Clauberg Test Research Articles

Antiimplantation activity of the ethanolic root extract of Momordica cymbalaria Fenzl in rats

<b>Objective</b> : To evaluate the antiimplantation activity of the ethanolic root extract of Momordica cymbalaria Fenzl.<br><b>Materials and Methods</b> : The acute oral toxicity study was performed according to the OPPTS guidelines. The ethanolic root extract was investigated for antiimplantation, estrogenic and progestrogenic activities at doses of 250 and 500 mg/kg body weight. Antiimplantation activity was studied on successive stages of embryogenesis. Estrogenic studies were carried out by examining uterine weight, histoarchitecture of uterus, vaginal cornification, uterine content of glucose, cholesterol and alkaline phosphatase levels in immature rats. Progestrogenic activity assay was performed by pregnancy maintenance in rats and Clauberg's test (endometrial proliferation assay) in immature rabbits.<br><b>Results</b> : Both doses of the ethanolic root extract exhibited highly significant (P < 0.001) antiimplantation activity. However, an investigation of the estrogenic activity did not show any increase in uterine weight or vaginal cornification. The histoarchitecture (uterotrophic changes) such as thickness of endometrium and height of endometrial epithelium was unaltered in treated rats. There were no increases in the uterine content of glucose, cholesterol or alkaline phosphatase levels when compared with the control group. Pregnancy was not maintained in the pregnancy maintenance test for progestrogenic activity. Uterine proliferation was not seen in Clauberg's test (endometrial proliferation assay) for progestrogenic activity in immature rabbits.<br><b>Conclusion</b> : The ethanolic root extract of Momordica cymbalaria Fenzl exhibited antiimplantation activity but this is not due to estrogenic or progestrogenic activities.

Gestodene: A novel synthetic progestin — characterization of binding to receptor and serum proteins

Gestodene, 17α-ethinyl-13-ethyl-17β-hydroxy-4,15-gonadien-3-one, is a new orally active progestational agent, which is available for clinical use in oral contraceptives. The aim of the present study is to make a broad characterization of gestodene at the receptor level and to discuss the results in comparison to those of established progestogens. Kinetic studies of H-gestodene uptake show a rapid increase in the amount of specific binding during the first three hours. After saturation, the amount of specifically bound 3H-gestodege remained almost constant up to 24 hours at 4°C. The dissociation of 3H-gestodene from the cytoplasmic myometrial progesterone receptor, measured by displacement of labeled steroid with dextran-coated charcoal treatment at 4°C at various times, showed a biphasic or two-component first order dissociation curve. As anticipated, sucrose gradient centrifugation analysis of the 3H-gestodene-labeled cytosol of human myometrial tissue showed that the gestodene binding components sedimented in the 4S and 8S region. A 200-fold molar excess of nonradioactive gestodene reduced only the 8S binding of 3H-gestodene. 4S binding of 3H-gestodene was not reduced, which indicate the existence of a second high capacity binding component. In biological test systems, such as the Clauberg test or Kaufmann test, gestodene has proved to be a very effective progestogen. Among nortestosterone derivatives it is one of the most potent and resembles progesterone biologically in its progestogenic effects. This biologically identical gestagenic activity of gestodene and progesterone is reflected by a very similar behavior in vitro in terms of binding to progesterone receptors of human uterus cytosol. Furthermore, competitive studies indicated that gestodene like other synthetic progestagens also displays some affinity for androgen and glucocorticoid receptors but no measurable affinity for the estrogen receptor. Remarkable is the high binding affinity of gestodene to the binding sites of the mineralocorticoid receptor of rat kidney with a RBA value of 350% compared to aldosterone. In contrast to progesterone, gestodene did not bind to CBG, but was bound with a relatively high affinity for SHBG, which is a typical behavior for all 19-nortestosterone derivatives.

Steroids and related products. LIII. 1 The synthesis of 11-oxa steroids. V. 2 The synthesis of 17-ethynyl-11-oxatestosterone 3

The synthesis of 17-ethynyl-11-oxatestosterone, both from 11-oxa-5α-pregnane-3, 20-dione and, via a 3, 17-dioxygenated 9-oxo 9, 12-seco 11-nor 5α-androstan-12-oic ester, from 3β-acetoxy-17-hydroxy-5α-pregnan-12-one - two products available from hecogenin - is reported. The new hormone analogue shows significant progestational activity in the Clauberg test and relatively weak activity in a post-coital antifertility assay.

