CGS 16949A Research Articles

The 2,2′,4,4′-tetrabromodiphenyl ether hydroxylated metabolites 5-OH-BDE-47 and 6-OH-BDE-47 stimulate estradiol secretion in the ovary by activating aromatase expression

The aim of the present study was to assess the effect of two hydroxylated BDE-47 metabolites, 5-OH-BDE-47 and 6-OH-BDE-47, on steroidogenesis in the ovary. Both metabolites failed to affect the production of androstenedione and testosterone but increased the secretion of estradiol at all concentrations tested. The increased secretion of estradiol was due to the stimulation of aromatase gene and protein expression. Direct assessment of aromatase activity by dibenzylfluorescein assay and indirect assessment of aromatase activity by measurement of the conversion of testosterone to estradiol confirmed that 5-OH-BDE-47 and 6-OH-BDE-47 stimulate aromatase activity. The aromatase inhibitor CGS 16949A abolished this stimulatory activity and reduced estradiol levels in the control and treatment groups.

Large and small cells of the porcine corpus luteum: differential capacity to secrete estradiol and aromatize exogenous androgen during mid- and late luteal phase.

It has been demonstrated previously that aromatization capacity of luteal cells increased as the luteal phase progresses, suggesting that estradiol secretion by regressing corpora lutea could play an active role in regulation of the estrus cycle. To determine whether a specific luteal subpopulation is responsible for this process, luteal cells collected from mature (8-10 days after ovulation) corpora lutea were separated at unit gravity into small (14-26 microns) and large (26-38 microns) cell types. Aliquots of separated cells were incubated overnight in M199 medium alone (= control) or in medium containing: 10 M-5 T, 10(-6) M or 10(-7) M T or 0.1 x 10(-3) M, 0.5 x 10(-3) M or 1 x 10(-3) M aromatase inhibitor CGS 16949A. Both cell types were capable of estradiol production without the addition of substrates. The endogenous content of estradiol was always higher in large granulosa-derived luteal cells (LC) than in small theca-derived luteal cells (SC), and was higher in the late (LLP) than in the mid-luteal (MLP) phase. Both types of cells responded to testosterone by increasing estradiol secretion. Adding aromatase inhibitor to LC isolated from MLP and LLP decreased testosterone-stimulated estradiol secretion. On the other hand, aromatase inhibitor had no effect on testosterone-stimulated estradiol secretion by SC isolated during MLP, and inhibited testosterone-stimulated estradiol secretion by SC isolated during LLP. These data are the first to show the ability of both populations of porcine luteal cells to aromatize exogenous testosterone. The observed lack of aromatase inhibitor influence upon basal secretion of estradiol by SC separated from mature corpora lutea, and the decrease of testosterone-stimulated estradiol secretion by SC isolated from regressing corpora lutea, indicate that cytodifferentiation during luteinization could cause expression of P450arom in theca-derived luteal cells.

In vitro exposure of porcine prepubertal follicles to 4-chlorobiphenyl (PCB3) and its hydroxylated metabolites: Effects on sex hormone levels and aromatase activity

The present study was undertaken to investigate the endocrine disrupting activity of PCB3 and its hydroxylated metabolites 4-OH-PCB3 and 3,4-diOH-PCB3 in ovarian follicle cells derived from prepubertal animals with special emphasis on cytochrome P-450-dependent aromatase (CYP19). Aromatase activity was assayed simply by measuring the production of 17β-estradiol in the culture medium after addition of the substrate (testosterone). Theca interna and granulosa cells were exposed in vitro to 6 ng/ml of test chemicals for 24, 48, 72 and 96 h. PCBs stimulated the conversion of testosterone to estradiol in all treatments. In addition, PCB3, and to a lesser extent its metabolites, decreased progesterone secretion by these cells. This increase in estradiol and simultaneous decrease in progesterone suggests that progesterone is converted to testosterone and further to estradiol. The rank order of potency in estradiol secretion was 3,4-diOH-PCB3 > 4-OH-PCB3 > PCB3. The non-steroidal aromatase inhibitor CGS 16949A abolished this stimulatory activity, and reduced estradiol levels in treatment and control groups below the control levels, verifying that the increased estradiol secretion by follicle cells was due to aromatase activity. We infer that: (1) metabolism of PCB3 does not protect against endocrine disruption and (2) the estrogenic effect of these compounds is due to stimulation of aromatase activity.

