The rise and fall of the British Drug Houses, Ltd.

Steroid research at BDH began in earnest in 1946-1948, when Hartley and Petrow joined the company. With the need to find new progestational agents to replace ethisterone and progesterone, the company began work. They were the first to discover the vital importance of 6-methylation in enhancing the hormonal effects of steroid hormones. Their progestational studies led them to work on antifertility agents and the development of ovulation inhibitors, the mini-pill, and preliminary studies on the postcoital pill. Their search for new steroids additionally resulted in synthesis for biological evaluation of new corticoids, anabolic agents, estrogens, and mineralocorticoids. In 1968 the company, then known as The BDH Group Ltd., was incorporated into the Glaxo Group and company research terminated.

Total suppression of spontaneous endometrial carcinoma in BDII/Han rats by melengestrol acetate.

Female virgin BDII/Han rats develop spontaneous endometrial carcinoma (EC) in incidences up to 90%. Our objective was to determine whether lifelong administration of the progestin melengestrol acetate (MGA) would suppress those tumors. Four groups of 20 rats aged 24-28 days were employed Group I animals were untreated controls. Groups II, III, and IV were fed 0.1, 0.2, and 0.4 mg MGA/kg daily in their diet during their lifetimes. All treated groups were free from EC during their lifetimes with an increased lifespan up to 30%. The controls, in contrast, had an EC incidence of 85%. Histologically, with one exception all tumors were classified as adenocarcinoma. While most of the control rats died from EC, nearly all animals of groups II and III died from age-related diseases. Rats in group IV showed side effects due to the glucocorticoid properties of MGA. Besides alopecia and obesity an acceleration of chronic progressive nephrosis was observed. The study establishes the validity of the prophylactic approach to spontaneous hormone-dependent cancers in a rat tumor model.

Structure-activity studies of 2,3,4,4a,5,9b-hexahydroindeno[1,2-c]pyridines as antispermatogenic agents for male contraception.

Analogs of (4aRS,5SR,9bRS)-2-ethyl-2,3,4,4a,5,9b-hexahydro-7-meth yl-5-p- tolyl-1H-indeno[1,2-c]pyridine (Sandoz 20-438, 10a; R1 = ethyl, R2 = R3 = methyl, R4 = H) have been synthesized and tested in mice for their ability to reduce testes weight and disrupt spermatogenesis. The activity was strongly dependent on stereoisomerism and chirality, consistent with a mechanism of action involving interaction with a specific macromolecule. It was affected by changes in the nitrogen substituent and most strikingly by changes in the p-substituent of the 5-aryl ring. A hydrogen, fluorine, hydroxy, or methoxy substituent led to loss of activity, whereas methyl (Sandoz 20-438, 10a), carboxylate (RTI-4587-054, 10k; R1 = ethyl, R2 = methyl, R3 = COOH, R4 = H), ester (RTI-4587-056, 12b; R1 = ethyl, R2 = methyl, R3 = COOMe, R4 = H), formyl (RTI-4587-030, 12i; R1 = ethyl, R2 = methyl, R3 = CHO, R4 = H), or hydroxymethyl (RTI-4587-055, 12g; R1 = ethyl, R2 = methyl, R3 = CH2OH, R4 = H) groups resulted in antispermatogenic compounds. Methyl ester 12b was an effective antifertility agent, without apparent effects on mating, when given orally to male mice at 7-15 mg/kg daily for 35 days. Further evaluation of these compounds as male contraceptive agents and probes for study of spermatogenesis appears warranted.

6-Methylene progesterone is cytotoxic to human cancer cell lines independent of its 5-alpha-reductase activity.

