrogenic molecules, oxidizing them to free radicals. In the present study the reactivity towards six species (estradiol, ethynylestradiol, estriol, estrone, pregnenolone and mestranol) was investigated by means of a NADH-coupled system. The enzyme activity towards estradiol, ethynylestradiol, estriol and estrone did not vary much, suggesting that the different substituents in the D-ring of the steroid had little effect on the reaction. A somewhat higher K _m-value was obtained with estriol; possibly because of a more effective splitting of the enzyme–substrate complex into products. Pregnenolone, without resonance in the A-ring, and a methyl group in 19-position, did not react with the enzyme, in spite of having the proposed essential hydroxyl group in 3-position. Mestranol, with a methoxy group in 3-position, did not react with the enzyme either, supporting the suggestion that lactoperoxidase reacts with the 3-hydroxyl group of the estrogens.     

Effects of age and weaning on the immature rat uterotrophic assay using ethynylestradiol.

The purpose of this study was to evaluate the most appropriate rat age for the start of administration, and the effect of weaning, in the immature rat uterotrophic assay using ethynylestradiol (EE). Animals weaned on postnatal day (PND) 20 were administered subcutaneously EE at doses of 0.06-6 micrograms/kg/day for 3 days beginning on PND 21, 23 or 25. EE at the same doses was also administered to rats weaned on PND 17 or 20 from PND 21 for 3 days. Although uterine weight was significantly increased in the rats given 0.6-6 micrograms/kg EE in both of the studies, the percentage increase relative to the control in each group given EE from PND 21 and weaned on PND 20 was higher than in those groups given EE from PND 23 or 25, and the group weaned on PND 17.     

The effect of sexual hormones on skin graft survival, thymic morphology and serum levels of glycoproteins and albumin in mice.

Inbred mice of the B10LP strain were injected subcutaneously over a period of 28 days with chloromadinone acetate, mestranol, both hormones together, and the carrier alone. Treatment with the gestagen chloromadinone acetate alone or in combination with mestranol caused a significant prolongation of the H-2 different skin graft survival. This was not seen after mestranol treatment alone. All the hormone-treated mice developed characteristic cysts in the thymic medulla and changes in the concentration of some plasma proteins, e.g. a fall of albumin level and a simultaneous increase of glycoprotein level. Therefore, it is necessary to look for such side-effects in humans also to prevent or to notice early alterations of immunological surveillance.     

Effects of intravulvo-submucosal cloprostenol injections on hormonal profiles and fertility in subestrous cattle

Eighty-two subestrous cattle were treated with different doses of cloprostenol through intramuscular (i.m.) and intravulvo-submucosal (i.v.s.m.) injections to study hormonal profile and fertility. The study was divided into two experiments. In Experiment I, 13 cows were treated with one of three doses of cloprostenol (500 mug i.m., 125 and 62.5 mug i.v.s.m.) to measure response of progesterone (P(4)) and estradiol (E(2)). P(4) decreased abruptly and E(2) levels increased from basal levels following injections of the two larger doses of cloprostenol. P(4) decreased to<5 nmol/l approximately 72 h after treatment. E(2) levels increased to >300 pmol/l 24 h after cloprostenol injections except in cows treated with 62.5 mug dose. Close agreement was observed between P(4) profiles and clinical findings following 500 and 125 mug of cloprostenol treatment. In Experiment II, 69 subestrous cows were treated with either 500 mug i.m. or 250, 125 or 62.5 mug i.v.s.m. doses of cloprostenol. The percent of cows in estrus 96 h following treatment were 60, 80, 67.8 and 18%, respectively. A total of 29 cows were artificially inseminated and 41.3% conceived. We concluded that i.v.s.m. injections of cloprostenol at the dosage of 125 mug and above causes luteolysis, induces estrus and establishes fertility in subestrous cattle. The method is economical but time consuming when compared to the intramuscular route.     

Synergistic effects of ethynylestradiol and norethisterone on the formation and lengthening of uterine lace in immature rabbits]

We have shown, through a test proceeding from the McPhail test, in the immature rabbit female, the existence of succession and simultaneity synergisms between ethynylestradiol and pure or impure norethisterone, given by oral route. The estrogen, administrated before the progestative, is able to potentialize the action of the latter on the building of the endometrial lace in 5 days, more than on the lengthening of it in 15 days. Ethynylestradiol, absorbed with norethisterone, strengthens the effects of the latter on the second phenomenon more than on the first one. Absolute and relative doses of the estrogen and of the progestative differ only lightly according as norethisterone is pure or not pure, because of the more potent estrogenicity of the impure norethisterone.     

