Comparison of the effect of 4-hydroxy-4-androstene-3,17-dione on aromatase activity in granulosa cells from preovulatory follicles of rats, rabbits, and humans

The effect of the aromatase inhibitor 4-hydroxy-4-androstene-3,17-dione (4-OH-A) on the synthesis of estradiol (1,3,5 (10)-estratriene-3,17 beta-diol) by granulosa cells from preovulatory follicles of rats, rabbits and humans was examined. Granulosa cells from all three species were incubated for 4 h without treatment (control) or in the presence of androstenedione (4-androstene-3,17-dione, 0.5 microM), 4-OH-A (5 microM), or both compounds together. Estradiol levels were determined in the medium and cells by radioimmunoassay. In all three species, estradiol synthesis was markedly increased by androstenedione and this increase was blocked by 4-OH-A. In the rabbit, however, 4-OH-A alone caused a small but significant increase in radioimmunoassayable estradiol. The apparent increase seen with 4-OH-A alone may be due to a metabolite of 4-OH-A that cross-reacts in the estradiol radioimmunoassay. With granulosa cells from humans, in which 4-OH-A is of potential therapeutic importance, no similar effect of 4-OH-A alone was observed.     

Superiority of gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol for monitoring of aromatase inhibitor therapy

Currently available radioimmunoassay methods for estradiol in serum lack sufficient sensitivity and precision to monitor estradiol levels in patients placed on third generation aromatase inhibitors. We recently validated a gas chromatography/tandem mass spectrometry assay (GC/MS/MS) for estradiol and determined estrogen levels in normal post-menopausal women and in women with breast cancer before and during administration of aromatase inhibitors. Validation of the GC/MS/MS assay in human plasma and human serum included determination of assay sensitivity (<0.63 pg/ml), precision (all CVs less than 17.8%), recovery (98-103%), and linearity of recovery (R=0.998). Levels of estradiol were lower when assayed by GC/MS/MS compared to RIA under all conditions (7.26+/-4.82 pg/ml versus 11.9+12.0 pg/ml in normal post-menopausal women; 5.88+/-3.43 pg/ml versus 13.8+/-7.5 pg/ml in breast cancer patients prior to treatment; and<0.63 pg/ml versus 5.8+/-4.1 pg/ml during aromatase inhibitor therapy). Fifty-five women treated either with atamestane/toremiphene or letrozole/placebo were monitored for estradiol levels at 4, 8 and 12 weeks of therapy. The mean levels of estradiol during aromatase inhibitor therapy was 5.8+/-4.1 pg/ml as measured by RIA and <0.63 pg/ml by GC/MS/MS. The degree of suppression with the aromatase inhibitors as detected by RIA was 58% versus >89% by GC/MS. These results suggest that most RIA methods detect cross-reacting estrogen metabolites and yield higher measured levels than GC/MS/MS. Several pharmacological and clinical considerations suggest that GC/MS/MS should become the preferred method for monitoring aromatase inhibitor therapy.     

Selection of 19-(ethyldithio)-androst-4-ene-3,17-dione (ORG 30958): a potent aromatase inhibitor in vivo.

19-Mercaptoandrost-4-ene-3,17-dione (ORG 30365) has been reported to be both a competitive and irreversible inhibitor of aromatase. In comparison to the known aromatase inhibitors 4-hydroxy-androst-4-ene-3,17-dione (4OH-AD) and 1-methyl-1,4-androstadiene-3,17-dione (SH 489), ORG 30365 was found to be, respectively, about 16 and 8 times more active in vitro using human placental microsomes. Although the activity profile of ORG 30365 is very attractive, this compound was not selected for further development because it has limited pharmaceutical stability, which is probably due to its free--SH group and therefore a number of more stable dithio-derivatives of ORG 30365 have been synthesized. These derivatives are considered to be converted to ORG 30365 before they become active. The in vivo aromatase inhibiting activity of these derivatives was determined in hypophysectomized rats treated with the estrogen precursor dehydroepiandrosterone sulphate (DHEAS) using inhibition of cornification of vaginal epithelium as parameter. The 19-(ethyldithio)-derivative (ORG 30958) appeared to be the most active inhibitor in this series being twice as active as ORG 30365 and about 8 times as active as inhibitors like 4OH-AD and SH 489. Besides inhibition of cornification of vaginal epithelium ORG 30958 decreased ovarian aromatase and plasma E2 levels in DHEAS-treated hypophysectomized rats. Plasma estradiol levels were also lowered by ORG 30958 in dogs which were treated with pregnant mare serum gonadotrophin in order to induce pro-estrus. ORG 30958 displayed much less than 1/400th of the androgenic activity of testosterone propionate in immature castrated rats and appeared to be devoid of estrogenic and anti-estrogenic activity in ovariectomized mature rats. A twice daily dose of 1.5 mg ORG 30958/kg postponed ovulation in mature female rats. In conclusion: ORG 30958 is a potent aromatase inhibitor in vivo. It probably becomes active after cleavage of the -S-S- bond yielding ORG 30365 a potent irreversible aromatase inhibitor. ORG 30958 does not display other hormonal activities making it an attractive candidate for the treatment of estrogen-dependent diseases.     

