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The influence of various experimental conditions, such as feeding schedule, composition of the diet, administration of a choleretic agent and vehicle used for its administration, on the development of indomethacin-induced intestinal ulcers in the rat has been studied.Independently of diet composition, incidence of ulcer formation is reduced by starvation and favored by the presence of food. Regardless of feeding schedule, animals on a regular diet are significantly more susceptible to ulceration than those on the fat free diet.The choleretic agent failed to enhance the ulcerogenic properties of indomethacin unless solubilized in sesame oil. Sesame oil alone significantly increased ulcer formation. These findings are discussed.

Cotton pellet-induced granuloma formation in intact animals is intimately dependent upon body growth and general anabolic processes. Deposition of connective tissue around the pellet continues up to (and probably beyond) the observation period of 90 days after implantation. Anorexic or catabolic agents, or both, or restricted food intake markedly impairs granuloma formation as a result of an impaired body growth. By expressing the amount of granuloma formed in mg/100 g body weight, the true anti-granulomatous properties of a drug can be easily dissociated from those attributable to an impaired body growth. The mechanism by which impairment in body growth affects granuloma formation is discussed.

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.

Ethynylestradiol-6,7-3H(EE) or its 1I-14C-cyclopentyl ether (EECPE) were administered to lactating rats, and the radioactivity ingested by the pups was measured during the following suckling periods: 0-4 hr; 0–24 hr; 24–48 hr. Only very small percentages of the administrered doses were found in the pups during the time intervals studied, the greatest amounts appearing during the first 4 hr after treating the mothers and the least during the 24–48 hr period. Only trace amounts of radioactivity were found in brains of the pups with most of the counts appearing in the carcasses. The radioactive compounds found in the pups were studied by column and thin-layer chromatography. In pups nursed by mothers treated with EE, EE was the only steroid which could be identified, while in pups suckled by EECPE-treated mothers, both EECPE and more polar metabolites were found. Both EE and EECPE extracted from pups were identified by crystallization to constant specific activity.

Splenectomized as well as intact mice responded to the injection of live Mycobacterium phleiHalpern with bimodal reticuloendothelial hyperfunction as measured by the intravascular clearance of colloidal carbon. The first stimulation persisted longer in splenectomized mice and the second stimulation endured longer in intact mice. Liver hyperplasia and lung inflammation were more severe after splenectomy, and made evident the manner in which the operated mice attempted to compensate for the extirpated organ. The phagocytic indices and histological findings indicate that the spleen suppresses the phagocytic activity of the liver and protects it from pathological change.

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.

The influences of dose level and route of administration on fat storage and excretion of ethynylestradiol-3-cyclopentyl ether (EECPE) were studied in rats. The EECPE was tagged with 3H in the steroid nucleus and with 14C in the cyclopentyl group in order to be able to trace the de-etherification process. Over a range of oral doses of 20 μg to 12.5 mg, there was a proportional increase in the concentration of compound in body fat and brain. The storage increment exceeded the dose increment suggesting that with increasing dose more of the compound escaped metabolic alteration. Nearly twice as much radioactivity was found in fat, brain, and blood of rats in which EECPE had been injected parenterally (iv, ip, sc) rather than administered orally. Evidence was sought but not found that EECPE is metabolized by the gastrointestinal tract; in fact, orally and intravenously administered EECPE were excreted in qualitatively similar patterns in bile and urine. Excretion of radioactivity following treatment of rats with cyclopentanol-14C led to the conclusion that EECPE follows two basic metabolic pathways: (i) de-etherification with urinary excretion of cyclopentanol derivatives; and (ii) conjugation and biliary excretion of metabolites in which the ether linkage is still intact.

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.

Uterine alkaline phosphatase and β-glucuronidase activity were assayed during the first 8 days of pregnancy in rats after unilateral tubal ligation and section. The results were as follows. Alkaline phosphatase increased in the pregnant horn from days 5 to 8 while the activity of the nonpregnant horn remained unaltered. This enhanced phosphatase activity was localized exclusively at the implantation sites. β-Glucuronidase steadily decreased during the first 8 days of pregnancy in both the gravid and non-gravid horns. The occurrence of a 'peak' in β-glucuronidase activity during the oestrogen surge on day 4 of pregnancy reported previously by Prahlad (1962) was not observed.The results are discussed in relation to an interaction between the implanting blastocyst and the hormonally conditioned uterus.

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.

An improved anti-inflammatory assay based on the reversal of endotoxin-induced lung inflammation in mice is described. Changes in design of the original apparatus have: a) increased the number of animals which can be exposed to endotoxin; b) decreased the amount of endotoxin needed for maximum inflammatory response. A direct correlation exists between concentration of endotoxin and the resulting increase in weight of the lung and degree of histopatho-logical changes. The advantages of this method for testing anti-inflammatory activity of steroid as well as non-steroid compounds are discussed in comparison with other methods described in the literature.We gratefully acknowledge the histopathological evaluations done by Mr. John A. Tornaben and Dr. John P. Manning, Dr. Robert L. Kroc is thanked for his support, interest and encouragement throughout this work.

1. The modification of pyruvate kinase activity in vitro was examined by altering the environmental [Mg2+]/[Ca2+] ratio with EDTA on the one hand and isolated rat liver mitochondria on the other. 2. Controlled additions of Ca2+ and EDTA caused pyruvate kinase activity to be alternately and rapidly switched on and off. 3. By being able to accumulate Ca2+ in preference to Mg2+ rat liver mitochondria were able to alter the [Mg2+]/[Ca2+] ratio in the vicinity of pyruvate kinase and thereby modify the activity of this enzyme. 4. The possible role of mitochondria in modifying pyruvate kinase and other ion-sensitive cytoplasmic enzyme activities is discussed.

The biological properties of some 19-nortestosterone-3-cyclopentyl enol ethers have been studied. The 17α-ethyl analogue resulted in decreased estrogenicity, increased progestational and pregnancy maintenance properties as compared to 17α-methyl and 17α-ethynyl derivatives. The methyl analogue, however, exhibited a considerable increase in androgenicity and greater anovulatory and gonadotropin inhibitory properties when compared to the other compounds, Both the methyl and ethyl substitutions resulted in masculinized female rat fetuses which in the case of the ethyl derivative demonstrated the lack of correlation between this effect and androgenic properties. Unlike the others, the 17α-methyl analogue, exhibited prolonged androgenic activity by either oral or subcutaneous administration. Our findings indicate that the nature of the parent ketone and not enol etheriftcation per segoverns the biological behavior of such compounds.The authors wish to thank Mr. Paul Nemith for his technical assistance and Mr. Neil Stasilli for his statistical analysis of the data. We also express our gratitude to Professors Dr. A. Ercoli and Dr. R. Gardi, Steroid Institute, Warner-Vister, 22064 Casatenova Brianza (Como) Italy, for the synthesis at our request of MCPE and ECPE.

When introduced directly into the small intestine of the rat, as oily solutions, ethynylestradiol (EE) or its 3-cyclo-pentyl ether (EECPE) were absorbed much more slowly than when administered as aqueous suspensions. These findings are in agreement with data in the literature on other steroidal compounds. Hydrolysis of the components of sesame oil (dependent upon the presence of bile and pancreatic secretions) seems to be necessary for release and subsequent absorption of EECPE but not EE from the lipid vehicle. Storage in body fat and brain of the unaltered EECPE is increased by its administration as an oily solution instead of an aqueous suspension. The nature of the vehicle of administration, however, had no influence on the degree of biological activity of EECPE as well as EE.