Estrogen regulates tyrosine hydroxylase expression in the neonate mouse midbrain

Estrogen plays an important role during differentiation of midbrain dopaminergic neurons. This is indicated by the presence of estrogen receptors and the transient expression of the estrogen-forming enzyme aromatase within the dopaminergic cell groups. We have previously shown that estrogen regulates the plasticity of dopamine cells through the stimulation of neurite growth/arborization. In this study, we have analyzed the capability of estrogen to influence the activity of developing mouse dopamine neurons. The expression of tyrosine hydroxylase (TH) was assessed by competitive RT-PCR and Western blotting. The developmental expression of TH in the ventral midbrain was studied from embryonic day 15 until postnatal day 15 and revealed highest TH levels early postnatally. This profile coincides with the transient aromatase expression in this brain area. Using cultured midbrain cells, we found that estrogen increased TH mRNA/protein levels. The application of the estrogen receptor antagonist ICI 182,780 resulted in a complete inhibition of estrogen effects. To verify these data in vivo, fetuses were exposed in utero from E15 until birth to the aromatase inhibitor CGS 16949A or to CGS supplemented with estrogen. CGS caused a robust reduction in TH mRNA/protein levels in the midbrain, which could be restored by estrogen substitution. Taken together, our data strongly suggest that estrogen controls dopamine synthesis in the developing nigrostriatal dopaminergic system and support the concept that estrogen is implicated in the regulation of ontogenetic steps but also in the function of midbrain dopamine neurons.

Effects of the aromatase inhibitor fadrozole on plasma sex steroid secretion, spermatogenesis and epididymis morphology in the lizard, Podarcis sicula.

Recently, increasing importance has been attached to the role of estrogens and their receptors in male reproduction, since they have been found to be abundant in the male reproductive tract. In the lizard, Podarcis sicula, a seasonal breeder, estrogens seem to be involved in the regulation of testicular activity. Particularly, it has been hypothesized that the block of spermatogenesis and the complete regression of the epididymis and other secondary sexual characters (SSCs) in autumn might be due to high estrogen levels. To investigate the role of estrogens in the reproductive process of male lizards, we utilized Fadrozole ((AI) [4-(5,6,7,8-tetrahydroimidazole [1,5-a] pyridin-5-yl)-benzonitrile monohydrochloride] (CGS 16949A)), a nonsteroidal inhibitor of aromatase, the enzyme involved in the aromatization of androgens to estrogens, evaluating its effects on plasma sex-hormone release, spermatogenesis and epididymis morphology. For this purpose, adult male lizards, captured during the autumnal recrudescence, were intraperitoneally injected with 0.5 microg and 5 microg/g/body weight of AI for 15 and 30 days. In the animals treated with the higher AI dose, estrogen levels decreased if compared to the control groups, whereas androgen levels increased. Furthermore, histologic sections of testes and epididymes showed that the 30-day treatment with AI-induced spermatogenesis resumption with release of sperms into the large lumen of the seminiferous tubules, and the epididymes appeared more developed with moderately secreting columnar canal cells. Therefore, it is proposed that failure of spermatogenesis in autumn might be due to high estrogen levels.

Estrogen stimulates brain-derived neurotrophic factor expression in embryonic mouse midbrain neurons through a membrane-mediated and calcium-dependent mechanism.