Inhibitory effect of a new androstenedione derivative, 14 alpha-hydroxy-4-androstene-3,6,17-trione (14 alpha-OHAT) on aromatase activity of human uterine tumors

The development of human uterine estrogen-dependent tumors is considered to be closely related to estrogen biosynthesis. This study examined whether or not 14 alpha-hydroxy-4-androstene-3,6,17-trione (14 alpha-OHAT), a new 4-androstene-3, 17-dione derivative synthesized microbiologically, inhibits estrogen biosynthetase (aromatase) activities of human uterine tumors (i.e. uterine endometrial cancer, uterine leiomyoma and uterine adenomyosis tissues). 14 alpha-OHAT inhibited aromatase activity in all uterine tumors, dose-dependently (0.1-10 microM). Moreover, 14 alpha-OHAT did not show the binding affinity to rabbit uterine cytosol-sex steroids, and it was not converted to estrogen in human placental preparations. Thus, 14 alpha-OHAT, an aromatase inhibitor, may be useful clinically as an endocrine chemotherapy for peri- or post-menopausal women with uterine estrogen-dependent tumors.     

Quantitative determination of 4-hydroxy-4-androstene-3,17-dione (4-OHA), a potent aromatase inhibitor, in human plasma, using isotope dilution mass spectrometry

An original method is described for the determination in human plasma of 4-hydroxy-4-androstene-3,17-dione (4-OHA), a potent aromatase inhibitor, by isotope dilution mass-spectrometry using 7,7-[2H2]-4-OHA as internal standard. This compound was synthesized starting from 7,7-[2H2]-4-androstene-3,17-dione. The procedure includes an extraction step using an Extrelut 1 column and a derivatization with N,o-bis(trimethylsilyl)trifluoroacetamide (BSTFA). The minimum detection level of the method is 0.650 pg and the coefficients of variation for the 0.5 ng/ml (plasma) and 5 ng/ml (plasma) concentrations are 3.2% (within assay) and 6.7% (between assay) and 1.86% (within assay) and 2.3% (between assay) respectively.     

Synthesis and evaluation of analogues of 4-androstene-3,17-dione as aromatase inhibitors

Twenty-three synthetic analogues of 4-androstene-3,17-dione (androstenedione) have been evaluated as inhibitors of human placental microsomal aromatase enzyme. Among the most potent of these compounds were the 4-hydroxy, 6 alpha-fluoro, 6 beta-fluoro, and 4-fluoroandrostenediones and 4-fluoro-19-nor-4-androstene-3,17-dione. 4-Hydroxy-4-androstene-3,17-dione (4HAD) is an irreversible inhibitor of aromatase in vitro, whereas the four fluoro analogues are reversible inhibitors. 4HAD and 4-fluoro-4-androstene-3,17-dione caused significant regression of the nitrosomethylurea-induced mammary tumor in rats, but the other fluoro derivatives were inactive.     

