Ronald E. Steele

In vitro and in vivo studies demonstrating potent and selective estrogen inhibition with the nonsteroidal aromatase inhibitor CGS 16949A.

CGS 16949A inhibited the conversion of [4-14C]androstenedione (A) to [4-14C]estrone by human placental microsomes in a competitive manner (Ki = 1.6 nM). Aminoglutethimide, also a competitive inhibitor, had a Ki = 0.7 microM in this assay system. The Km for the aromatization of A was 0.11 microM. Using ovarian microsomes from immature rats primed with pregnant mares serum gonadotrophin and using [4-14C]testosterone conversion to [4-14C]estradiol as a measure of aromatase activity, the Km was 42 nM. At a substrate concentration 3-fold the Km, CGS 16949A was 180 times more potent as an inhibitor than aminoglutethimide, exhibiting half-maximal inhibition at 1.7 nM as compared to 0.3 microM. In vivo CGS 16949A lowered ovarian estrogen synthesis by gonadotropin-primed, androstenedione treated, immature rats by 90% at a dose of 260 micrograms/kg (PO). A dose of 100 mg/kg of aminoglutethimide was needed to produce this same effect. CGS 16949A at a dose of 4 mg/kg (PO) induced uterine atrophy (aromatase inhibition) without inducing adrenal hypertrophy - indicating a lack of inhibition of corticosterone secretion, while aminoglutethimide at 40 mg/kg (PO) induced adrenal hypertrophy without inducing uterine atrophy. CGS 16949A was neither androgenic nor estrogenic in rats using standard bioassays. The data suggest that CGS 16949A may serve as a potent and selective agent for modulating estrogen-dependent functions.

Reduction of cardiovascular and thyroxine-suppressing activities of L-T3 by liver targeting with cholic acid

This study was designed to determine whether the conjugation product of L-T3 with cholic acid would result in a liver-targeted compound (CGH 509A) with hypocholesterolemic (HC) activity significantly dissociable from cardiovascular (CV) and thyroxine-suppressing (TS) effects normally observed with thyroid hormone. Evaluation of HC activity in lipemic rats showed that CGH 509A was 6 times less potent than L-T3 with ED25 values estimated at 150 and 25 nmol/kg, respectively. CV function measured as changes in atrial rate, atrial tension and heart weight was determined in euthyroid rats. CGH 509A was at least 64 times less cardio-stimulant than L-T3 with minimum effective doses estimated at 2350 and 37 nmol/kg, respectively. TS activity was assessed in euthyroid rats as the potency of any compound to reduce plasma T4 levels. CGH 509A was 50 times less potent than-L-T3 with ED50 values estimated at 900 and 18 nmol/kg, respectively. From these results, it is clear that, while L-T3 was equally potent on HC, CV and TS activities, the HC potency of CGH 509A was at least 15 and 6 times greater than its CV and TS potencies, respectively.

Novel aromatase inhibitors

Aminoglutethimide (AG), an inhibitor of the aromatase enzyme, inhibits the biosynthesis of estrogens and displays well-documented anti-tumor efficacy in breast-cancer. However, this efficacy is accompanied by a relative lack of specificity in inhibiting aromatase and moderate tolerability. We report on two new non-steroidal aromatase inhibitors (CGS 16949A and CGS 18320B) which are more potent, selective and efficacious in their inhibition of aromatase than AG. Both compounds inhibit aromatase more potently in vitro and in vivo (over 400 and 1000 times respectively) than AG. They are both more selective in their inhibition of aromatase with CGS 18320B showing an improved selectively over CGS 16949A. When administered to adult female rats, both compounds elicit responses in serum hormones similar to those seen after ovariectomy. The duration of action of CGS 18320B, however, appears to be longer than that of CGS 16949A. CGS 18320B and CGS 16949A cause almost complete regression of DMBA-induced mammary tumors in adult female rats and almost completely suppress the appearance of new tumors. Thus CGS 16949A and CGS 18320B represent significant advances in the search for novel aromatase inhibitors which are more potent, selective and efficacious than aminoglutethimide.

Demonstration of potent lipid-lowering activity by a thyromimetic agent devoid of cardiovascular and thermogenic effects

A potent lipid-lowering thyromimetic (CGS 26214) devoid of cardiac and thermogenic activity was identified based on its ability to preferentially access and bind the nuclear fraction of hepatocytes over that of myocytes in culture. The difference in access achieved with CGS 26214 was at least 100-fold better for hepatocytes than for myocytes. This in vitro hepatoselectivity resulted in a compound with unprecedented in vivo lipid-lowering potency with a minimal effective dose of 1 microgram/kg in rats and dogs (approximately 25x that of L-T3). At the same time, CGS 26214 was free of any cardiovascular effects up to the highest dose tested of 25 mg/kg and 100 micrograms/kg in rats and dogs, respectively.