This investigation examined the effects of 6-methylene progesterone (6MP), an irreversible inhibitor of 5-alpha-reductase, on prostatic cancer (PC) cell lines. Dose titration microculture tetrazolium assays were used to evaluate cytotoxicity in cultures treated for 72 hr with 6MP (0-20 micrograms/ml). An androgen-sensitive cell line, LNCaP, was drug-sensitive with a mean 50% lethal dose value (LD50) of 2.632 +/- 0.103. Hormone-resistant PC cell lines 1-LN, DU 145, and PC3 also demonstrated sensitivity with LD50 values between 0.8579-1.110 micrograms/ml with a group average of 1.023 +/- 0.082 micrograms/ml. Increasing dosages of dihydrotestosterone in the growth media did not alter 6MP cytotoxicity in androgen-insensitive prostatic cancer cell lines. No correlation between androgen-responsiveness and 6MP-induced cytotoxicity was observed. In nonprostatic malignancies, 6MP inhibited adenocarcinoma cell lines with a mean group LD50 value of 0.7772 micrograms/ml +/- 0.110. J82, a transitional cell carcinoma cell line of bladder origin, exhibited an average LD50 value of 1.041 +/- 0.260. In an epidermoid cervical cancer cell line, ME180, an LD50 value of 0.5356 micrograms/ml +/- 0.010 was noted. In a melanoma cell line, Du Mel 6, a mean LD50 of 0.7428 +/- 0.023 micrograms/ml was achieved with 6MP. We conclude that 6MP, a novel 5-alpha-reductase inhibitor, has potential as a cytotoxic agent in prostatic carcinoma and additional human malignancies. Further study is justified.

Effects of D-ring substituents on antiprogestational (antagonist) and progestational (agonist) activity of 11 beta-aryl steroids.

The discovery of antiprogestational steroids by the Roussel-Uclaf group not only was a major scientific advance but also opened the way to new methods of fertility control and new therapies for such conditions as cancer. RU486, the prototype of the series, is distinguished by a p-(N,N-dimethylaminophenyl) substituent at the 11 beta- position of the steroid framework, a 4,9-dien-3-one system and 17 beta-hydroxy-17 alpha-propynyl substituents. We examined the effect of varying the 17 alpha- substituent in 17 beta-hydroxy compounds analogous to RU486, the effect of introducing a progesterone side chain at C-17, and the effects of further substitution at C-17 alpha and C-16 alpha on the activity of these latter compounds. These studies indicate an important role for D-ring substituents in determining the balance of agonist/antagonist activity in this series. For example, 17 alpha-acetoxy-17 beta-acetyl substitution gave a potent antagonist, whereas 16 alpha-ethyl-17 beta-acetyl substitution resulted in a compound with potent progestational (agonist) activity. The compounds present opportunities for further interesting and useful biological investigations.

Angiostatic activities of medroxyprogesterone acetate and its analogues.

The effects of medroxyprogesterone acetate (MPA) (I) and related compounds (II-VI) upon angiogenesis induced by basic fibroblast growth factor (bFGF) or transforming growth factor-alpha (TGF-alpha) were investigated using a rabbit corneal system for assay of angiogenesis. Dexamethasone (Dex) was used as a positive control. The MPA analogues tested were 6,6'-dehydro-MPA (II), megestrol acetate (III), 1-dehydromegestrol acetate (IV), melengestrol acetate (V), and 1-dehydromelengestrol acetate (VI). The inhibitory activities of these steroids using bFGF were in the order: Dex = MPA = (VI) = (V) > (IV) > (III). Steroid (II) was inactive. 5 alpha-dihydrotestosterone was weakly active, while estradiol-17 beta and progesterone were inactive. The angiostatic activity of MPA was completely abolished by mefipristone (RU 486) which showed no anti-angiogenic activity in this assay. With TGF-alpha, the order of angiostatic activities was Dex = (VI) > (IV) > (III) > (V). Steroid (II) was again inactive. Dex, MPA, and all the MPA analogues except steroid (II) markedly inhibited the activity of plasminogen activator secreted by cultured calf pulmonary artery endothelial cells, but did not inhibit growth of these cells. The binding affinities of MPA and its analogues to glucocorticoid, progesterone and androgen receptors were determined, but were found not to be correlated with their angiostatic activities.

19-Aldehydo-4-androstene-3,17-dione: an estrogen precursor that inhibits sebaceous secretion in a rat model.