Structure-activity relationships of 2-, 4-, or 6-substituted estrogens as aromatase inhibitors

Aromatase, which is responsible for the conversion of androgens to estrogens, is a potential therapeutic target for the selective lowering of estrogen levels in patients with estrogen-dependent breast cancer. To develop a novel class of aromatase inhibitors, we tested series of 2- and 4-substituted (halogeno, methyl, formyl, methoxy, nitro, and amino) estrones (7 and 9), as well as series of 6alpha- and 6beta-substituted (alkyl, phenalkyl, and alkoxy) estrones (13 and 14), and their estradiol analogs (8, 10, 11, and 12) as aromatase inhibitors. All of the inhibitors examined blocked the androstenedione aromatization in a competitive manner. Introduction of halogeno and methyl functions at C-2 of estrone as well as that of a phenalkyl or methyl function at the C-6alpha or C-6beta position markedly increased affinity to aromatase (apparent K(i) value=0.10-0.66 microM for the inhibitors versus 2.5 microM for estrone). 6alpha-Phenylestrone (13c) was the most powerful inhibitor among the estrogens studied, and its affinity was comparable to that of the androgen substrate androstenedione. Estradiol analogs were much weaker inhibitors than the corresponding estrone compounds in each series, indicating that the 17-carbonyl group plays a critical role in the formation of a thermodynamically stable enzyme-inhibitor complex.     

Antitumor effect of a specific aromatase inhibitor, 1-methyl-androsta-1,4-diene-3,17-dione (atamestane), in female rats bearing DMBA-induced mammary tumors

Atamestane is a potent competitive inhibitor of estrogen biosynthesis (aromatase) in several species, in vitro and in vivo, and has no endocrine side effects. In this study, the efficacy of atamestane in suppressing tumor growth was evaluated in comparing with that of a non-steroidal aromatase inhibitor (CGS 16949A, Ciba-Geigy) and ovariectomy. Female Sprague-Dawley rats bearing DMBA-tumors were treated s.c. once daily either with 30 or 150 mg/kg atamestane or with 0.1 or 0.5 mg/kg CGS 16949A for 4 weeks. At these biologically equivalent doses both aromatase inhibitors effectively inhibited tumor growth: at the end of treatment they caused a marked reduction in tumor size (up to 70%), while ovariectomy led to a complete remission of tumor growth. The histo-morphological pictures of the mammary tumors from treated animals were qualitatively almost similar to those of the control. In hosts, neither compound exerted any influence on the weight of genital organs (ovary, uterus and vagina), although the peripheral LH levels were significantly elevated by the higher dose of the aromatase inhibitors. This effect on LH levels is probably due to the elimination of the negative feed-back effect of estrogens on gonadotropin secretion (counter regulation). The serum prolactin levels were decreased by the aromatase inhibitors, indicating a diminution of estrogen levels in the treated animals. The present results clearly demonstrate that, in spite of the counter regulation, a pure aromatase inhibitor such as atamestane in sufficiently high doses is able to inhibit the growth of DMBA-induced mammary tumors in intact female rats.     

Rainbow trout, Oncorhynchus mykiss, as a model for aromatase inhibition.

The feasibility of utilizing rainbow trout, Oncorhynchus mykiss, as an alternative model for studying the inhibition of aromatase (CYP 19) was investigated. The suppression of estrogen-dependent tumors by aromatase inhibitors has been important in the treatment of breast cancer. Estrogens, estrogen precursors and xenoestrogens have been found to promote liver cancer in the trout model. A steroid, 4-hydroxy-4-androstene-3,17-dione (4-OHA), and non-steroids, aminoglutethimide (AG) and Letrozole (CGS 20267), all of which are known aromatase inhibitors in rats and humans, were examined in vitro for activity in trout ovarian microsomes. Aromatase activity was quantified as the release of 3H2O from the conversion of [3H]-4-androstene-3,17-dione to 17beta-estradiol and estrone. Trout ovarian microsomes exhibited activity between 39-60 fmol mg(-1) min(-1) with a calculated Vmax of 71.1 fmol mg(-1) min(-1) when incubated at 25 degrees C with 200 nM 4-androstene-3,17-dione (K(M) = 435 nM). Significant inhibition by 4-OHA up to 80% was seen at 1.5 microM. At 2000 microM, AG decreased aromatase activity by up to 82%. Letrozole reduced aromatase activity a maximum of 90% in a dose-dependent manner, but the Ki (2.3 microM) was 1000-fold higher than reported in human trials. Indole-3-carbinol and some of its derivatives, two DDE isomers and four flavones (except alpha-naphthoflavone) at 1000 microM did not significantly inhibit aromatase in vitro. Letrozole and clotrimazole, fed to juvenile rainbow trout at doses up to 1000 ppm for 2 weeks, were not effective in suppressing dehydroepiandrosterone (DHEA) induced increases in vitellogenin and 17beta-estradiol levels. These results document that trout aromatase is sensitive to inhibition in vitro by known inhibitors of the mammalian enzyme. The mechanism(s) for lack of inhibition in vivo is currently unknown and must be further investigated in order to develop a trout model for studying the role of aromatase in carcinogenesis.     

