Automation of a chloramphenicol acetyltransferase assay.

Accurate quantification of chloramphenicol acetyltransferase (CAT) enzyme activity in a large number of samples has been achieved through robotization of a CAT assay on a laboratory workstation (Biomek 1000). The basic principle of this CAT assay relies on the selective diffusion of [3H]acetylchloramphenicol into a water-immiscible liquid scintillation cocktail. This methodology gives unique characteristics to this robotized protocol by allowing complete control over the kinetics of the CAT enzymatic reaction which is a critical parameter in the CAT assay. Thus it has been possible to optimize the CAT assay for every processed sample, through real time monitoring of the enzymatic reaction, and to achieve maximum accuracy in CAT quantification. Moreover the sensitivity of this automated assay is high (detection threshold; 10(-4) CAT unit), and the sample processing is fast (approximately 125 samples per hour). Compared to other CAT assay protocols currently used, our robotized technique offers major advantages in terms of CAT quantification, and sets new standards for CAT assay productivity.

How the study of the biological activities of antiandrogens can be oriented towards the clinic.

Antiandrogens can be used in various androgen-dependent diseases. Depending upon the therapeutic indication, they can be administered systemically or topically. Systemic treatment with an antiandrogen will inhibit androgen action not only in the desired target site but also in all other target tissues; thus, it will block the androgen-dependent feedback regulating the secretion (hypothalamo-pituitary-testis axis) or the action (protein factors) of androgens. In contrast, topical treatment (acting through cutaneous receptors or local metabolism) should not produce systemic side effects especially in man. Pharmacological assays which can select antiandrogens irrespective of the mechanism measure changes in the final androgenic response, but they consume a great deal of time and test compound and bear little relation to therapeutic activity. Therefore, the biological strategy that we report here and which, at Roussel-Uclaf, has led to the selection of a systemic and a topical antiandrogen (RU 23908 and RU 38882) has consisted in successively performing: (1) in vitro assays which measure an effect at a specific level in the mechanism of antiandrogen action, e.g. interaction with the androgen receptor. Assessing interactions with other classes of steroid hormone receptor can be used to predict possible hormonal side-effects, (2) in vitro determinations of agonist or antagonist activity, e.g. in pituitary cells (LH response to LHRH) or mammary tumor cells (induction of androgen-dependent proteins), (3) in vivo antiandrogen assays after a single treatment (induction of mouse kidney proteins, rat prostatic binding protein) or after repeated treatment (inhibition of the growth of rat accessory glands or of hamster sebaceous glands), to determine the active dose of the compound and possibly the absence of systemic effects by the topical route, (4) assays in animal models designed to mimic a therapeutic context e.g. for prostate cancer: inhibition of the "flare-up" effect of LHRH-A or of the trophic effect of perfused adrenal androgens on rat prostate, antitumoral activity in experimental cancer models. For hyperseborrhoea and acne: histological and stereological analysis of rat skin biopsies to measure the volume density of the smooth endoplasmic reticulum vesicles of the differentiating cells of the sebaceous gland.

Inhibition by glucocorticoids of PGE2 and ACTH secretion induced by phorbol esters and EGF in rat pituitary cells.

In rat pituitary cells in primary culture glucocorticoids specifically inhibit PGE2 and ACTH secretions induced by TPA, a potent phorbol ester derivative (triamcinolone acetonide greater than dexamethasone greater than cortisol greater than or equal to corticosterone). However, while PGE2 secretion can be inhibited up to 80%, ACTH secretion can only be inhibited up to 40%. Similar inhibitory effects are observed with mepacrine, an inhibitor of phospholipase A2 (PLA2). Glucocorticoids having also been described as PLA2-inhibitors, their inhibitory effect on TPA-induced secretions could thus be related to their anti-PLA2 activity. Their inhibitory effect on PLA2 has been attributed to their ability to induce the synthesis of lipocortin, the activity of which could be regulated by activation of kinase C or EGF-receptor kinase. Since in our model, EGF-induced PGE2 secretion is also inhibited by dexamethasone, these results suggest that a lipocortin-like protein could be present in pituitary cells and involved in the effect of TPA and EGF on PGE2, and, at least partly, on ACTH release.

Antiestrogens inhibit the mitogenic effect of growth factors on breast cancer cells in the total absence of estrogens.

The antiproliferative effect of antiestrogens in breast cancer is believed to be entirely due to the inhibition of estrogen induced growth. We show here that non-steroidal antiestrogens inhibit the growth of the human breast cancer MCF7 cells in the complete absence of estrogens (phenol-red-free medium) when cell proliferation is stimulated by insulin or epidermal growth factor. This non-antiestrogenic effect of antiestrogens is, however, mediated by accessible estrogen receptor sites, as it is not observed in receptor negative hormone-independent breast cancers, and is rescued by estradiol but not by insulin. We conclude that antiestrogens inhibit cell proliferation by inhibiting growth factor action as well as estrogen action and that in both cases, accessible estrogen receptors are required.