19-aldehydo-4-androstene-3,17-dione (A; R = CHO), a biogenetic precursor of estrone in the body, has been found to suppress sebum secretion in the ovariectomized testosterone-treated Wistar rat at 1/2000 times the dose of cyproterone acetate required to produce an equivalent effect. The action of this steroid must therefore be analogous to that of an estrogen even though, in striking contrast to estradiol, it is without effect on uterine weight or vaginal cornification. It is postulated that 19-aldehydo-4-androstene-3,17-dione (A; R = CHO) is converted locally into estrone (B) by aromatase present in skin but only in low concentrations in vagina and uterus. The potential of biogenetic precursors (I) of estrogens for therapy of acne or alleviation of the worst effects of skin aging is worth investigation.

Reversal of activity profile in analogs of the antiprogestin RU 486: effect of a 16 alpha-substituent on progestational (agonist) activity.

16 alpha-Ethyl-17 beta-acetyl substitution in the D-ring of steroids having an 11 beta-aryl-4,9-dien-3-one structure resulted in compounds with strong progestational activity. These compounds caused endometrial proliferation in the uterus of estrogen-primed rabbits with a potency greater than that of progesterone and had no detectable antiprogestational activity in this model. This is in stark contrast with the marked antiprogestational activity in rabbits, rats and humans reported for most 11 beta-aryl-4,9-diene-3-keto steroids such as RU 486 and its 17 beta-acetyl-17 alpha-acetoxy analog, 17 alpha-acetoxy-11 beta-(4-N,N-dimethylaminophenyl)-19-norpregna-4,9-diene- 3,20-dione. Examination of structure activity relationships in combination with computer aided molecular modelling suggests that a binding interaction of the 16 alpha-ethyl group with the progesterone receptor (PR) or the PR-progestin response element complex may play the major role in this reversal of activity profile.

LY207320 (6-methylene-4-pregnene-3,20-dione) inhibits testosterone biosynthesis, androgen uptake, 5 alpha-reductase, and produces prostatic regression in male rats.

LY207320 is an in vitro inhibitor (estimated IC50 = 0.06 microM) of steroid 5 alpha-reductase that catalyzes the conversion of testosterone (T) to dihydrotestosterone (DHT). In contrast, LY207320 was only moderately active against rat prostatic 5 alpha-reductase in vivo (32% inhibition at 50.0 mg/kg single dose). LY207320 did, however, inhibit the in vivo uptake of [3H]-T by the prostate. The antiprostatic and endocrine effects of this agent were evaluated following daily (21 days) administration to castrated, androgen-supplemented castrate, and intact rats. LY207320, which has modest progestational competitive binding activity, does not bind to rat prostatic androgen or uterine estrogen cytosolic receptors. In the castrated male rat, subcutaneously (s.c.) administered LY207320 had no androgen agonist activity, as evidenced by a lack of accessory sex organ weight gains. Administration of s.c. LY207320 to intact rats for 21 days at doses greater than 5.0 mg/kg-day produced significant (P < 0.05) reductions of seminal vesicle and ventral prostatic weights (maximal regression = -65% and -40% from control values, respectively at 50.0 mg/kg-day). The compound had no regressive activity on male accessory sex organs when administered orally. LY207320 did not alter circulating prolactin, LH, or corticosterone levels, but at high doses (> or = 50.0 mg/kg-day), lowered circulating T[-67% from intact control levels (P < 0.05)]. Histological analysis of the rat ventral prostates (RVPs) in LY207320-treated rats was consistent with an androgen-deprived state. Decreased circulating androgens and prostatic regression are associated with inhibition of testicular 17 alpha-hydroxy/C17,20-lyase enzyme activity (IC50 = 0.06 microM). These findings support the contention that LY207320 is a physiological antagonist of androgen action in male rats, and that its effects are mediated primarily through inhibition of testicular androgen production rather than accessory sex organ 5 alpha-reductase.

1-dehydro-melengestrol acetate inhibits the growth and protein kinase C activity of androgen-independent Dunning rat prostatic tumors.

