T. Giannina

Pathway of Absorption of Orally Administered Ethynylestradiol-3-Cy-clopentyl Ether in the Rat as Influenced by Vehicle of Administration.

Summary The pathway of transport of labelled ethynylestradiol (EE) or its 3-cyclo-pentyl ether (EECPE) has been studied following oral administration to rats. The data on lymphatic transport of EE, whether administered as aqueous suspension or dissolved in a lipid vehicle, agree well with previous findings on other steroidal compounds which similarly indicate that: a) the portal system is the major, if not the exclusive pathway of transport of many steroids and b) the pathway of transport is independent of the vehicle of administration. By contrast, the lymphatic transport of EECPE could be significantly increased by use of a lipid vehicle indicating that not only the chemical nature of the compound but also the nature of the vehicle plays an important role in determining pathway of transport.

Studies on Biliary Metabolites of Orally Administered Ethynylestradiol (EE) and its 3-Cyclopentyl Ether (EECPE-Quinestrol)

Summary Large amounts of radioactivity appeared in the bile at 21/2 and 5 hours after a single oral dose of radiolabeled EECPE or EE. Conjugated metabolites (glucuronides and sulfates) predominated in bile although their pattern of distribution differed markedly from that observed in plasma of the same rats. Intestinal washings obtained from treated rats with bile duct cannulas suggested that some alteration of both EECPE and EE may occur in the gut. Thin layer chromatograms of extracts of bile obtained from EE- or EECPE-treated rats did not reveal the presence of unaltered EE or EECPE. However, several of the TLC fractions exerted uterotrophic activity when administered orally to immature rats. The dose response curves were similar to those of EE, suggesting loss of lipophilic properties in the case of EECPE metabolites. None of the fractions were as potent as EE or EECPE. When bile from EECPE-treated donor rats was infused into the small intestine of recipient rats, radioactivity appeared in the plasma and bile of the recipients providing direct evidence of active enterohepatic circulation of metabolites. Little or no radioactivity accumulated in body fat confirming the loss of lipophilic properties following biliary cycling.

Biological and Chromatographic Evidence for the Storage of Ethynylestradiol-3-Cyclopentyl Ether in Rat Brain

Summary The possible storage of estrogenic substances in brains of rats treated with ethynylestradiol (EE) or its 3-cyclopentyl ether (EECPE) was investigated. Twenty-four or 72 hours after a single oral administration of 2 mg of the test steroids, brains were removed and extracted with methylene chloride. The extracts were bio-assayed for uterotrophic potency or subjected to thin layer chromatography. Uterotrophic activity, corresponding to 2.3 and 0.5 μg of the standard, was detected 24 and 72 hours after administration of EECPE. This material corresponded to authentic EECPE in two TLC systems. Similar chromatographic analyses showed that the estrogenic substance, previously detected biologically in body fat of rats treated with EECPE(1), likewise corresponded to the unaltered ether. Neither bioassay nor TLC detected any estrogenic substances in brains or fat of rats treated with EE. The evidence supports the view that EECPE, but not EE, is stored unaltered in brain and body fat and that such storage may account for the enhanced anti-gonadotrophic and uterotrophic potency of the etherified form of the steroid.

Biological Profile of Quingestanol Acetate

Summary Like other 3-enol ethers of δ4δ3-ketosteroids, 17α-ethynyl-19-nor-testo-sterone acetate-3-cyclopentyl enol ether (EN TACP) possesses unusual biological properties. ENTACP is approximately twice as active as its parent ketone (ENTA) in producing endometrial proliferation in the immature estrogen-primed rabbit. In stimulating uterine growth ENTACP behaves like a true estrogen whereas ENTA or 17α-ethynyl-19-nor-testosterone (ENT) act as impeded estrogens, androgens, or progestogens, yielding a shallow dose-response curve. While ENTACP was significantly more androgenic than ENTA or ENT, the three compounds were equipotent in producing masculinization of female fetuses. Although adrenal hypertrophy was noted, neither ENT or ENTACP affected the corticoidogenic response of the adrenals to ACTH. However, when steroid output was corrected for differences in adrenal weight, both compounds either reduced the capacity of the adrenal cortical tissue to respond to ACTH or the increase in adrenal size was not due to an increase in corticosteroid producing tissue. When administered postcoitally, ENTACP was slightly more effective than ENTA in interfering with pregnancy. This may have been related to the inherent estrogenicity of each compound. Unlike methyltestosterone-3-cyclopentyl enol ether, orally administered ENTACP is not stored in body fat and does not have prolonged biological activity.

Studies on the Transport of Estrogens by the Rat Small Intestine in Vivo

Summary Rats intraduodenally administered 17β-estradiol-6,7 -3H (E), ethynylestradiol-6, 7 -3H (EE) or ethynylestradiol-6, 7 -3H-3-cyclopentyl-1-14C ether (EECPE) were killed at 7.5-, 30- or 120-min intervals. Intestinal lumen contents, intestinal wall homogenates and portal plasma were analyzed for free and conjugated steroids. After 7.5 min, more than 80% of the administered dose of E or EE had been absorbed. Characterization of the steroids present in the various ether extractable fractions (free steroid) indicated that, unlike EE, E had undergone rapid metabolic transformation with estrone accounting for 70% of the total radioactivity in the intestinal wall and portal blood. This transformation to the less potent estrone could be responsible for the reduced biological activity of orally administered E.