We have provided evidence that 17beta-estradiol (E) synthesized in the midbrain promotes the differentiation of midbrain dopamine neurons through nonclassical steroid action. Because these developmental effects resemble those reported for brain-derived neurotrophic factor (BDNF), we hypothesized that E influences dopaminergic cell differentiation through a BDNF-dependent mechanism. Competitive RT-PCR and ELISA techniques were employed to study first the developmental pattern of BDNF and trkB expression in the mouse midbrain. BDNF protein/mRNA levels peaked postnatally, whereas trkB did not fluctuate perinatally. To prove the hypothesis that E regulates BDNF expression in vivo, fetuses and newborns were treated with the aromatase antagonist CGS 16949A. CGS 16949A exposure reduced midbrain BDNF mRNA/protein levels. The coapplication of CGS 16949A and E abolished this effect. Midbrain cultures from mouse fetuses were used to investigate intracellular signaling mechanisms involved in transmitting E effects. Estrogen increased expression of BDNF but not of other neurotrophins. As concerns the related signaling mechanism, these effects were antagonized by interrupting intracellular Ca(2+) signaling with BAPTA and thapsigargin but not by the estrogen receptor antagonist ICI 182,780. Insofar as E effects on BDNF mRNA expression were inhibited by cycloheximide, it appears likely that other, not yet characterized intermediate proteins take part in the estrogenic regulation of BDNF expression. We conclude that E exerts its stimulatory effect on the differentiation of dopaminergic neurons by coordinating BDNF expression. This particular E effect appears to be transmitted through Ca(2+)-dependent signaling cascades upon activation of putative membrane estrogen receptors.

Expression of AMH, SF1, and SOX9 in gonads of genetic female chickens during sex reversal induced by an aromatase inhibitor.

Aromatase inhibitors administered prior to histological signs of gonadal sex differentiation can induce sex reversal of genetic female chickens. Under the effects of Fadrozole (CGS 16949A), a nonsteroidal aromatase inhibitor, the right gonad generally becomes a testis, and the left gonad a testis or an ovotestis. We have compared the expression pattern of the genes encoding AMH (the anti-Müllerian hormone), SF1 (steroidogenic factor 1), and SOX9 (a transcription factor related to SRY) in these sex-reversed gonads with that in control testes and ovaries, using in situ hybridization with riboprobes on gonadal sections. In control males, the three genes are expressed in Sertoli cells of testicular cords; however, only SOX9 is male specific, since as observed previously AMH and SF1 but not SOX9 are expressed in the control female gonads. In addition to testicular-like cords, sex-reversed gonads present many lacunae with a composite, thick and flat epithelium. We show that during embryonic and postnatal development, AMH, SF1 and SOX9 are expressed in the epithelium of testicular-like cords and in the thickened part but not in the flattened part of the epithelium of composite lacunae. AMH and SF1 but not SOX9 are expressed in follicular cells of ovotestes. Coexpression of the three genes, of which SOX9 is a specific Sertoli-cell marker, provides strong evidence for the transdifferentiation of ovarian into testicular epithelium in gonads of female chickens treated with Fadrozole.

In Vitro Effects of Estradiol and Aromatase Inhibitor Treatment on Sex Differentiation in Xenopus Laevis Gonads

Gonads, still in the indifferent stage, were taken from tadpoles of Xenopus laevis and maintained in organ culture. These gonads were kept in good condition by frequent replacement of the culture medium and then underwent sexual differentiation. When the indifferent gonads were cultured in estradiol for 14 days, 90% of the explants showed female histological characteristics. The estradiol treatment of gonads in vitro gave results identical to those obtained from tadpoles treated in vivo. The gonads of X. laevis were successfully maintained for 14 days in vitro in a medium containing 20 μg/ml aromatase inhibitor (CGS 16949A). All the gonads treated with aromatase inhibitor showed the histological characteristics of the male phenotype. These results suggest that estradiol is important for ovarian differentiation in X. laevis.