Androgen-independent Dunning R3327-AT3 rat prostate tumors are considered an appropriate model of advanced prostate cancer in humans. We recently reported that the progestational steroid melengestrol acetate (MGA) inhibited growth of these tumors on oral administration but also induced a marked involution of adrenals and androgen target organs (prostate, seminal vesicles, and testes). We report herein that the 1-dehydro derivative of melengestrol acetate (dMGA) fed to rats for 21 days also inhibited the growth of Dunning AT3 tumors by approximately 55% without causing a significant regression of adrenals or androgen-dependent tissues. Thus, tumor-growth inhibition was induced by dMGA in the absence of glucocorticoid activity. Cytosolic AT3 tumor fractions obtained by diethylaminoethyl (DEAE)-Sephacel batch chromatography were assayed for lipid- and Ca(2+)-dependent (PKC) and -independent protein kinase activities. Prostatic cytosols had equivalent activity levels of both types of kinases (approximately 2 nmol gamma-[32P]-adenosine 5'-triphosphate (ATP) incorporated mg protein-1 min-1. The PKC activity recovered from the cytosol of untreated AT3 tumors was approximately 4 times higher. Oral administration of dMGA reduced this activity by > 95%. The relationship between protein-kinase activity levels and dMGA-induced growth inhibition of androgen-independent tumors in this animal model is discussed.

Effects of 6-methylenetestosterone acetate on spontaneous mammary tumourigenesis in SHN mice.

The effects of 6-methylenetestosterone acetate (MTA), an androgen derivative, on spontaneous mammary tumourigenesis were studied in a high mammary tumour strain of SHN virgin mice. AT 5-6 months of age female litter mates were divided into the experimental and the control mice. The experimental mice were given subcutaneous implantations of Silastic capsules containing MTA (MTA1 group) and non-tumourous mice received additional Silastic capsules after 2 months (MTA2 group) followed by MTA pellet implantation to non-tumourous animals a further 2 months later (MTA3 group). The control mice received Silastic capsules containing cholesterol and cholesterol pellets with the same design as in the experimental groups. In the MTA1 group, there was no effect on mammary tumourigenesis when treated mice were compared to the controls. In the MTA2 group, mammary tumour incidence tended to be lower in the experimental mice than in the controls, while in the MTA3 group mammary tumourigenesis was significantly suppressed in the experimental mice compared to the controls. Associated with these effects, normal mammary gland growth was inhibited and serum prolactin level was reduced in both the MTA2 and MTA3 treated mice. The ovarian weights were decreased in the experimental mice of the MTA1 group, while the adrenal weights were lower in the MTA2 or MTA3 group when compared to respective control values. Ovaries and adrenals did not differ histologically between any of the groups. A prolonged oestrous/metoestrous stage was observed only in the experimental mice of the MTA1 group. The results indicate that MTA has two opposing effects on mammary tumourigenesis according to its dose; thus a low dose has little effect on mammary tumourigenesis, while higher doses inhibited both normal and neoplastic mammary gland growth probably through the inherent androgenicity of MTA.

Effects of 6-methylene progesterone on growth, morphology, and blood flow of the Dunning R3327 prostatic adenocarcinoma.

Copenhagen x Fisher F1 rats were implanted with the androgen-dependent Dunning R3327 prostatic adenocarcinoma. When the tumors had median volumes of ca 470 mm3, the rats were castrated and/or treated with 6-methylene-4-pregnene-3,20-dione (6MP) in different doses. Tumor growth inhibition occurred in all castrated and treated groups, with decrease in volume of the epithelial compartment in the intact group. Tumor volumes at the highest dose level of 6MP equalled those observed in the castrate group. Plasma levels of testosterone were within the normal range. The administration of 6MP surprisingly induced an increment of tumor blood flow in the castrate group. Also in castrated and testosterone-supplemented animals, 6MP induced a reduction of prostatic tumor growth. Through the castration-like effect on tumor growth, the use of 6MP may represent an attractive alternative to castration for treatment of androgen-responsive prostate cancer.

Growth modulatory effects of some 6-methylenic steroids on human and hamster pancreatic adenocarcinoma cells in vitro.