Aromatase activity after a short-course of letrozole administration in adult men at sea level and at high altitude (with or without excessive erythrocytosis)

Men living at high altitudes in Peru compared to sea level counterparts have erythrocytosis (hemoglobin 16-21?g/dl) or excessive erythrocytosis (hemoglobin>21?g/dl). High testosterone (T) levels in men at high altitude (HA) were associated with excessive erythrocytosis. High androgen levels could be due to a low aromatase activity or to an elevated rate of conversion from precursors to testosterone. The aim of this study was to evaluate aromatase activity and rate of conversion from precursors to testosterone before and after administration of the aromatase enzyme inhibitor letrozole (5?mg/day) for a 5-day period to men at HA and at sea level (SL). The response to short term aromatase inhibition was assessed in 30 adult men living at sea level, 31 native men at HA with erythrocytosis (Hb 16-21?g/dl), and 35 men at HA with excessive erythrocytosis (Hb>21?g/dl). Serum hormone levels, estradiol/testosterone, testosterone/androstenedione, and testosterone/dehydroepiandrosterone sulfate (DHEAS) ratios were measured. Men with erythrocytosis had lower basal serum T/androstenedione ratios than men with excessive erythrocytosis at HA and men at sea level. Men at HA with excessive erythrocytosis had higher T/DHEAS ratios than men with erythrocytosis and than those at sea level before and after letrozole administration. After letrozole administration, both groups of men at high altitude (with erythrocytosis or with excessive erythrocytosis) showed lower aromatase activities than those at sea level. In conclusion, higher serum testosterone levels in men with excessive erythrocytosis were associated with an increased rate of conversion from DHEAS to testosterone rather than to a lower aromatase activity.     

Growth suppression of MCF-7 human breast cancer cells by aromatase inhibitors: A new system for aromatase inhibitor screening

In our previous study we found that MCF-7 cells possess aromatase activity and stimulate estrogen receptor-mediated growth. The pathways through which androgens are converted to estrogens by aromatase and estrogens interact with estrogen receptors contribute significantly to growth stimulation. The administration of aromatase inhibitor results in suppression of growth stimulation by androgens. This system enabled us to assess directly the biological activities of aromatase inhibitors. Aromatase activity was inhibited in a dose-dependent manner by the addition of aminoglutethimide and CGS 16949A, competitive inhibitors, and of 14-hydroxy-4-androstene-3,6,17-trione and 4-hydroxy-androstenedione, mechanism-based inhibitors. After preincubation with mechanism-based inhibitors, aromatase activity was significantly suppressed, whereas after preincubation with competitive inhibitors, it was adversely increased. These effects were concentration- and time-dependent. Preincubation with competitive inhibitors resulted in augmentation of subsequent androgen stimulation of thymidine incorporation, while preincubation with mechanism-based inhibitors resulted in diminished stimulation by subsequent androgen administration. These results suggest that in MCF-7 cells competitive inhibitors adversely induce aromatase and accelerate the subsequent androgen stimulation of DNA synthesis. Suicide inhibitors are more effective than competitive inhibitors. This system will be useful for aromatase inhibitor screening.     

