Larry John Black

Antagonism of estrogen action with a new benzothiophene derived antiestrogen

A new benzothiophene derived antiestrogen, LY139481, inhibited the uterotropic action of estradiol in a dose related fashion, and at 1 mg per day suppressed more than 90 percent of estradiols activity in immature rats. LY139481 induced minimal uterotropic activity, and that activity declined in relation to dose. The relative binding affinity of LY139481 for rat uterine cytosol estrogen receptors was greater than that of estradiol in competitive assays and increased in relation to temperature (2.9 +/- 0.5 x estradiol at 30 degrees C). LY139481 caused estradiol-induced uterine hypertrophy to regress in a manner similar to that which resulted from withdrawal of estradiol treatment. Three successive daily injections of LY139481 slightly increased uterine weight, and blocked additional uterotropic action in response to estradiol and LY139481 administration on subsequent days. Furthermore, ten daily injections of estradiol alone did not increase uterine weight in animals pretreated with LY139481 for three days. In contrast, LY139481 did not prevent the partial uterotropic action of tamoxifen administration.

Effects of a new antiestrogen, keoxifene (LY156758), on growth of carcinogen-induced mammary tumors and on LH and prolactin levels

Keoxifene (LY156758) is a new benzothiophene-derived antiestrogen with an extremely low degree of estrogenicity. Administration of 1-20 mg/kg per day inhibited the growth of 7, 12-dimethylbenzanthracene (DMBA)-induced mammary tumors in rats. The degree of inhibition of mammary tumor growth was similar to that observed with tamoxifen treatment.

Uterine bioassay of tamoxifen, trioxifene and a new estrogen antagonist (LY117018) in rats and mice

Abstract Dose response uterotrophic and antiuterotrophic activity of antiestrogens was examined in immature rats, immature mice and adult ovariectomized mice. LY117018 was the most active antagonist and the least estrogenic, while tamoxifen induced the greatest uterine growth and the weakest antagonism. The reported estrogenic activity of tamoxifen in mice (1) was found to be related to maturity. All compounds caused uterotrophic changes in immature mice similar to those observed in immature rats. However, in adult mice tamoxifen was devoid of antagonism, and trioxifene was active only at a very high dose as both were extremely estrogenic in this model. LY117018 activity in adult mice was comparable to that observed in immature rats and mice. Results depict significant agonist and antagonist advantages of LY117018 over tamoxifen and trioxifene.

Treatment of mammary cancer

brought about by disturbances in the control of hepatic function due to change in functional activity of one of the endocrine glands, with or without concomitant or compensatory changes in other glands or even in the same gland; or it may be due to changes in the central nervous system. In a very large proportion of cases of obesity, however, there is absence of definite collateral evidence of disease of an endocrine gland or of focal disease of the nervous sysem. It might therefore be supposed that -in such cases the disorder was one of the neuro-endocrine system as a whole, whereby automatic control of constancy of body weight became thrown out of gear. At the same time-it is difficult to conceive of a specific anomaly of metabolism affecting all parts of such a complicated mechanism simultaneously, in the absence of some central co-ordinating mechanism. Such a mechanlism may perhaps be found in the metabolic centsres in the hypothalamic region of the brain or in the neuro-hypophyseal mechanism considered as a functional unit. A functional derangement of this mechanism may be at the root of many cases of obesity-perhaps the majority. Obesity of this variety would be classified as neuro-endocrine.

Inhibition of central nervous system aromatase activity: A mechanism for fenarimol-induced infertility in the male rat

Fenarimol (alpha-(2-chlorophenyl)-alpha(4-chlorophenyl)-5-pyrimidine-methanol), a pyrimidine carbinol agricultural fungicide, was previously reported to cause a dose-related decrease in fertility in rats (K. S. Hirsch, E. R. Adams, D. G. Hoffman, J. K. Markham, and N. V. Owen (1986), Toxicol. Appl. Pharmacol. 86, 391-399). Based on the results of a number of reproduction studies (K. S. Hirsch, E. R. Adams, D. G. Hoffman, J. K. Markham, and N. V. Owen (1986), Toxicol. Appl. Pharmacol. 86, 391-399), the infertility appeared to be associated with an impairment of male sexual behavior. When [14C]fenarimol was administered to the dam, high concentrations of radioactivity were observed in the neonatal hypothalamus, which functions in the development and subsequent expression of male sexual behavior. In the present studies fenarimol exhibited neither antiandrogenic nor antiestrogenic activities. The compound did, however, prevent the increase in nuclear estrogen receptors in the brain which normally occurs in the male during the early postnatal period. These results suggested that fenarimol might be acting to inhibit estrogen biosynthesis (via the aromatase enzyme complex) within the central nervous system. [3H]Testosterone was administered to neonatal rats, and the tritiated metabolites were isolated. Testosterone and dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) concentrations were similar in all treatment groups. Tritiated estrogens were detected in the brain cell nuclei from control neonates but not in neonates exposed to fenarimol. Fenarimol was also observed to inhibit rat ovarian aromatase activity in vitro. These data indicate that the decrease in male sexual behavior and the infertility associated with exposure to fenarimol were, most likely, due to inhibition of aromatase activity within the central nervous system.

