Wilbur K. Sawyer

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.

Effect of Matrix Metalloproteinase Inhibition on Progression of Atherosclerosis and Aneurysm in LDL Receptor‐Deficient Mice Overexpressing MMP‐3, MMP‐12, and MMP‐13 and on Restenosis in Rats after Balloon Injury

ABSTRACT: The broad‐spectrum MMP inhibitor CGS 27023A was tested to determine its potential as a therapy for atherosclerosis, aneurysm, and restenosis. LDL receptor‐deficient (LDLr −/−) mice fed a high‐fat, cholic acid‐enriched diet for 16 weeks developed advanced aortic atherosclerosis with destruction of elastic lamina and ectasia in the media underlying complex plaques. Lesion formation correlated with a 4.6‐ to 21.7‐fold increase in MMP‐3, ‐12, and ‐13 expression. Treatment with CGS 27023A (p.o., b.i.d. at 50 mg/kg) had no effect on the extent of aortic atherosclerosis (36 ± 4% versus 30 ± 2% in controls), but both aortic medial elastin destruction and ectasia grade were significantly reduced (38% and 36%, respectively, p < 0.05). In the rat ballooned‐carotid‐artery model, CGS 27023A (12.5 mg/kg/day via osmotic minipump) reduced smooth muscle cell migration at 4 days by 83% (p < 0.001). Intimal lesions were reduced by 85% at 7 days (p < 0.001), but intimal smooth muscle proliferation was unaffected, and inhibitory efficacy was lost with time. At 12 days, intimal lesion reduction was less potent (52%, p < 0.01). At 3 and 6 weeks, reductions of 11% and 4%, respectively, were not significant. This demonstrates that it is essential to include late time points when the ballooned‐carotid‐artery model is employed to ensure that lesion size does not “catch up” when a compound solely inhibits smooth muscle cell migration. In summary, MMP inhibitor therapy delayed but did not prevent intimal lesions, thereby demonstrating little promise to prevent restenosis. In contrast, MMP inhibitor therapy may prove useful to retard progression of aneurysm.

Enhanced Expression of Matrix Metalloproteinase-3, -12, and -13 mRNAs in the Aortas of Apolipoprotein E-deficient Mice with Advanced Atherosclerosis

Increased expression of matrix metalloproteinases (MMPs) has been reported at sites of advanced atherosclerosis and aneurysm in humans. 1,2 An animal model that has been reported to develop advanced atherosclerosis and medial elastin degradation with progression to ectasia and aneurysm is the apolipoprotein E–deficient (apoE -/) mouse. 3–5 Using immunohistochemistry, a variety of MMPs have been demonstrated at sites of atherosclerosis and elastin degradation in apoE -/mice on a high-fat diet. 5 However, little information is available on MMP expression in apoE -/mice on a normal diet or in mice that are not as susceptible to atherosclerosis on either normal or high-fat diet. The present study compares mRNA expression of MMP-3, -9, -12, and -13 in mice that are highly susceptible (apoE -/), moderately susceptible (C57BL/6J and B6129F1/J), or resistant (C3H/HeJ and Balb/cJ) to diet-induced atherosclerosis. 6

On the mechanism of prostaglandin F2α-induced abortion in hamsters

Abstract The mechanisms by which prostaglandins (PGs) induce abortion in hamsters remain poorly understood. PGs may effect one or more of these factors: a) corpus luteum (CL) structure and/or function; b) ovarian blood supply; c) pituitary function; and d) uterine motility. A minimum s.c. dose of 12.5 μg of PGF 2α is required to abort hamsters on day 5 of pregnancy. Radioimmunoassays revealed a drop of 71% in serum progesterone (P) levels 3 hours after dosing. Pregnancy was maintained indefinitely in hamsters receiving PGF 2α on day 5 by injections of progesterone on days 4, 5, and 6. In a related experiment, pregnancy was maintained with 5 mg doses of 17α-ethyl-19-nortestosterone (ENT) before and after PGF 2α injection, thereby permitting assessment of endogenous P levels (ENT does not cross-react with antiprogesterone antisera). Serum P levels were depressed by PGF 2α in both ENT and control hamsters. Finally, PGF 2α was injected on day 6 in hypophysectomized hamsters in which pregnancy was maintained with ovine prolactin (1 mg) and ovine FSH (5 μg). Serum P levels were decreased 70% 3 hours post-injection, although pregnancy was maintained in 37% of the animals. PGF 2α thus interferes directly with P secretion by the ovary in the absence of the pituitary and in the presence of injected luteotrophic complex. A sustained drop of 85–90% in serum P levels appears to be required for PGF 2α to induce abortion in 100% of the hamsters. We conclude that PGF 2α does not permanently damage CL when pregnancy is maintained by exogenous hormones, and the involution observed by others is most likely a consequence rather than a cause of pregnancy loss.