Effects of Embryonic Treatment with Fadrozole on Phenotype of Gonads, Syrinx, and Neural Song System in Zebra Finches

Previous studies have found that treatment of zebra finch embryos with an aromatase inhibitor on Day 5 or 8 of incubation caused partial sex reversal of gonadal phenotype in females. These females possessed both testicular and ovarian tissue, and the development of the neural circuit for song remained feminine. The present study attempted more complete gonadal reversal by treating zebra finch embryos earlier, on Day 3 of incubation, with Fadrozole (CGS 16949A), an aromatase inhibitor, or with saline. We examined the phenotype of the syrinx (androgen-dependent vocal organ), the gonads, and the telencephallic neural song system in 100-day-old birds. Treated females typically possessed a left ovotestis and a right testis, and significantly larger syringes than control females. The histology and steroid synthetic enzyme activity of the testicular tissue in treated females were quite masculine and similar to that of control males. At the time of sacrifice, the plasma concentrations of testosterone and estradiol for fadrozole-treated females did not differ from those of control females, but dihydrotestosterone was lower in treated females. Despite the large amount of functional testicular tissue and a masculine syrinx, the volumes and soma sizes of song system nuclei (HVC, RA) in treated females remained feminine. These results suggest that testicular secretions masculinize the syrinx, but are not sufficient to masculinize the song system in zebra finches.

Gonadal development and growth of chickens and turkeys hatched from eggs injected with an aromatase inhibitor

It was the purpose of these experiments to describe gonadal development and posthatching growth of genetic female chickens and turkeys following in ovo injection of the aromatase inhibitor Fadrazole (CGS 16949A) prior to incubation. In ovo injection of Fadrazole (CGS 16949A) resulted in the development of testes-like gonads in the majority of day-old genetic female chickens and turkey poults. Ninety-eight to 99% of these birds have masculine-type male genitalia at 1 d of age. Microscopic examination of the gonads of day-old genetic female chicks hatched from Fadrazole-treated eggs showed the presence of atypical seminiferous tubules in 3 of 18 individuals and the presence of ovarian follicles in 3 of 18 individuals. No germinal elements were seen in 12 individuals. The gonads in the majority (8/11) of day-old female poults from treated eggs showed the presence of atypical seminiferous tubules. Three of 11 individuals had structures characterized as disorganized or degenerate follicles. Between the day of hatch and 6 wk, gonads in an increasing proportion of female chickens from Fadrazole-treated eggs had normal appearing ovarian follicles. A similar trend was seen in the female turkeys between hatch and 12 wk of age. There were no differences in BW of female chickens hatched from Fadrazole-treated eggs and those from control eggs between the day of hatch and 6 wk of age. The pectoral muscle mass and fat pad weights of these birds did not differ. In one experiment, the BW of female turkeys hatched from Fadrazole-treated eggs was significantly greater than that of controls and equal to that of males at 3 and 6 wk of age. Thereafter, both types of females were of equal weight and significantly lighter than males. Fadrazole treatment did not affect pectoral muscle mass of either sex of turkeys.

Hormonal Reversal and the Genetic Control of Sex Differentiation in Xenopus

Abstract Administration of exogenous estradiol between stages 50 and 52 completely feminized the developing gonads of Xenopus laevis. However, when tadpoles were injected or cultured during the critical period with an inhibitor (CGS 16949A) of aromatase that prevents synthesis of estradiol from androgen, there were no detectable effects on the sexual differentiation of the gonads. Aromatase transcription in Xenopus gonads was then studied by the reverse-transcription polymerase chain reaction (PCR) method. In embryos at the beginning of the estradiol-sensitive period (stages 49 and 50), expression of the aromatase gene was not detected in the gonad. These results show that the period between stages 50 and 52 is the time when Xenopus is sensitive to sex reversal by estradiol and critical for sex determination, although estradiol synthesis may not be naturally involved in the gonad at this step.

Effect of the potent aromatase inhibitor fadrozole hydrochloride (CGS 16949A) in postmenopausal women with breast carcinoma.