Apolipoprotein L-I is positively associated with hyperglycemia and plasma triglycerides in CAD patients with low HDL

Apolipoprotein L-I (apoL-I) is present on a subset of HDL particles and is positively correlated with plasma triglycerides (TGs). We measured plasma apoL-I levels in coronary artery disease (CAD) subjects with low HDL who were enrolled in an angiographic CAD prevention trial. At baseline, apoL-I levels (n = 136; range, 2.2-64.1 mug/ml) were right skewed with a large degree of variability. Multivariate analysis for biological determinants of apoL-I revealed that the log of VLDL-TG (+0.17; P < 0.05) and hyperglycemia (HG; +0.26; P < 0.005) independently predicted apoL-I level. Hyperglycemic patients (n = 24) had mean apoL-I levels >50% higher than normoglycemic subjects (n = 112; 13.2 vs. 8.3 mug/ml, respectively; P < 0.001). No relationship between apoL-I level and change in CAD was found (r = 0.06, P = 0.49). Simvastatin-niacin therapy did not alter apoL-I levels (n = 34; P = 0.27), whereas antioxidant vitamins alone increased apoL-I by >50% (n = 36; P < 0.01). Genotyping of a known apoL-I polymorphism (Lys166Glu) did not independently account for any of the variability in apoL-I levels. In conclusion, we found TG and HG to be the strongest predictors of apoL-I within a dyslipidemic CAD population. These data provide further characterization of the novel HDL-associated apoL-I.     

Intrauterine injection of 15(S)-15-methyl-prostaglandin F2alpha for termination of early pregnancy in out-patient.

15-me-PGF2alpha was administered as single intrauterine injection for interruption of very early pregnancy in 30 out-patients. After 2 weeks, abortion was complete in 60% induced with 125 or 200 mug and 80% induced with 300 mug. After 3 weeks, abortion was complete in 90% induced with 125 mug, in 70% induced with 200 mug and in 100% induced with 300 mug. One failure occurred in patients treated with 200 mug and 2 curettages were performed because of incompleteness of abortion. No serious complications occurred. Compared with our previous results it appears that 15-me-PGF2alpha is as effective as natural PGF2alpha in inducing abortions during very early pregnancy but causes somewhat fewer side-effects.     

Peripheral vasodilator and cardiac properties of the 1,5-benzothiazepine calcium antagonist analog, TA-3090, in dogs

Diltiazem, a 1,5-benzothiazepine, has demonstrated efficacy in the treatment of numerous cardiovascular diseases. TA-3090, a newly synthetized 1,5-benzothiazepine compound was studied in open-chest anesthetized dogs to characterize its hemodynamic properties, to compare it with diltiazem, and finally to correlate hemodynamic properties and plasma level concentrations. Anesthetized open-chest dogs were instrumented with electronic devices and fluid-filled catheters to monitor cardiac, coronary, and peripheral hemodynamic changes. A cumulative intravenous bolus administration of TA-3090 (n = 16) or diltiazem (n = 15) (15, 50, 200, and 400 micrograms/kg) was carried out, and blood samples were taken before and 5 min following each dose administration. Hemodynamic changes were followed for 30 min after each administration, at which time most hemodynamic parameters were back to baseline levels. The results indicate that both TA-3090 and diltiazem elicit slight peripheral and coronary vasodilator properties at low doses (15 and 50 micrograms/kg). With higher dosage, hemodynamic effects were maximal: coronary blood flow increased by 75%, arterial pressure decreased by 25%, and reflex positive inotropic effects were also observed. Heart rate was significantly reduced (10%). Comparison between TA-3090 and diltiazem indicates that both drugs elicit coronary vasodilator selectivity and TA-3090 has a prolonged duration of action compared with that of diltiazem. A straightforward relationship is demonstrated between vasodilator properties and plasma levels of either TA-3090 or diltiazem. Our data suggest that with plasma levels between 40 and 80 ng/mL, significant hemodynamic changes were observed with TA-3090. Changes of heart rate were not correlated with plasma levels.(ABSTRACT TRUNCATED AT 250 WORDS)