Endocrine characterization of a human ovarian carcinoma (BG-1) established in nude mice

The steroid receptor-positive human ovarian cancer (BG-1) was evaluated to determine its usefulness as a tumor model. This tumor grows in intact male and female nude mice without hormone supplements. Moreover, its growth was significantly accelerated in ovariectomized mice, and the increased growth rate could be reversed by estradiol administration. Evaluation of tumor growth following endocrine therapy revealed that, while antiandrogens did not affect the tumor growth, both an aromatase inhibitor and a luteinizing hormone-releasing hormone agonist significantly impaired growth of this human ovarian tumor. Estradiol was also shown to up-regulate both estrogen and progesterone receptors in tumors grown in ovariectomized mice. Therefore, the BG-1 human ovarian carcinoma grows without hormonal supplements and yet responds to specific forms of endocrine therapy. Moreover, the steroid receptors present in this tumor respond to exogenous steroids. In conclusion, this tumor may serve as an ideal model for the study of hormonal regulation of ovarian tumor growth.     

Cyclooxygenase-2 directly regulates gene expression of P450 Cyp19 aromatase promoter regions pII, pI.3 and pI.7 and estradiol production in human breast tumor cells

The present studies evaluated the direct effects of the presence of human cyclooxygenase-2 (Cox-2) on gene expression of specific promoter regions of the P450 Cyp19 enzyme aromatase enzyme and its product, estradiol, in Cox-2 null estrogen-dependent MCF-7 breast tumor cells and in a stable clone of MCF-7 cells containing transfected Cox-2 cDNA, designated as MCF-7/Cox-2 Clone 10. Clone 10 human breast tumor cells have significantly increased gene expression of total mRNA of the P450 Cyp19 enzyme aromatase, with high levels of gene expression of specific aromatase promoter (p) regions pII, pI.3, and p1.7, with no significant change in mRNA levels of p1.4. Clone 10 human breast tumor cells produced significantly increased amounts of both prostaglandin E2 (PGE2) derived from Cox-2 enzyme activity and estradiol derived from aromatase enzyme activity (p<0.01), compared to MCF-7/vector control cells. The greatest inhibition of PGE2 or estradiol production was observed by the combination of the selective Cox-2 inhibitor celecoxib (25 microM) and the aromatase inhibitor, formestane (10nM) (p<0.01). The greatest anti-proliferative effect in Cox-2 null MCF-7/vector control cells was observed with the combination of 25 microM celecoxib and 10nM formestane but not with 10 microM celecoxib, suggesting that there are Cox-2-independent mechanisms involved in the anti-proliferative effect of this agent at doses greater than 10 microM. Celecoxib (25 microM) also significantly inhibited proliferation of MCF-7/Cox-2 Clone 10 human breast tumor cells, with no further anti-proliferative activity with the addition of 10 nM formestane observed at either 24 or 48 h of treatment. These studies demonstrate that Cox-2 directly regulates gene expression of specific aromatase promoter regions and regulates aromatase enzyme activity. Agents that inhibit Cox-2 or block the biological effects of PGE2 may be useful in significantly limiting aromatase activity and proliferation of human breast tumor cells regardless of the presence of Cox-2. In addition, the unique human breast tumor cell model used in these studies may be a useful tool in identifying the spectrum of activities of agents that block the biological effects of PGE2 and estradiol.     

Expression of P450(arom) in Malaclemys terrapin and Chelydra serpentina: a tale of two sites.

The formation of estrogens from androgens in all vertebrates is catalyzed by the "aromatase" complex, which consists of a membrane bound P(450) enzyme, P(450) aromatase (which binds the androgen substrate and inserts an oxygen into the molecule), and a flavoprotein (NADPH-cytochrome P450 reductase). Among vertebrates, the two major sites of aromatase expression are the brain and gonads. Given the importance of estrogen in reptile sex determination, we set out to examine whether P450arom was involved in the initiation and/or stabilization of sex determination in turtles. We examined the expression of aromatase activity in the brain and gonads of two turtle species exhibiting temperature dependent sex determination (TSD), the diamondback terrapin (Malaclemys terrapin), and the common snapping turtle (Chelydra serpentina). Estradiol when applied at stage 14 of the terrapin induces expression of aromatase in the gonad of embryos incubated at male temperatures (26.5 degrees C). The level of expression is similar to that of a normal embryonic ovary. When applied at stage 22, estradiol does not induce aromatase expression in the terrapin. The xenoestrogen, nonylphenol, sex reverses terrapin embryos at 26.5 degrees C. Letrazole, a nonsteroidal aromatase inhibitor, suppresses aromatase activity in the brain at either incubation temperature. Ovotestes are produced by letrazole administration in the terrapin when incubated at 30.5 degrees C. In the snapping turtle at stage 23, gonadal and brain aromatase activity in embryos incubated at female temperatures (30.5 degrees C) is nearly half that exhibited in terrapin embryos at the same temperature. Moreover, letrazole administration suppresses aromatase expression to nearly basal levels. At male incubation temperatures (26.5 degrees ), brain aromatase expression is nearly three times higher than at female temperatures, while gonadal expression levels are nearly one third lower. However, the gonadal expression levels at male temperatures in the snapping turtle are nearly 25 times higher than that found in the terrapin. Estradiol administration elevates this level nearly three fold. These data suggest that is not merely the expression of aromatase that is important for ovarian development, but that the level of expression may be more important.     