Strategy in drug reseabch. Synthesis and study of the psogestational and ovulation inhibitory activity of a series of 11β-substituted-17α-ethynyl-4-estren-17β-ols

Using the strategy based on the Hansch method which analyses effects of substituents on biological activity in terms of their hydrophobic, electronic and steric effects we selectively synthesised a series of 11β-substituted-17α-ethynyl-4-estren-17β-ols that combine ease of synthesis with good discrimination between these factors aiming at finding the compounds with optimum biological activity in that series. The compounds were tested quantitatively in the Clauberg test (rabbit) and the ovulation inhibition test (rat). The differences in biological activity could reasonably be correlated with two steric effects introduced by the 11β-substituent. These were a change in the overall shape of the 11β-substituent and the angular methyl group, and direct steric hindrance of the steroid-receptor protein binding. Some exceptions were found possibly due to metabolic conversion of these compounds to the corresponding 11β-substituted-17α-ethynyl-l,5,5(10)-estratriene-5, 17β-diols.

11-Oxa and 17α-hydroxymethyl analogues of steroid hormones and their derivatives

In view of the markedly reduced activity in the Clauberg test of 11-oxaprogesterone and of its significantly enhanced anovulatory activity in rabbits, ovulation stimulated with copper acetate, the synthesis and biological evaluation of other 11-oxa hormone analogues was undertaken. The syntheses of 11-oxa-17-acetoxyprogesterone, 11-oxa-17α-ethynyltestosterone, 11-oxa-estradiol, 11-oxa-17α-ethynyl-estradiol, and of 11-oxatestosterone are reported and some of their biological activites are discussed. In general, the replacement of the 11-methylene group by oxygen results in a diminution of the progestational, androgenic-anabolic, and estrogenic activities, this effect being the least pronounced in the case of the progestational activity of 11-oxa-ethisterone and particularly strong in the case of the uterotropic activity of 11-oxa-estradiol. On the other hand, the observation that the introduction of bulky 17α-substituents into progesterone and related products results in general in an increased progestational activity and that acylation of the progestationally inactive 17-hydroxyprogesterone leads to orally active progestogens and antifertility agents, prompted us to synthesize, by two pathways, 17α-acetoxymethylprogesterone and 17α-hydroxymethylprogesterone. It is shown that one of these pathways can be advantageously applied also to the synthesis of 17α-acyloxymethyl and 17α-hydroxymethyl glucocorticoids. Interestingly, 17α-acetoxy-methylprogesterone proved inactive in the Clauberg test even at high dose levels. The primary objective of the investigations which we should like to present in the framework of this Symposium was, and is indeed, a contribution to the domain of structure-activity relationships of hormonal steroids. However, this lecture will be concerned to the greatest extent with results pertaining to the structural component of this tandem field—to the synthesis of new analogues of steroid hormones—and we shall touch its activity component only in a fragmentary and preliminary way; this, quite simply, because we are not yet in the possession of the full biological data. Still, we hope that the chemical aspects discussed will not be completely devoid of interest and we also hope that even some of the fragmentary biological results will not be meaningless to the intricate and problematic field of structure-activity relationships. We should like to divide our presentation into two parts: one dealing with 11-oxa analogues of steroidal sex hormones, the other with 17α-hydroxymethyl analogues of hormones of the progester-one-corticoid group, with the emphasis on derivatives of progesterone.

Kinetic study of the enzymatic inactivation of progestogens by rat liver microsomes.

The kinetics of enzymatic hydrogenation of synthetic progestogens by female rat liver microsomes with NADPH as hydrogen donor were investigated by means of an optical test. Steroids with high progestational activity (Clauberg test) showed a low hydrogenation rate (Vmax). There also seemed to be a certain correlation between molecular structure and Vmax. The metabolites from incubation with the progestogens were isolated from the micro-preparations, and identified by infrared spectroscopy. It was found that the hydrogenation of the olefinic and carbonylic groups involved the uptake of hydrogen from NADPH.

A quantitative reexamination of structure-activity relationships in the delta6-6-substituted progesterone series.

14 6-substituted-Delta6-16-methylene-17alpha-hydroxy-4,6-prenadiene-3, 20-dione 17-acetate derivatives were analyzed by multiparameter regression techniques for quantitative structure-activity relationships. The parameters were published values for hydrophobic bonding power (pi), inductive effect (F), resonance effect (R), and molar refraction of the substituent (MR); the activity was progestational activity by Clauberg test. Least squares analysis showed that pi gave the best fit with single parameters. With 2 parameters, pi and F accounted for 69% of the variance. F and pi squared gave the best 3-term results, suggesting that pi values up to .50 permit the maximum progestational activity, but perhaps larger substituents decrease activity. The 6-methyl derivative was an exception, having 4 standard deviations' higher activity than computed by the 3-term equation, pointing to unusual metabolism.