Effect of a novel series of macrocyclic hypolipidemic agents on plasma lipid and lipoprotein levels of four non-primate species

The ansamycins are structurally novel hypolipidemic agents derived from rifampicin, but lacking antibacterial activity. Oral or intravenous administration resulted in rapid lowering of plasma cholesterol in rats, hamsters, guinea pigs and dogs. In the chow-fed rat, three related compounds (CGP 43371, CGS 23810 and CGS 24565) exhibited ED50 values of 13.7, 3.1 and 0.18 mg/kg, respectively. A feature common to the lipid lowering documented in these four species was the concomitant reduction of low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol. In the chow-fed rat, however, apolipoprotein AI (apo AI) levels were much less affected than were those of HDL cholesterol. CGP 43371 at 3 and 10 mg/kg, lowered HDL cholesterol by 20% and 39%, respectively, whereas plasma apo AI was reduced by only 1% and 12%. Similarly, in lipoprotein fractions separated by ultracentrifugation, apo AI was unchanged in the d = 1.019-1.21 g/ml fraction after treatment with 3 or 10 mg/kg of CGP 43371, but HDL cholesterol was reduced 12% and 26% in this fraction at the two dose levels. Plasma and lipoprotein apo B levels, on the other hand, were reduced to a level equivalent to that of the reduction in cholesterol. The ansamycins thus represent a new structural series which may possess a novel mechanism of action as well, involving differential effects on HDL cholesterol and protein.

Effects of Aminoglutethimide on Cervical Dilatability and Serum Immunoreactive Relaxin in Pregnant Rats

Abstract Administration of aminoglutethimide (AG) at a daily sc dose of 20 mg/kg to rats from the 10th to 20th days of pregnancy resulted in fetal wastage, increased placental weights, and decreased placental alkaline phosphatase. In an effort to determine the endocrine problems relating to these abnormalities, serum relaxin (R) and progesterone (P) levels were determined by RIA and cervical distensibility was measured: All three parameters were subnormal in AG-treated rats. Therapy with estrogen (E) or (P) failed to correct any of the physiological problems associated with AG treatment, but a combination of these steroids increased fetal survival to control levels. However, cervical dilation in preparation for parturition failed to occur. Relaxin therapy alone increased cervical dilation but did not enhance fetal survival. A combination of E, P, and R maintained a normal complement of fetuses and provided normal cervical dilation. None of the treatments prevented placental enlargement but P treatment tended to maintain normal placental alkaline phosphatase.

Post-implantation termination of pregnancy in rats

Abstract Several types of antifertility drugs have been tested for abortifacient activity in pregnant rats. The interceptive 1 1Interception is defined as termination of pregnancy after implantation (1). action of the estrogens and anti-estrogens appeared to be due to their uterotropic effects and was not prevented by concomitant progesterone administration. The effect of estradiol on the uterus was augmented by theophylllne but not influenced by indomethacin. The prostaglandins and the steroid synthesis inhibitor, aminoglutethimide (AG), interfered primarily with biosynthesis of progesterone. Sub-threshold doses of AG and estradiol or AG and prostaglandin F 2α (PGF 2α ) were highly effective interceptives when given in combination. An estrogen (fenocyclin), PGF 2α and AG each partially inhibited trauma-induced uterine growth in pseudopregnant rats but did not prevent the concomitant rise in progesterone-dependent uterine alkaline phosphatase which signals decidualization. This unexpected finding suggest that once decidualization is initiated, progesterone may no longer be required for continued activation of enzyme, which may explain the high doses of compounds required for (post-implantation) interception as compared with (pre-implantation) contraception.

CGP 43371 paradoxically inhibits development of rabbit atherosclerotic lesions while inducing extra-arterial foam cell formation

Ansamycins are hypolipidemic compounds which, when administered to various animal species, dramatically lower high density lipoprotein (HDL) cholesterol levels, in addition to reducing the levels of other lipoprotein classes. The current study tested one of these ansamycins (CGP 43371) for its hypolipidemic and anti-atherosclerotic activity in cholesterol-fed rabbits. Rabbits were fed a 0.25% cholesterol-enriched diet with or without admixed CGP 43371, equivalent to 30 mg/kg per day for 16 weeks. Compared with control rabbits, CGP 43371 treatment lowered total cholesterol levels (46%, P<0.05) and lipoprotein cholesterol levels (HDL, 58%; VLDL, 49% [both P<0.05]; LDL, 28% [not significant]). Despite the dramatic lowering of HDL cholesterol levels, aortic atherosclerosis, assessed by grossly visible sudanophilia, was significantly inhibited versus controls (total aorta=38%; aortic arch=32%; thoracic aorta=60%). Of particular note in CGP 43371-treated rabbits was a striking splenomegaly, which correlated with the presence of massive accumulations of macrophage foam cells in the splenic red pulp. We speculate that CGP 43371 inhibits the development of atheroselerotic lesions in rabbits by both a hypolipidemic mechanism, and by a mechanism(s) in which macrophage foam cells accumulate in the spleen.

Studies on the prostate glands of adult inbred LSH hamsters.

Summary We have studied several parameters of prostate function in two inbred lines (2.4 and SsLak) of young and old LSH hamsters. These included weight, acid phosphatase, and [3H]testosterone uptake as influenced by age, castration, and androgen treatment. In the hamster, prostatic acid phosphatase concentration was found to vary inversely with androgen levels, contrary to the usual assumption that this enzyme is androgen dependent. Prostatic uptake of tritiated testosterone was enhanced by castration and by treatment of castrates with doses of androgen which induced a moderate increase in gland size. With advancing age, the prostates of LSH hamsters (both strains) became atrophic rather than hyperplastic, in contrast with a previous report (1). This atrophy appeared to be a consequence of decreased testicular function. The LSH hamsters appear to be a suitable model for the study of senescent changes in the male reproductive system.

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.