Binding characteristics of [3H]delta(5)-androstene-3beta,17beta-diol to a nuclear protein in the rabbit vagina

In this study, we investigated the binding characteristics of [3H]Delta(5)-androstene-3beta,17beta-diol to rabbit vaginal cytosolic and nuclear extracts and in freshly excised intact tissue strips. [3H]delta(5)-Androstene-3beta,17beta-diol bound to a protein(s) in the vaginal nuclear extract with high affinity (K(d)=3-5 nM) and limited capacity (50-100 fmol/mg protein). No specific binding was detected in the cytoplasmic extracts. Competitive binding studies showed that binding of [3H]delta(5)-androstene-3beta,17beta-diol was effectively displaced with unlabeled delta(5)-androstene-3beta,17beta-diol but not with dehydroepiandrosterone, testosterone, dihydrotestosterone, triamcinolone acetonide, or progesterone. However, estradiol at high concentrations partially displaced bound [3H]delta(5)-androstene-3beta,17beta-diol. Incubation of freshly excised vaginal tissue strips with [3H]delta(5)-androstene-3beta,17beta-diol in the absence or presence of excess unlabeled delta(5)-androstene-3beta,17beta-diol for 1h at 37 degrees C resulted in specific binding to a soluble macromolecule in the nuclear KCl extracts. In addition, quantitative measurement of estrogen receptor, androgen receptor and delta(5)-androstene-3beta,17beta-diol binding protein was performed by equilibrium ligand binding assays using extracts of distal vaginal tissue from intact animals or ovariectomized animals treated for 2 weeks with vehicle, estradiol, testosterone, or estradiol plus testosterone. These changes in steroid hormone levels resulted in opposing trends between the estrogen receptor and delta(5)-androstene-3beta,17beta-diol binding protein, suggesting that delta(5)-androstene-3beta,17beta-diol binding protein is regulated differently by the hormonal milieu than the estrogen receptor. These data suggest that rabbit vaginal tissue expresses a novel binding protein which specifically binds delta(5)-androstene-3beta,17beta-diol and is distinct from the androgen and estrogen receptors.     

Effects of aromatase inhibitors and different doses of testosterone on gonadotropins in one year old male Atlantic salmon (Salmo salar)

The effects of different doses of testosterone (T), the aromatase inhibitors 1,4,6-androstatriene-3,17-dione (ATD) and 4-hydroxy-4-androstene-3,17-dione (4OH), and the combined treatment of T and ATD on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) at the onset of puberty in juvenile Atlantic salmon males were investigated. T always increased pituitary LH. Also, ATD increased pituitary LH, though to a lesser extent than T. However, ATD combined with T diminished pituitary LH levels compared to T alone, indicating an aromatase-dependent positive feedback of T on LH in immature males. 4OH, which was less effective than ATD as an aromatase inhibitor, increased LH content. ATD treatment resulted in increased pituitary FSH levels, similar to those of mature controls. Positive effects of ATD on plasma FSH were found, indicating the presence of an aromatase-dependent negative feedback. The 4OH effects on FSH levels were inconsistent. T exerted both positive and negative effects on pituitary FSH and testes growth, depending on dose and season, with the positive effects being more pronounced with the low doses and the negative effects with the high doses. The treatment of T combined with ATD did not affect the positive effect of T alone on pituitary and plasma FSH, indicating the presence of an aromatase-independent positive feedback on FSH. There was a positive correlation between FSH and gonadosomatic index, especially during summer when gonadal development occurs.     