Role of calcium on TPA-induced secretion of ACTH and PGE2 by pituitary cells: effect of dexamethasone.

In pituitary cells in primary culture, ACTH and PGE2 secretions can be simultaneously stimulated by TPA, in the presence of Ca2+. However, whilst PGE2 secretion is under an absolute requirement for Ca2+, ACTH secretion is not. Both secretions are inhibited by dexamethasone but to various extents: PGE2 release is abolished in the presence of dexamethasone whilst only 35% of the TPA-induced ACTH release is sensitive to dexamethasone. Similar inhibitory effects are observed with mepacrine, a PLA2-inhibitor, suggesting that PLA2-activation could be related to these secretory process. Since PLA2-inhibition by dexamethasone is claimed to be mediated via lipocortin, these results suggest that a lipocortin-like protein is present in pituitary cells and could be involved in the TPA-induced secretions of PGE2 and ACTH.

Effect of a new topically active antiandrogen (RU 38882) on the rat sebaceous gland: comparison with cyproterone acetate.

RU 38882 is a new antiandrogen. When given by subcutaneous or oral route, RU 38882 is about 25 times less potent than cyproterone acetate. However, when applied topically to the intact rat skin, RU 38882 (0.25-25 mg/rat/day for 5 days or 3 weeks) decreases, in a dose-related manner, the volume density of the smooth endoplasmic reticulum vesicles of the differentiating cells of the sebaceous gland, a structure directly involved in sebum lipid synthesis. Under these conditions RU 38882 is about 100 times more potent than cyproterone acetate and unlike cyproterone acetate, does not modify the prostate weight. The lack of efficacy of cyproterone acetate on the sebaceous gland could be due to its partial androgenic activity while RU 38882, under these conditions, acts as a pure antiandrogen which inhibits the nuclear androgen-receptor translocation.

Antiandrogenic activity of a series of des-A-steroid derivatives.

In the search for new antiandrogens, a number of des-A-steroids were prepared by condensation of Grignard reagents with lactone 3. From the resulting key intermediates 5, various structural modifications were performed such as the introduction of an additional unsaturation to afford dienones 8 and aromatic derivatives 10 or the introduction of an alkyl substituent mostly in position 10 (11-13) but also in some cases in position 16 (22). In addition, 13-ethyl analogues were also prepared from lactone 4. The relative binding affinities (RBAs) for the androgen receptor of these compounds were determined under various conditions. Some compounds exhibit a capacity to interact with the receptor comparable to that of testosterone. One of the most potent compounds is 17beta-hydroxy-des-A-androsta-9,11-dien-5-one (8b), RBA value 73% of that of testosterone. More interestingly, several compounds were found to have an antiandrogenic profile in vitro and in vivo. One of the most effective compounds is 10-ethyl-17beta-hydroxy-des-A-estra-9-en-5-one (5c), which exhibits a strong local antiandrogenic activity in hamsters, without any significant systemic antiandrogenic effects. The corresponding 17beta-acetyl derivative (RU 38882) has been selected for extended pharmacological studies.

Transient transcriptional inhibition of the transferrin gene by cyclic AMP.

Transferrin mRNA content and gene transcription rate were measured in the liver of rats submitted to iron overload or depletion, castration, treatment with sexual steroid hormones, glucagon and cyclic AMP. The influence of puberty in males and females and of pregnancy was also analysed. Glucagon and cyclic AMP reduced mRNA level by about 50% at the 12th hour of treatment and transferrin gene transcription by as much as 95% at the 30th minute of drug infusion, with a secondary increase of the transcription rate for a protracted treatment. None of the other hormones tested had any detectable effect on transferrin gene expression, the same being true for iron overload or depletion.

RU 16117, an orally active estriol-like weak estrogen.

RU 16117, the 11 alpha-methoxy derivative of ethynyl estradiol, is an orally active weak estrogen potentially effective in the treatment of estrogen-deficiency in postmenopausal women (climacteric symptoms and severe osteoporosis). Biochemical studies have shown that RU 16117, like estriol, possesses the properties characteristic of a partial estrogen agonist/antagonist. RU 16117 binds to the cytosol estrogen receptor (ER) to form a complex which dissociates much faster than the estradiol complex. This explains its lower nuclear uptake. Furthermore, the nuclear RU 16117 complex also dissociates faster than the estradiol complex. Consequently, although low doses of RU 16117 can induce the majority of the effects of estradiol (increased polymerase A and B activities, cytosol ER replenishment, progestin receptor induction, increased uterine weight), these effects are long-lived only if the dose is considerably increased or if the compound is administered repeatedly or continuously. Since RU 16117 transiently occupies available estrogen binding sites, it can prevent the full response of estradiol. Thus, under appropriate kinetic conditions, it acts as an estrogen antagonist on the above parameters and also on DMBA-induced mammary tumors in the rat. At a daily dose of 24 micrograms for a period of 4 weeks RU 16117 led to 65% reduction in the number of already-established tumors. RU 16117 inhibits basal gonadotropin secretion and decreases the LH response to LHRH. Injection of 5 micrograms s.c. to the rat in estrus markedly inhibited the spontaneous peaks of LH, FSH and PRL measured on the afternoon of expected proestrus. Low doses which block ovulation by 100% had no detectable effect on vaginal cornification, thus suggesting a greater sensitivity at the hypothalamo-pituitary level.(ABSTRACT TRUNCATED AT 400 WORDS)