Aldosterone synthase inhibition for the treatment of hypertension and the derived mechanistic requirements for a new therapeutic strategy.

Context: We describe the clinical investigation of the first generation aldosterone synthase inhibitor, LCI699, in patients with essential, uncontrolled, resistant, or secondary hypertension. LCI699 competitively reduced blood pressure at lower doses yet counterintuitive effects were observed at higher doses. Objective and method: An extensive endocrine biomarker analysis was performed to better understand the pharmacological mechanism of the drug. Results: The interference of LCI699 in the renin–angiotensin–aldosterone system occurred with limited target selectivity, as a dose-dependent compensatory stimulation of the hypothalamic-pituitary-adrenal feedback axis was discovered. Thus, LCI699 affected two endocrine feedback loops that converged at a single point, inhibiting the 11&bgr;-hydroxylase reaction in the adrenal gland, leading to supraphysiological levels of 11-deoxycortiscosterone. The accumulation of this potent mineralocorticoid may explain the blunted blood pressure response to LCI699. Conclusion: Future aldosterone synthase inhibitors may improve their target selectivity by sparing the 11&bgr;-hydroxylase reaction and preferentially inhibiting one of the two other enzymatic reactions mediated by aldosterone synthase.

Synthesis and biological activity of phenoxyphenyl oxamic acid derivatives related to L-thyronine.

The synthesis of substituted phenoxyphenyl oxamic acid derivatives related to L-thyronine (L-T3) is described. The in vitro and in vivo cholesterol lowering and cardiovascular effects of these compounds are presented and discussed.

Relative importance of 5α reduction for the androgenic and LH-inhibiting activities of Δ4-3-ketosteroids

Abstract The significance of 5α reduction of C 19 , Δ 4 -3-ketosteroids in regulating growth of the rat ventral prostate (VP) was examined. The androgenic and LH-inhibiting activities of a C 19 , Δ 1,4 -3-ketosteroid which does not undergo appreciable 5α reduction were compared with those of its 5α reduced analogue and those of testosterone (T). In intact rats M (17β-hydroxy-17α-methyl-androsta-1:4-dien-3-one) caused a suppression of VP weights and plasma testosterone concentrations, and in castrated rats suppressed plasma LH concentrations. M was considerably less androgenic and moderately less potent as an inhibitor of LH secretion than either T or the 5α reduced analogue of M [17β-hydroxy-17α-methyl-5α-androst-1-ene-3-one; (5αM)]. 5αM was found to be at least as androgenic and as active as an inhibitor of LH as T, suggesting that the weak activity of M may be attributable to a lack of reduction to 5μM. Following incubation of 3 H-M with VP minces, over 96% of the radioactivityecovered corresponded with M by TLC. Under identical conditions 32–48% of the radioactivity recovered from incubations with 14 C-T corresponded with 5α reduced metabolites of T. This study demonstrates the importance of 5α reduction for both the androgenic and LH-inhibiting activities of Δ 4 -3-ketosteroids.

Identification of Potential Antiatherosclerotic/Hypolipidemic Agents by their Effect on Hepatic Conversion of Androst-4-ene-3,17-dione to Etiocholanolone and Androsterone

Clinical observations have shown that hypercholesterolemia is associated with abnormal androgen metabolism, viz. an increased excretion of etiocholanolone (E) relative to androsterone (A). Substances which restore the A/E ratio to normal likewise lower serum cholesterol. Postulating that the abnormal steroid and sterol metabolism may be either causally related or dependent on the same metabolic defect, we have developed in vitro and in vivo models to select drugs which favorably effect the ratio of A to E produced from [4-14C]androst-4-ene-3,17-dione [4-14C]A-dione). The in vitro model employs a mixture of rat liver microsomal delta 4-3-ketosteroid-5 alpha-reductase and cytosolic 3 alpha-hydroxysteroid dehydrogenase and delta 4-3-ketosteroid-5 beta-reductase. Kinetic and mechanistic studies have been performed on active compounds using this in vitro assay. The in vivo model employs i.v. injection of [4-14C]A-dione followed by collection of bile in anesthetized, hypophysectomized female rats. Many compounds preselected in the in vitro assay likewise reduced the A/E ratio in vivo. One of these compounds (CGS 10614A) also lowered serum cholesterol and reduced the incidence and severity of atherosclerotic lesions in aortas of cholesterol-fed rabbits.