Steroids and related products. XXXV: The synthesis of 11-oxa steroids. II.

The synthesis of 3,20-dioxygenated 11-oxa steroids is reported. As starting materials, 11-nor 9,12-seco 9-oxo 12-acids were used. One method for the elaboration of the 11-oxa steroid structure consisted in the treatment of 11-nor 9,12-seco 9,12-diols, derived from such acids, with acylating agents, in particular with p -toluenesulfonyl chloride in pyridine, a second one in the reduction of the carbonyl function of lactones of 11-nor 9,12-seco 9β-hydroxy 12-acids. These procedures were applied to the synthesis of the first 11-oxa analogue of a steroid hormone, 11-oxaprogesterone, which shows, in comparison with progesterone, significantly enhanced ovulation inhibiting activity and markedly reduced activity in the parenteral Clauberg test.

Secosteroids as progestational agents

This paper describes the synthesis of a few secosteroids. Their proton magnetic resonance spectra in CDCl3 and C6H6 were investigated to evaluate the Δ values of the C-18, C-19, and C-6 methyl groups. The progestational activity of these compounds as evaluated in the Clauberg test is also described.

STUDIES ON THE INTERACTIONS OF ETHYNYL OESTRADIOL AND NORGESTREL (dl-13β-ETHYL-17α-ETHYNYL-17β-HYDORXYGON-4-EN-3-ONE; WY 3707)

ABSTRACT The interactions of norgestrel (dl-13β-ethyl-17α-ethynyl-17β-hydroxygon-4-en-3-one; Wy 3707) and ethynyl oestradiol (EO) have been examined in a variety of experiments. Norgestrel is an antagonist of EO in mouse uterine growth and vaginal smear tests; in a rat uterine growth test the progestagen appeared to potentiate the oestrogen. EO was an antagonist of norgestrel in the Clauberg test for progestational activity. In tests for pregnancy maintenance in spayed rats, the oestrogen was largely antagonistic to the progestagen, although a suggestion of potentiation was observed at certain dose combinations. The pituitary effects of EO, as judged from studies in hemicastrated rats, were not modified by the simultaneous administration of norgestrel. Cohabitation experiments in rats failed to demonstrate a »contraceptive« effect of norgestrel alone, whereas EO and the combinations were effective; essentially normal fertility and fecundity were recovered after termination of treatment. None of these results differed significantly from what would be expected from data on other oestrogen/progestagen combinations.

Distribution pattern of a non-steroid compound with endocrine actions, 14C-bis(p-hydroxyphenyl)cyclohexylidenemethane (compound F6060) and its diacetate (compound F6066).

ABSTRACT While testing a series of unsymmetrical diphenylalkenes, it was observed that bis(p-hydroxyphenyl)cyclohexylidenemethane (compound F6060) and its diacetate (compound F6066) had an oestrogenic activity of about 1/1000 that of oestradiol benzoate. Further pharmacological investigations showed antioestrogenic (chicken oviduct test) and antigestagenic (Clauberg test) properties. The compounds had a low toxicity both in acute and chronic tests. In order to further elucidate the physiology of the compounds they were labelled with 14C and their distribution in the body was investigated by whole body autoradiography in male and female (pregnant and nonpregnant) mice. A rapid and selective accumulation of both compounds was observed in the corpora lutea. A selective and progressively increasing accumulation was also seen in the visceral yolk sac epithelium. A slight accumulation was also found in the hypophysis, the adrenal cortex, the endometrium, the interstitial tissue of the testis and ovary, the epididymis and the mucosa of the seminal vesicles. No accumulation was found in the prostate. There was a very low concentration in the foetus during the whole course of observation. In addition to the radioactivity in the liver, bile and intestinal lumen, the only organs showing any retention 24 hours after administration were the corpora lutea and yolk sac epithelium. The distribution pattern is in agreement with the physiological findings of an antigestagenic effect. The concentration in the yolk sac epithelium suggests further studies on the localization of the different steps in gestagen biosynthesis.

THE BIOLOGICAL PROFILE OF PROGESTERONE 3-CYCLOPENTYL ENOL ETHER AS COMPARED WITH THAT OF PROGESTERONE.