Similarities between pancreatic, prostate and mammary tumors in possession of steroidal receptors and enzymes led to investigation of the responsiveness of pancreatic cancer to steroids with potential for tumor inhibition. The compounds were tested in vitro against human (HPAF and PANC-1) and hamster (HP-1) pancreatic ductal tumor cell lines using a colorimetric enzyme-based assay (MTT) to assess both cytotoxic and cytostatic effects and the 3H-thymidine uptake assay for cytostatic effects. Only certain 6-methylenic steroidal 3-ketones and the anti-estrogen tamoxifen citrate exerted appreciable anti-tumor effects. Marked cytotoxic and cytostatic activity was shown by some 6-methylenic congeners of progesterone, testosterone and its acetate, and 4-androstene-3,17-dione on both human and hamster pancreatic tumor cell lines. In contrast to prostate cancer, testosterone, but not 5 alpha-dihydrotestosterone, enhanced growth of the well differentiated HPAF cell line as well as the poorly differentiated PANC-1 cell line. It is therefore surprising that the 6-methylene derivative of testosterone acetate, which is both a potent androgen and 5 alpha-reductase inhibitor, is a very active tumor inhibitor in our assay.

Quantitative analysis of the development of genital organs from the urogenital sinus of the fetal male mouse treated prenatally with a 5 alpha-reductase inhibitor.

The role of 5 alpha-dihydrotestosterone (DHT) in the development of the genital organs and in the differentiation of the genital tract into prostate, coagulating gland (CG), bulbo-urethral gland (BUG) and seminal vesicle (SV) in male mice exposed prenatally to the 5 alpha-reductase inhibitor 6-methylene-4-pregnene-3,20-dione (6-MP) has been examined quantitatively. Female ICR mice were given 7 daily s.c. injections of the inhibitor (400 mg/day) starting on day 12 of gestation and the experiment was terminated on day 19 when the fetuses were removed by Caesarean section. In the prenatally 6-MP-exposed male mice the anogenital distance was significantly shorter than in the controls. Feminization of the nipples and hypospadias of the phallic urethra were noted. Development of prostate, CG and BUG was significantly suppressed. SV and testis development were not affected. These results lend further support to the conclusion that DHT is necessary for the development of the urogenital sinus (prostate, CG and BUG) and penis, and for the regression of the nipples in male mice. Reproductive abnormalities were not found in 90-day-old mice of both sexes exposed to 6-MP in utero. The 6-MP-exposed male and female mice had a normal reproductive capacity when mated with normal mice. These results show that 6-MP-induced growth retardation of reproductive organs is evident on day 19 of gestation, but that such retardation is no longer apparent in the adult.

In vivo assay for conversion of testosterone to dihydrotestosterone by rat prostatic steroid 5 alpha-reductase and comparison of two inhibitors.

An in vivo assay for steroid 5 alpha-reductase in rat ventral prostate has been developed and used to compare the inhibitory activity of N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide (4-MA) and 6-methylene-4-pregnene-3,20-dione (LY207320). Immature rats (70-80 g) received test compounds 30 min prior to s.c. injection of [3H]-T. The rats were sacrificed 30 min later and the ventral prostates were analyzed for [3H]-T metabolites. Intraprostatic [3H]-T and [3H]-DHT reached peak levels within 5 min after injection of [3H]-T and declined to about 25% of peak levels after 2 hr. 4-MA was a very potent inhibitor of [3H]-DHT formation with an estimated IC50 of 0.2 mg/kg. LY207320, an inhibitor of 5 alpha-reductase in vitro, was weakly active in vivo and did not achieve greater than 45% inhibition at high doses (greater than 200 mg/kg, s.c.). Tissue uptake of [3H]-T was also inhibited by LY207320, which may contribute to its inhibitory activity on accessory sex organ growth in the rat.

Melengestrol acetate and megestrol acetate are prostatic tumor inhibiting agents.