Effects of altered endocrine function on biliary metabolites of [4-14C]androst-4-ene-3,17-dione in rats: Possible utility as a model for identifying anti-atherosclerotic agents

Atherosclerosis, coronary artery disease and elevated serum cholesterol are frequently associated with an abnormal pattern of androgen metabolites, especially an elevation of etiocholanolone (E) and/or epiandrosterone (EA) relative to androsterone (A). Therapeutic correction of these metabolic defects may lower serum cholesterol. We have attempted to reproduce this metabolic syndrome in rats by altering their endocrine status. Intact male rats excreted very little A or E in their bile; more than 80% of the [4-14C]A-dione was excreted as unknown polar compounds. Adrenalectomy, thyroidectomy or streptozotocin diabetes induced little or no change in the excretion of both E and A and did not alter the A/E ratio. Hypophysectomy (hypox), however, resulted in a huge increase in E excretion and a 10-fold decrease in the A/E ratio. Treatment of hypophysectomized males with bovine growth hormone (bGH) but not testosterone or thyroxine restored the pattern of androgen metabolites to that of intact male rats. Intact female rats excreted mainly A, and this was decreased by ovariectomy. Hypophysectomy, however, resulted in a marked increase in E and a corresponding large decrease in A excretion. Treatment of hypox females with estradiol or triiodothyronine did not correct the metabolic defects in A and E production, whereas GH resulted in a pattern of A-dione metabolism resembling that of intact males; i.e., primarily polar metabolites with low A and E. Hypophysectomy thus results in a dramatic increase in 5 beta-reductase activity in male and female rats. GH therapy restores the metabolic pathway to that seen in intact males. Our objective had been to find a model capable of detecting substances which would increase A and decrease E production. The male rat (regardless of endocrine status) has little 5 alpha-reductase activity. The intact female rat, however, has high 5 alpha-reductase activity, and retains significant 5 alpha-reductase in the absence of the ovaries. In hypox females, 5 alpha-reductase was much reduced while 5 beta-reductase was increased. Furthermore, serum cholesterol was elevated in hypox females but could be lowered by exogenous androsterone. Thus the hypox female rat appears to offer the best model for identifying non-hormonal agents which could enhance the production of A and/or decrease the production of E. Such agents might favorably influence cholesterol metabolism.

1.P.213 Beneficial effects of a novel thyromimetic on lipoprotein metabolism

Although L-triiodothyronine (L-T3) lowers cholesterol, this hormone is not used to treat hypercholesterolemia because of its cardiotoxic effects. Thyromimetics, such as the novel compound CGS 23425, that mimic the beneficial but lack the detrimental effects of T3, may be useful in the treatment of hypercholesterolemia. To show that CGS 23425 has no cardiotoxicity, atrial contractility and force were both measured and found to be unchanged in rats treated with up to 10 mg/kg drug. The lipid lowering actions of this drug resulted in a 44% decrease in low-density lipoprotein (LDL) cholesterol in hypercholesterolemic rats treated with 10 microg/kg of the compound. Normal rats required a higher dose of 1000 microg/kg to elicit a similar 50% reduction in LDL cholesterol. Both CGS 23425 or T3 (10 nM) increased the specific binding of 125I-labeled LDL to Hep G2 cells and increased LDL receptor number by 44 and 49%, respectively. These data indicate that CGS 23425 enhances hepatic clearance of serum LDL cholesterol. Normal and fat-fed animals treated with the drug showed a dose-dependent increase in apolipoprotein AI, a protein that promotes the efflux of cholesterol from peripheral tissues. Transient transfection of a rat apolipoprotein AI promoter-chloramphenicol acetyltransferase construct, in human hepatoma cells, showed a dose-dependent increase in chloramphenicol acetyltransferase activity with EC50 values of 2 x 10(-12) M and 10(-10) M for thyroid hormone receptors beta1 and alpha1, respectively, with maximal responses at 10(-7) M. These data indicate that CGS 23425 is a thyromimetic that increases apolipoprotein AI expression via thyroid hormone receptor. In summary, CGS 23425 ameliorates hypercholesterolemia by increasing apolipoprotein A1 and the clearance of LDL cholesterol. Therefore, a compound like CGS 23425 may be useful for the prevention and reversal of atherosclerosis.