Progesterone and its 3-cyclopentyl enol ether (PCPE) have been compared in a battery of biological tests. Orally or intraperitoneally administered PCPE was respectively 1/80th and 1/20th as active as subcutaneously injected progesterone in the Clauberg test. Oral doses of PCPE, 10–20 times those of subcutaneous progesterone were unable to maintain pregnancy in ovariectomized rats. Three to four times as much “pregnanediol” was detected in the urine of rabbits following oral administration of progesterone than a comparable dose of PCPE. Orally or intraperitoneally administered PCPE at doses 20–40 times those of effective doses of progesterone failed to potentiate the anesthetic effect of a subhypnotic dose of sodium hexobarbital. These data demonstrate that PCPE and progesterone have a markedly different biological profile and cast serious doubt on the possibility that biologically active amounts of PCPE are converted to and/or metabolized in the same way as progesterone, as claimed by others. Our data strongly support the view that if any transformation of PCPE to progesterone occurs, the likeliest site of this conversion is the gut.

BIOLOGISCHE WIRKUNGEN DES 20β-HYDROXY-PREGN-4-EN-3-ON

ABSTRACT The biological effects of 20β-hydroxy-pregn-4-en-3-one were investigated in the rodent by means of the Clauberg test, the Allen-Doisy test, the vaginal opening test, the uterine growth test, the levator ani test and the prostate test. In the human subject the actions of 20β-hydroxy-pregn-4-en-3-one were investigated with regard to its effects on the endometrium, delay of menstruation, production of withdrawal bleeding, cessation of dysfunctional bleeding, cervical mucus, vaginal smear, basal body temperature, excretion of gonadotrophins and pregnanediol in the urine and protein metabolism. 20β-hydroxy-pregn-4-en-3-one is a true progestational hormone. With regard to its different physiological actions it is nearly halve as active as progesterone in the human subject.

BIOLOGICAL PROPERTIES OF 9,10-ISOMERIC STEROIDS

ABSTRACT Progestational activity of four 9β,10α steroids (»retro-steroids«) has been studied. These new compounds have the hydrogen atom on carbon atom 9 in the β position, and the 19-methyl group on carbon atom 10 in the α position, which is the reverse of the normal 9α,10β structure. The following compounds were subjected to the Clauberg test, both after subcutaneous and oral administration: I retro-progesterone II 6-dehydro-retro-progesterone* III 17α-acetoxy-retro-progesterone IV 6-dehydro-17α-acetoxy-retro-progesterone. All four compounds were orally active. In the subcutaneous route the maximum effect (comparable total dose of progesterone 10 mg), was obtained for the compounds I, II, III and IV with total doses of 5 mg, 1 mg, 1 mg and 0.5 mg, respectively. In the oral route these figures were respectively 50 mg, 10 mg, 2.5 mg and 1.25 mg (compared with progesterone 10 mg s. c.). Compound IV was within the 95% confidence limit 3.5–12.6 times orally more active than progesterone s. c.

Studies on the Biological Action of 19-Norsteroids

Various biological activities of 19-Norsteroids, Methyl 19-Nortestosterone (MNT), Ethinyl 19-Nortestosterone (EtNT), Ethyl 19-Nortestosterone (ENT), 19-Nortestosterone (NT) and their esters were investigated. 1) Androgenic activity : According to Eisenberg and Gordan's test, 19-Norsteroids corresponded to 1/1C-1/20 of Testosterone propionate (TP). Ethinyltestosterone (ET) was the weakest (1/40 of TP) and MNT was a little stronger than EtNT. 2) Anabolic activity : By means of Eisenberg and Gordan's test, they corresponded to 1/5-1/20 of TP in anabolic activity, ET was the weakest (1/40 of TP). EtNT was a little weaker than MNT, where as NT was equal to TP, ENT was the strongest among them (1/2, 5 of TP). 3) Estrogenic activity : On Allen-Dolsy test, they corresponded to 1/1,000-1/4,000 of Estradiol (OH2) in estrogenic activity, EtNT was two times stronger than MNT. In uterotrophic activity on Lauson's test, thay had only 1/3,000-1/40,000 of action of OH2. EtNT was the strongest of all. 4) Progestational activity : On Clauberg test by injection they corresponded to 1/2-1/30 of Progesterone (P). MNT and EtNT was 1/30 of P. By oral administration they were distinctly activer than ET, in fact at least two-ten times. Both MNT and ENT were ten times activer than ET. As for the duration of Clauberg's progestational action following a single injection of 20mg. their esters were stronger than free compounds. The effects of both MNT-enanthate (MNTE) and EtNT-enanthate (EtNTE) were recognized even on 10th day after injection. 5) Inhibiting activity on pituitary gonadotrophic potency : Of inhibiting activity on pituitary gonadotrophic potency by testes weight decrease test in rats, EtNT was the strongest of others, MNT and their esters were not so active. By means of gonadotrophic potency test in mice following injection with 19-Norsteroids, EtNT inhibited its action and was the strongest of all. 6) Pregnancy maintenance activity : Activity in Clauberg test and pregnancy maintenance effect in rats were not always pararrel. The 19-Norsteroids were all quite active in even if given by mouth, but pregnancy maintenance effect was rather weak. On Clauberg test 19-Norsteroids were generally stronger when given by mouth than by injection, while in pregnancy maintenance actlvity parenteral application was better than peroral treatment. 7) Effect on sexual cycle of rats : MNT and EtNT inhibited the estrus when given for a long time. In generally the sexual cycle became irregular. 8) Chemical structures and biological activities : There seemed to exist a definite relation between the chemical structures and the biological activities of 19-Norsteroids.