Fadrozole hydrochloride (CGS 16949A) is a highly potent, nonsteroidal aromatase inhibitor that significantly lowers estrogen levels in postmenopausal women and can be effective therapy for patients with advanced hormone-dependent breast carcinoma. Circulating estradiol, estrone, and estrone sulfate are reduced to undetectable levels within weeks of the initiation of therapy. Before this study, it was not known whether this decrease in serum estrogen levels results in altered parameters associated with cardiovascular disease. The authors examined the levels of several critical blood parameters that are important to cardiovascular risk for heart disease and thromboembolic disorders in patients treated with fadrozole. Cholesterol, triglyceride, low density lipoprotein (LDL), high density lipoprotein (HDL), very low density lipoprotein (VLDL), antithrombin III, protein C, protein S, and fibrinogen were serially measured in 21 postmenopausal women with advanced breast carcinoma treated with various doses of fadrozole (1.8 mg/day, n=3; 2.0 mg/day, n=13; 4.0 mg/day, n=5) over 3-24 months (mean, 15.8 months). A repeated measure analysis of variance was applied to each cardiovascular variable to assess changes in the response over time. Analyses were performed separately for each dose group and were also pooled over the dose groups. There was no statistically significant change over time in lipid parameters, namely, total cholesterol (P=0.57), triglyceride (P=0.27), LDL (P=0.99), HDL (P=0.30), and VLDL (P=0.43), over the 24 months of therapy. There were also no significant changes in coagulation factors, namely, antithrombin III (P=0.41), protein C (P=0.49), or protein S (P=0.31), over the 24 months. However, an increase in fibrinogen that occurred over time did reach statistical significance (P=0.011). With the exception of acute phase reactant fibrinogen, this study did not identify an increase in parameters associated with cardiovascular disease in women treated with fadrozole, a potent aromatase inhibitor.

Effects of the Aromatase Inhibitor Fadrozole on Gonadal Development in Coho Salmon, Oncorhynchus kisut

The effects of the aromatase inhibitor fadrozole on oogenesis, spermatogenesis and gonadal development in coho salmon Oncorhynchus kisutch were investigated. Both fe. males and males were implanted intraperitoneally with single silicone pellets containing the aromatase inhibitor fadrozole (CGS 16949A) at dosages of 10 mg•kg·1, 100 mg•kg·1 and 500 mg• kg·1, respectively. After 44 d, histological examination showed that females treated at dosages of 100 mg•kg·1 and 500 mg•kg· 1 had gonads with atretic oocytes. Conversely, control females had ovaries filled with synchronously developing oocytes. Both average ova­rian cross-sectional areas and average oocyte diameters in the treated groups were smaller than those in the control. It is suggested that the aromatase inhibitor fadrozole affected oo­genesis and ovarian development in coho salmon. The results did not show any significant differences in spermatogenesis and testicular development between treated and control males, except for the males which received a dosage of 100 mg·kg·1, a small number of which had testes which were further developed than those in the control.

Synthesis of 14C-labelled CGS 16949A (Fadrozole HCl), a potent aromatase inhibitor

CGS 16949A (Fadrozole HCl) is an aromatase inhibitor currently used in Japan for the treatment of estrogen-dependent cancer. A 14C-labelled form of this compound was synthesized for metabolism and pharmacokinetic studies. The synthesis pathway includes six steps, which are shown in synthetic Scheme 2. Initially, 4-bromotoluene was reacted with [14C]CuCN to yield 4-tolunitrile-[14C]-cyano. The nitrile was brominated on the α carbon to give an intermediate that was used to N-alkylate a known imidazole analogue. The product was treated with thionyl chloride to convert a 3-hydroxy propyl moiety to its chloro analogue. Treatment with potassium butoxide gave [14C] fadrozole as the free base via ring closure. Hydrogen chloride gas was used to give the desired salt [14C]CGS 16949A. Using this procedure, [14C]CGS 16949A was prepared with 97% radiochemical purity. The overall radiochemical yield for the synthesis was 1% from [14C]KCN. © 1997 John Wiley & Sons, Ltd.