Biological characterization of 10-(2-propynyl) estr-4-ene-3, 17-dione (MDL 18,962), an enzyme-activated inhibitor of aromatase

MDL 18,962 was shown to be a highly specific, potent (Ki = 3-4 nM), enzyme-activated inhibitor of aromatase with minimal intrinsic endocrine properties. The affinity of MDL 18,962 was higher for human and baboon placental aromatase than for rhesus placental or rodent ovarian aromatase. These species differences necessitated the development of a novel model of peripheral aromatase utilizing human enzyme. Human choriocarcinoma trophoblast xenografts in athymic nude mice were used for pharmacologic and pharmacokinetic evaluation of MDL 18,962. The ED50 for inhibition of aromatase activity in these trophoblast tumors at 6 h post-treatment was 1.4 mg/kg, s.c. and 3.0 mg/kg, oral. Preliminary results indicated that the ED50 for inhibition of peripheral aromatization of androgen by MDL 18,962 in female baboons was 0.01 mg/kg, i.v. and 4 mg/kg, oral.     

Covalent modification of aromatase by a radiolabeled irreversible inhibitor

7 alpha-Substituted 4-androstene-3,17-diones are effective inhibitors of aromatase. The microsomal enzyme complex has a greater affinity for several of these inhibitors than for the substrate androstenedione, with 7 alpha-(4'amino)phenylthio-4-androstene-3,17-dione being the most potent competitive inhibitor of the series. A potential affinity analog, the bromoacetamide derivative of the amino compound, has been synthesized in both unlabeled and 14C-labeled forms via a condensation of bromoacetic acid with the amino compound using DCC. Inactivation studies with the unlabeled inhibitor showed a time-dependent, first-order inactivation of aromatase enzymatic activity. Androstenedione, when incubated in varying concentrations with the irreversible inhibitor, provided protection from inactivation. Binding studies with radiolabeled inhibitor and microsomal aromatase preparations showed that irreversible binding had occurred. SDS-electrophoresis, followed by fluorography, identified four major microsomal proteins that were radiolabeled, with the protein band at 52,000 mol. wt predominating. Similar studies with a solubilized aromatase preparation decreased the amount of nonspecific binding. Thus, covalent bonds between the irreversible inhibitor and the aromatase cytochrome P450 molecule were formed.     

Synthesis of 2,2-dimethyl-4-hydroxy-4-androstene-3,17-dione as an inhibitor of aromatase

2,2-Dimethyl-4-hydroxy-4-androstene-3,17-dione (4) has been synthesized and has been shown to be a powerful competitive inhibitor of aromatase (Ki = 11.4 nM). However, compound 4 does not cause time-dependent loss of enzyme activity, in contrast to the unmethylated parent compound, 4-OHA.     

The measurement of 4-androstene-3, 11, 17-trione (11-oxo-androstenedione) by radioimmunoassay in human plasma

A radioimmunoassay (RIA) method is described for the determination of 4-androstene-3, 11, 17-trione (11-oxo-androstenedione) in human plasma. 4-androstene-3, 11, 17-trione 3-(0-carboxymethyl) oxime-bovine serum albumin conjugate was used to generate highly specific antiserum in rabbits. Cross reactivities of several other steroids with the antiserum were less than 4%. [1,2-3H] 4-androstene-3, 11, 17-trione was synthesized from [1,2-3H] 17 alpha, 21-dihydroxy-4-pregnene-3, 11, 20-trione. The intra- and interassay variation was 7.3% and 9.8%, respectively. The mean serum 4-androstene-3, 11, 17-trione level for healthy young subjects was 2.37 +/- 0.56 nM (X +/- SD) in males and 3.16 +/- 0.43 nM in females at 8 a.m. During the night, there was a marked decrease in serum level, giving at 11 p.m. 0.87 +/- 0.33 and 1.15 +/- 0.52 nM, respectively. During ACTH stimulation tests, 4-androstene-3, 11, 17-trione increased from 1.81 +/- 0.58 to 2.32 +/- 0.69 nM, while in dexamethasone suppression tests a decrease from 3.20 +/- 0.03 nM was seen. In contrast, HCG administration on 3 consecutive days did not influence plasma concentrations of 4-androstene-3, 11, 17-trione.