Comparative potency of several progestogenic compounds in a battery of different biological tests.

This report compares the effectiveness of various compounds with progestogenic activity in a series of tests involving the chick the rat the mouse and the rabbit. The inhibition of oviduct growth stimulation of decidual growth and the Clauberg test were used for measurement of activity of 17-alpha-hydroxyprogesterone 17-alpha-hydroxyprogesterone caproate 11 dehydroprogesterone 17-alpha-ethyl-19-nortestosterone and 17-alpha-ethinyl-19-nortestosterone. In the chick oviduct test 16-day-old female white Leghorns were treated for 7 days and sacrificed on Day 8. The entire genital tract from the infundibulum to the cloaca was included in the oviduct weight. Injection of stilbestrol alone gave a typical sigmoid dose-response curve with a sharp increase in weight from 13 mg % of body weight at .05 mg daily to 1048 mg percent at 1 mg daily. A standard dose of 1 mg a day of stilbestrol was then selected for further experiments. Progesterone produced a marked inhibition of the stilbestrol-stimulated oviduct in the chick particularly when dissolved in oil; all the compounds tested showed some inhibition ability. Adult female Holtzman rats were castrated treated with 1 mg of estrone for 4 days followed by 9 days of progesterone or one of the test compounds. The uterus was exposed on Day 5 of test-drug treatment and 1 mg of histamine dihydrochloride injected into the left horn. A marked deciduoma was apparent with .5 mg progesterone daily. The degree of deciduomatogenesis was evaluated by comparing the percentage of increase of the histamine-treated horn with the other. At .2-2 mg daily progesterone gave a good dose response curve up to 400% increase when suspended in oil. Suspension of the hormone in saline with "Tween 80" decreased activity by 50%. 11-dehydroprogesterone was twice as effective as progesterone. The other compounds gave little or no response even in large doses. In mice similar tests showed the decidual response in the mouse is a more sensitive index of progestone-like activity than in the rat. No significant activity was observed with any of the test compounds except 17-alpha-ethyl-19-norethisterone. The Clauberg test in rabbits gave similar results to previously reported work. 11-dehydroprogesterone was effective at .15 mg twice the activity of progesterone. It had only a third the activity of progesterone in the Hooker-Forbes test. Both the 17-alpha-ethyl-19-nortestosterone and the 17-alpha-ethinyl-19-nortestosterone appeared to be superior to progesterone in Clauberg and chick-oviduct tests; the ethynyl derivative was superior to the ethyl derviative in the chick test; only 11-dehydroprogesterone mirrored all the activities of progesterone and was consistently superior to the hormone.

The preparation and progestational activity of some alkylated ethisterones.

Abstract The preparation and progestational activity of some 6-alkyl, 21-alkyl and 6:21-dialkyl derivatives of ethisterone are described. 6α:21-Dimethylethisterone (XIV), the most potent compound of the series, given by mouth proved to have approximately twelve times the activity of ethisterone in the Clauberg test.

Progestational activity of halogenated corticosteroids and related compounds in rabbit and monkey.

SummaryProgestational activity of 9 alpha-chlorohydrocortisone acetate, 9 alpha-fluorohydrocortisone acetate and 9 alpha-fluoro-11-beta-hydroxyprogesterone has been demonstrated using progestational proliferation of the rabbit endometrium (Clauberg test) as index of activity. Hydrocortisone acetate and cortisone acetate show a lack of activity in the same test. Progestational activity of 9 alpha-fluorohydrocortisone is further demonstrated by prevention of estrogen withdrawal bleeding and characteristic histological changes in the endometrium of the ovariectomized rhesus monkey pre-treated with estrogen.