Mechanisms of the actions of aromatase inhibitors 4-hydroxyandrostenedione, fadrozole, and aminoglutethimide on aromatase in JEG-3 cell culture

Selective inhibition of estrogen production with aromatase inhibitors has been found to be an effective strategy for breast cancer treatment. Most studies have focused on inhibitor screening and in vitro kinetic analysis of aromatase inhibition using placental microsomes. In order to determine the effects of different inhibitors on aromatase in the whole cell, we have utilized the human choriocarcinoma cell line, JEG-3 in culture to compare and study three classes of aromatase inhibitors, 4-hydroxyandrostenedione, fadrozole (CGS 16949A), and aminoglutethimide. Fadrozole is the most potent competitive inhibitor and aminoglutethimide is the least potent among the three. However, stimulation of aromatase activity was found to occur when JEG-3 cells were preincubated with aminoglutethimide. In contrast, 4-OHA and fadrozole caused sustained inhibition of aromatase activity in both JEG-3 cells and placental microsomes, which was not reversed even after the removal of the inhibitors. 4-OHA bound irreversibly to the active site of aromatase and caused inactivation of the enzyme which followed pseudo-first order kinetics. However, 4-OHA appears to be metabolized rapidly in JEG-3 cells. Sustained inhibition of aromatase induced by fadrozole occurs by a different mechanism. Although fadrozole bound tightly to aromatase at a site distinct from the steroid binding site, the inhibition of aromatase activity by fadrozole does not involve a reactive process. None of the inhibitors stimulated aromatase mRNA synthesis in JEG-3 cells during 8 h treatment. The stimulation of aromatase activity by AG appeared to be due to stabilization of aromatase protein. According to these results, 4-OHA and fadrozole would be expected to be more beneficial in the treatment of breast cancer patients than AG. The increase in aromatase activity by AG may counteract its therapeutic effect and might be partially responsible for relapse of breast cancer patients from this treatment.

A novel in vitro and in vivo breast cancer model for testing inhibitors of estrogen biosynthesis and its action using mammary tumor cells with an activated int-5/aromatase gene

We recently showed that the cellular gene int-5/aromatase in BALB/c mammary alveolar hyperplastic nodule (D2 HAN/D2 tumor cells) is activated as a result of mouse mammary tumor virus integration within the 3′ untranslated region of the aromatase gene. In the present study, we evaluated the effect of various aromatase inhibitors on androstenedione-mediated tumor cell growth. Also, we compared the effect of the non-steroidal aromatase inhibitor (CGS 16949A) on the inhibition of tumor growth. Our results show that D2 tumor cells respond well to various aromatase inhibitors and antiestrogens. We examined the usefulness of this model by using D2 tumor cells to simulate postmenopausal breast cancer employing both in vitro cell culture and in vivo ovariectomized (OVX) nude mouse. Unlike DMBA-induced tumors or other models, D2 tumor cells form very rapid tumors within a few days in intact mice or OVX nude mice with androstenedione supplementation and respond well to an aromatase inhibitor. This model with its known mechanism of aromatase activation should be useful for studying the role of intra-tumoral estrogen in mammary cancer, for evaluating the effects of aromatase inhibitors and antiestrogens, and for comparing breast cancer treatments.

Effect of Estrogen Deprivation on the Reproductive Physiology of Male and Female Primates

The availability of CGS 16949A, CGS 20267 and CGP 47645, a series of aromatase inhibitors (AIs) having high specific activity and specificity, made possible this study wherein the need for estrogen (E) for regulating (a) follicular maturation/ovulation, luteal function and pregnancy establishment, and (b) testicular function of the bonnet monkey ( Macaca radiata ) has been examined. Generally these compounds, used in the range of 500 μg to 2.5 mg/day did not inhibit follicular maturation although they did reduce E levels. Although low doses had no effect on ovulation it appears that relatively high doses of CGS 20267 and CGP 47645 could be inhibiting it. Three oral doses of letrozole (CGS 20267, each dose of 2 mg) during the follicular phase resulted in the formation of multiple follicles in cycling females, and these could be ovulated by exogenous hCG (1000 IU) treatment. Although administration of AI during the early luteal phase had no effect on progesterone (P) production, it prevented pregnancy establishment. Whereas AI administration in the female had no significant effect on luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels (except at high drug dosages), it significantly increased serum testosterone (T) levels in the male. Sustained high levels of T (30–50 ng/ml) could be maintained for 100 days by administering 2.5 mg of CGP 47465 orally once in 5 days. Blockade of E synthesis in the male led to the disruption of testicular germ cell transformation, which in turn resulted in a significant reduction in sperm production. These studies with aromatase inhibitors in the monkey suggest that these compounds have a potential for use as fertility regulating agents in both the male and female primate.