Substituted derivatives of A-nor-5 alpha-androstane and A-nor-5 alpha-estrane--structure and affinity for hormonal receptors.

The interactions of A-nor-5 alpha-androstane and A-nor-5 alpha-estrane derivatives with the estrogen and androgen receptors, have been evaluated by measuring their relative binding affinities (RBAs), under two sets of incubation conditions in order to discriminate between potent agonists from weak agonists with potential antagonist activities. Surprisingly some of these compounds which do not possess a phenolic hydroxyl group interact somewhat markedly with the estrogen receptor. This interaction is characteristic of weak estrogens, with potential anti-estrogenic activity (RBA values decreasing when increasing time and temperature of incubation). Results are in good agreement with data obtained in vivo. Moreover, some of these compounds interact also to some extent with the androgen receptor. Results will be discussed in order to outline some structure-activity relationships in these series.

Effects of RU16117, an orally active weak oestrogenic compound, in postmenopausal women.

Daily oral administration of 1, 3 or 10 mg of RU16117 (11 alpha-methoxy ethinyl oestradiol) to normal postmenopausal women led to a progressive decrease of basal serum LH levels to 60.4 +/- 17.0, 35.1 +/- 9.1 and 20.1 +/- 2.8% of control (pretreatment values, P less than 0.01), respectively, after 4 wk of drug administration. Although the pattern was similar, the inhibitory effect of RU16117 was even more pronounced on FSH than LH levels: a 50% decrease of basal LH and FSH levels was obtained at the daily 1.8 and 1.2 mg doses of RU16117, respectively. No significant change of basal serum gonadotrophin levels was observed with the daily 0.3 mg dose. Administration of 1 mg of RU16117 every second day or 10 mg once a week led to a relatively small but significant (P less than 0.05) 20--25% decrease of basal serum LH levels after 4 wk of treatment in four out of five women. While daily 0.3 and 1.0 mg doses of RU16117 had no significant effect on the LH response to 100 microgram LHRH, the 3.0 mg dose delayed the response up to 90 min. The 10 mg dose, on the other hand, led to a markedly delayed and reduced response. Treatment for the same period (4 wk) with 1 mg RU16117 every second day or 10 mg once a week led to a small (20--25%, P less than 0.05) inhibition of the LH response to LHRH. At the dose of 10 mg once a week, RU16117 had no or minimal effect on endometrial histology. Since RU16117, an orally active weak oestrogenic compound, has been shown to have anticarcinogenic activity in the rat, the present findings suggest that this new steroid could be useful for the treatment of climacteric symptoms.

PIP: Daily oral administration of 1, 3, or 10 mg of RU16117 (11alpha-methoxy ethiyl estradiol) to normal postmenopausal women led to a progressive decrease of basal serum LH levels to 60.4 + or - 17.0, 35.1 + or - 9.1 and 20.1 + or - 2.8% of control (pretreatment values, P 0.01), respectively, after 4 weeks of drug administration. Although the pattern was similar, the inhibitory effect of RU16117 was even more pronounced on FSH than LH levels: a 50% decrease of basal LH and FSH levels was obtained at the daily 1.8 and 1.2 mg doses of RU16117, respectively. No significant change of basal serum gonadotrophin levels was observed with the daily 0.3 mg dose. Administration of 1 mg of RU16117 every 2nd day or 10 mg once a week led to a relatively small but significant (P 0.05) 20-25% decrease of basal serum LH levels after 4 weeks of treatment in 4 out of 5 women. While daily 0.3 and 1.0 mg doses of RU16117 had no significant effect on the LH response to 100 mcg LHRH, the 3.0 mg dose delayed the response up to 90 minutes. The 10 mg dose, on the other hand, led to a markedly delayed and reduced response. Treatment for the same period (4 weeks) with 1 mg RU16117 every 2nd day or 10 mg once a week, led to a small (20-25%, P 0.05) inhibition of the LH response to LHRH. At the dose of 10 mg once a week, RU16117 had no or minimal effect on endometrial histology. Since RU16117, an orally active weak estrogenic compound, has been shown to have anticarcinogenic activity in the rat, the present findings suggest that this new steroid could be useful for the treatment of climacteric synptoms.