Differential interaction of antiestrogens with cytosol estrogen receptors

Interaction of tamoxifen, trioxifene and LY117018 with cytosol-estrogen receptors from immature rat uteri was compared. Determination of relative binding affinity (RBA) by competition with [3H] estradiol under various assay conditions revealed that the RBA of LY117018 increased with temperature while that of trioxifene declined. Furthermore, the RBA values of tamoxifen and trioxifene observed after 24 h of incubation at 4 degrees C were significantly lower than those obtained with 1-h incubations. However RBA values obtained with 1- or 24-h incubations of LY117018 at 4 degrees C were similar. The complex formed by estradiol or LY117018 at 4 degrees was relatively stable for 24 h, while significant dissociation of tamoxifen and trioxifene was detected under these conditions. At 30 degrees C estradiol displayed a biphasic pattern of dissociation, but tamoxifen and trioxifene dissociated rapidly and little evidence of a stable phase was apparent. By contrast, the complex formed by LY117018 exhibited greater stability than that of estradiol at 30 degrees C. These results establish a relationship between shifts in competition curves (RBA) and rates of dissociation relative to estradiol; and clearly reveal that LY117018 has different binding characteristics than tamoxifen and trioxifene.

Complimentary, sequential antifertility effects of chlormadinone and norethindrone in the rabbit: implications in progestin only fertility control.

Abstract The antifertility activity of chlormadinone and norethindrone, administered preovulatory, postovulatory or continuously was examined in the rabbit. Chlormadinone was considerably more potent than norethindrone by preovulatory treatment. With each compound a specific dose was found to be active by preovulatory treatment, but inactive when the dose was given continuously. Postovulatory treatment revealed a striking separation of action in that norethindrone inhibited fertility, while chlormadinone was totally inactive. When chlormadinone was given preovulatory, and norethindrone postovulatory, at doses that alone were inactive, a significant inhibition of fertility was observed. Sequential administration of these progestins appeared to enhance specificity, and broaden the total spectrum of antifertility influence.

The effect of estrogen priming on the uptake of radioactive estradiol.

The action of estrogens on target organs has been amply reviewed (1-3). Since the report by Jensen and Jacobson (4), demonstrating accumulation and retention of estradiol in rat uterine tissue, many investigators have turned to the use of tritiated estradiol with high specific activity as a tool for studying mechanisms of estrogen action. The effect of estrogen pretreatment on the uptake of radioactive estradiol has been studied in several laboratories with variable results (4-8). This report defines the conditions under which various estrogens stimulate the uptake of tracer estradiol by the mouse uterus. Materials and Methods. Five to ten female Cox mice weighing 11-13 g were used per group. At sacrifice, the uteri were quickly removed, dissected free of extraneous tissue, and weighed after uniform blotting to remove excess fluid. The uteri were transferred directly to counting vials and dissolved in 1.0 ml NCS Reagent (Nuclear Chicago) with gentle warming. Diatol was added and radio-activity was determined by liquid scintillation spectrometry. Disintegrations per minute were determined by using an internal standard. Estradiol-6,7 3H (42.5 Ci/mM) was obtained from New England Nuclear. Tracer injection solutions were prepared in 5% ethanol-saline and contained 1 μCi/0.1 ml (6.4 ng estradiol/μCi). In all cases the tracer dose of 1 μCi estradiol was given exactly 1 hr prior to sacrifice. Priming doses of estrogen were prepared in corn oil or saline, and the concentration was adjusted so that the injection volume was 0.1 ml. Results.When 0.03 μg estradiol-17β was given subcutaneously in corn oil or intravenously in saline at various times up to 6 hr prior to sacrifice, the uptake oftracer estrogen was increased at 3 hr after priming with no furher increase at 6 hr (Fig. 1). The response developed somewhat faster with intravenous injection, but the magnitude was not so great.

The Pharmacology of a New Antiestrogen

Presently, attempts to pharmacologically antagonize estrogen action are impeded by the weak estrogenic activity of the currently available estrogen antagonists. Most antiestrogens, rather than being pure antagonists, possess varying degrees of estrogen agonist activity. Thus, the action of estrogens can be antagonized only to the point where the agonist activity of the antiestrogen begins to be expressed.