Effect of Estrogen and Progesterone on Uterine Acid and Alkaline Phosphatase and β-Glucuronidase Activity in Mated Ovariectomized Rats

Summary The influences of ovariectomy and hormone replacement therapy on nidation and on uterine phosphatases and β-glucuroni-dase were studied on the eighth day after mating in rats with unilateral section and ligation of the fallopian tubes (“USLT” rats). No implantation sites were found in either horn of untreated USLT animals which had been ovariectomized on day 1 or day 4. Implantation did occur in the non-ligated uterine horns of intact and ovariectomized USLT rats when the latter were injected daily with suitable doses of 17β-estradiol and/or progesterone. In all of these animals alkaline phosphatase activity was 10-17 times greater in implantation sites than in inter-implantation areas or in the ligated (non-pregnant) horns. A slight rise in acid phosphatase activity was also observed in implantation sites, whereas β-glucuronidase activity was not influenced by nidation. In ovariectomized USLT animals treated only with the estrogen (not implanted), uterine alkaline phosphatase and β-glucuronidase activities were significantly higher than in similar rats which received progesterone in addition. The data support the view that the alkaline phosphatase in the implantation sites is not the same enzyme as that which is increased by estrogens.

Effect of aminoglutethimide on serum progesterone and estrogen in the pregnant baboon (Papio SP).

Summary AG was administered orally twice daily for 1 or 3 days to six baboons whose duration of pregnancy ranged from 31 to 99 days. Serum progesterone levels were reduced to as little as 3.2% of the initial concentration in one animal and to 20 % or less in four of the five remaining baboons. Serum estrogen levels were depressed by 50 % or more in three of the four animals in which they were measured. However, pregnancy ensued for at least 3 wk after treatment in all animals. The data suggest that peripheral blood levels of sex steroids may not reflect the critical concentration of hormones required at the uteroplacental juncture for successful pregnancy maintenance. The authors thank Professor Hilton Salhanick of Harvard Medical School for his continuing interest and helpful discussions during the course of this work.

On the mechanism of prostaglandin F2α-induced abortion in hamsters

Abstract The mechanisms by which prostaglandins (PGs) induce abortion in hamsters remain poorly understood. PGs may effect one or more of these factors: a) corpus luteum (CL) structure and/or function; b) ovarian blood supply; c) pituitary function; and d) uterine motility. A minimum s.c. dose of 12.5 μg of PGF 2α is required to abort hamsters on day 5 of pregnancy. Radioimmunoassays revealed a drop of 71% in serum progesterone (P) levels 3 hours after dosing. Pregnancy was maintained indefinitely in hamsters receiving PGF 2α on day 5 by injections of progesterone on days 4, 5, and 6. In a related experiment, pregnancy was maintained with 5 mg doses of 17α-ethyl-19-nortestosterone (ENT) before and after PGF 2α injection, thereby permitting assessment of endogenous P levels (ENT does not cross-react with antiprogesterone antisera). Serum P levels were depressed by PGF 2α in both ENT and control hamsters. Finally, PGF 2α was injected on day 6 in hypophysectomized hamsters in which pregnancy was maintained with ovine prolactin (1 mg) and ovine FSH (5 μg). Serum P levels were decreased 70% 3 hours post-injection, although pregnancy was maintained in 37% of the animals. PGF 2α thus interferes directly with P secretion by the ovary in the absence of the pituitary and in the presence of injected luteotrophic complex. A sustained drop of 85–90% in serum P levels appears to be required for PGF 2α to induce abortion in 100% of the hamsters. We conclude that PGF 2α does not permanently damage CL when pregnancy is maintained by exogenous hormones, and the involution observed by others is most likely a consequence rather than a cause of pregnancy loss.

Distribution of Radioactivity in Body Fat and Organs of Rats Treated with Labeled Quinestrol, Ethynylestradiol or 17β-Estradiol.

Summary The distribution of radioactivity in body fat and organs, 24 hours after administration of radiolabeled ethynylestradiol-3-cyclopentyl ether (EECPE), ethynylestra-diol (EE) and 17 β-estradiol (E2) has been studied in castrated female rats. Radioactivity was highest in various types of fat of EECPE-treated animals. Only 1/10-1/30 as much tritium was detected in fat of rats treated with EE or E2. Each steroid exhibited an individual preference for each particular type of fat. Concentration of steroid in the various organs did in general correspond to the amount of fat present. Free (ether soluble) metabolites were in general more concentrated in organs than in plasma whereas water soluble metabolities (presumably conjugates) were generally higher in plasma than in the various organs. The data on the 3H/14C ratio suggests that the 14C labeled cyclopentyl group is cleaved from EECPE and eliminated via the kidney at a faster rate than the steroid moiety. The distribution of radioactivity in different brain areas following administration of radiolabeled EECPE was studied in male albino rats. EECPE is not uniformly distributed throughout the brain, but rather localized to specific areas. Rate of release of stored EECPE seems to be dependent upon metabolic turnover rather than binding capacity of the components of the different areas.

Post-implantation termination of pregnancy in rats

Abstract Several types of antifertility drugs have been tested for abortifacient activity in pregnant rats. The interceptive 1 1Interception is defined as termination of pregnancy after implantation (1). action of the estrogens and anti-estrogens appeared to be due to their uterotropic effects and was not prevented by concomitant progesterone administration. The effect of estradiol on the uterus was augmented by theophylllne but not influenced by indomethacin. The prostaglandins and the steroid synthesis inhibitor, aminoglutethimide (AG), interfered primarily with biosynthesis of progesterone. Sub-threshold doses of AG and estradiol or AG and prostaglandin F 2α (PGF 2α ) were highly effective interceptives when given in combination. An estrogen (fenocyclin), PGF 2α and AG each partially inhibited trauma-induced uterine growth in pseudopregnant rats but did not prevent the concomitant rise in progesterone-dependent uterine alkaline phosphatase which signals decidualization. This unexpected finding suggest that once decidualization is initiated, progesterone may no longer be required for continued activation of enzyme, which may explain the high doses of compounds required for (post-implantation) interception as compared with (pre-implantation) contraception.