C. David Jones

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.

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.

Discovery and development of a novel class of nonsteroidal aromatase inhibitors

Efforts to develop a novel class of nonsteroidal aromatase inhibitors began with the discovery that the infertility in male rats exposed to high levels of the agricultural fungicide, fenarimol (alpha-(2-chlorophenyl)-alpha-(4-chlorophenyl)-5-pyrimidine-methanol), was attributable to the inhibition of aromatase activity within the central nervous system during the critical neonatal period. Although fenarimol was not particularly potent in inhibiting rat ovarian microsome aromatase activity in vitro (50% inhibition (IC50) = 4.1 microM). Subsequent testing of a number of analogues led to the identification of LY56110 (alpha,alpha-bis(4-chlorophenyl)-5-methylpyrimidine) which exhibited an IC50 of 29 nM. LY56110 was orally active, blocking the testosterone-induced increase of uterine weight and ovarian estrogen biosynthesis in immature female rats. In rats with established DMBA-induced mammary carcinoma, complete tumor regression was observed in 80% of the animals. Development of LY56110 was, however, stopped because of its effects on hepatic microsomal enzymes and an unacceptably long half-life. Structural modifications resulted in the development of the indenopyrimidines. LY113174 (8-chloro-5-(4-chlorophenyl)-5H-indeno less than 1, 2D greater than pyrimidine) was highly effective in vitro (IC50 = 24 nM) and in vivo but was far less potent than LY56110 with respect to induction of hepatic microsomal enzymes. LY113174 exhibited an acceptable biological half-life and had no effect on cholesterol side-chain cleavage. The indenopyrimidines appear to be a novel class of nonsteroidal aromatase inhibitors which may prove useful in the treatment of estrogen-dependent diseases.