We had previously reported that 6-methylene progesterone, an inhibitor of 5 alpha-reductase, the enzyme which converts testosterone to dihydrotestosterone, markedly inhibited growth of the androgen-dependent Dunning R3327-H rat prostatic tumors. We now find that the progesterone derivatives melengestrol acetate (MGA) and megestrol acetate (MA) inhibit both the androgen-dependent (Dunning R3327-H) and the androgen-independent (Dunning R3327-AT3) prostatic tumors. Growth of the AT3 tumors was suppressed by approximately 53% after 9 days of daily s.c. injections with MGA at 10 mg/kg body weight. MGA also caused a 54% weight reduction of the ventral prostate and a 53% reduction of the seminal vesicles. Adrenal weights were reduced by 42%. A 24-day oral treatment with MGA (at approximately 15-17 mg/(kg.day)) inhibited AT3 tumor growth by 59% and caused a weight reduction in the following tissues: prostate (46%), seminal vesicles (19%), testes (12%), and adrenals (52%). Under the same protocol, MA inhibited AT3 tumor growth by 32% and reduced the weight of the ventral prostate by 49% and the weight of the adrenals by 18%, but had no effect on the seminal vesicles and testes. The extent of the MGA-induced prostatic regression was accompanied by cytological changes similar to those effected by 6-methylene progesterone, i.e., shrinking of the acinar epithelium. The AT3 tumors in MGA-treated rats displayed a limited degree of apoptosis. Atrophy of the adrenal cortex and lowered plasma levels of corticosterone and dihydroepiandrosterone were also observed. A therapeutic role for MGA and MA against androgen-independent prostatic neoplasms in man is forecast by these observations.

Inhibition of experimentally induced mouse prostatic hyperplasia by castration or steroid antagonist administration.

Mouse prostatic hyperplasia has been induced experimentally by implanting fetal urogenital sinus tissue into the prostate gland of syngeneic mice. We compared the effects of castration and steroid antagonist administration on the growth of the prostate gland during both the early (15 days) and late (30 days) phases of prostatic enlargement. Castration at the time of induction of prostatic hyperplasia is by far the most effective method of inhibiting prostatic overgrowth. A comparison of castration for 7 days with the short-term (7 days) administration of steroid antagonists showed that during the early phase of prostatic enlargement castration is more effective than antiandrogen, which is more effective than 5 alpha-reductase inhibitors. In the late phase of mouse prostatic enlargement, castration for 7 days is less effective than treatment with either antiandrogen or a 5 alpha-reductase inhibitor. Our data indicate that treatment with a combination of an antiestrogen (keoxifene) with a 5 alpha-reductase inhibitor (in particular, 6-methylene progesterone) is the most effective combination for reducing prostatic overgrowth. The antiestrogen (keoxifene) treatment alone was ineffective in both the early and late phases of prostatic overgrowth.

Growth modulation effects of synthetic steroids on rat prostatic adenocarcinoma and human breast adenocarcinoma cells in vitro.

Treatment of carcinoma of the reproductive organs using both an antiproliferative agent to inhibit cell growth and a cytotoxic agent to kill the neoplastic cells represents an attractive approach to therapy. By using steroids to achieve both effects, it may be possible to avoid some of the dose-limiting side effects of conventional antiproliferative or cytotoxic agents. We screened in vitro 23 selected steroids against four rat prostatic carcinoma cell lines and one human breast carcinoma cell line. Effects of the steroids were measured using a tritiated thymidine uptake assay to assess antiproliferative activity and a colorimetric enzyme-based assay to assess cytotoxicity. Several of the steroids had marked cytostatic and cytotoxic properties rendering them worthy of further evaluation, both separately and in combination.

The effect of some antiprostatic steroids upon cortisol production by guinea-pig adrenal cells stimulated by ACTH.

The antiprostatic steroids 6-methylene-4-pregnene-3,20-dione (6-MP) (I), 17-alpha-acetoxy-6, 16-dimethylene-4-pregnene-3,20-dione (II), and melengestrol acetate (MGA) (III) were incubated with guinea-pig adrenal cells, both alone and maximally stimulated with ACTH. Cortisol output was then measured by RIA. Increased cortisol-like secretion was obtained with 6-MP in the absence of ACTH. In the presence of ACTH, cortisol-like steroid secretion was the sum of that seen with ACTH and 6-MP alone. It follows that 6-MP stimulates in vitro a cortisol-like steroid cross reacting with the cortisol antibody by a mechanism that by-passes ACTH. Steroid (II) weakly inhibited cortisol output. MGA, in contrast, proved to be a strong inhibitor of cortisol output (ID50 of 2.3 mumol/l). Its site of action was established by adding it to adrenal cells incubated with precursor steroids on the cortisol pathway. Conversion of 3 beta-hydroxysteroids to cortisol was inhibited whereas conversion of 3-keto steroids was not affected. It follows that MGA inhibits 3 beta-hydroxysteroid dehydrogenase.