Effect of estrogen deprivation on the reproductive physiology of male and female primates

The availability of CGS 16949A, CGS 20267 and CGP 47645, a series of aromatase inhibitors (AIs) having high specific activity and specificity, made possible this study wherein the need for estrogen (E) for regulating (a) follicular maturation/ovulation, luteal function and pregnancy establishment, and (b) testicular function of the bonnet monkey (Macaca radiata) has been examined. Generally these compounds, used in the range of 500 μg to 2.5 mg/day did not inhibit follicular maturation although they did reduce E levels. Although low doses had no effect on ovulation it appears that relatively high doses of CGS 20267 and CGP 47645 could be inhibiting it. Three oral doses of letrozole (CGS 20267, each dose of 2 mg) during the follicular phase resulted in the formation of multiple follicles in cycling females, and these could be ovulated by exogenous hCG (1000 IU) treatment. Although administration of AI during the early luteal phase had no effect on progesterone (P) production, it prevented pregnancy establishment. Whereas AI administration in the female had no significant effect on luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels (except at high drug dosages), it significantly increased serum testosterone (T) levels in the male. Sustained high levels of T (30–50 ng/ml) could be maintained for 100 days by administering 2.5 mg of CGP 47465 orally once in 5 days. Blockade of E synthesis in the male led to the disruption of testicular germ cell transformation, which in turn resulted in a significant reduction in sperm production. These studies with aromatase inhibitors in the monkey suggest that these compounds have a potential for use as fertility regulating agents in both the male and female primate.

Fourteen-day oral combination dose toxicity study of CGS 16949 A (aromatase inhibitor) with 5-fluorouracil or tamoxifen in rats.

CGS 16949A, an aromatase inhibitor, was administered orally to female rats at doses of 1 and 10 mg/kg/day alone and in combination with tamoxifen (0.5 or 5 mg/kg/day) or 5-fluorouracil (20 mg/kg/day) for 14 days. CGS 16949A and tamoxifen combination: Increased food intake and body weight noted after CGS 16949A treatment were also observed following combination treatment, though to a lesser degree. Most of the clinical pathological features noted following combination treatment were similar to those induced by single compound treatment. Gross pathological and histopathological changes ascribed to the antiestrogenic action of CGS 16949A, such as increased ovarian weight, decreased uterine weight, cystic follicles and atrophied uterus and vaginal epithelium, were alleviated by combination treatment, and were comparable in severity to those caused by tamoxifen alone. No severe toxic changes were induced by combination treatment. CGS 16949A and 5-fluorouracil combination: Increased body weight noted after CGS 16949A treatment was also observed following combination treatment, though to a lesser degree. Most of the changes caused by single compound treatment, including the aforementioned effects of CGS 16949A on the genital organs, were also noted following combination treatment. There was no evidence of enhancement of the effects by combination treatment.

Are ovine Leydig cells able to aromatize androgens?

The conversion of testosterone into oestradiol by ovine Leydig cells cultured in vitro was studied using the non-steroidal aromatase inhibitor CGS 16949A. Additionally, aromatase activity was detected by immunohistochemical staining of cultured Leydig cells or cryosections. The cells were obtained from testes of Polish Mountain rams 5-6 months old (immature) or 12-15 months old (mature). Leydig cells were cultured alone (controls) or incubated for 6 h in the presence of testosterone. Aromatase inhibitor was then added to the cultures which were incubated for a further 18 h. After a 24-h incubation period, testosterone and oestradiol secretion were determined by testing the culture medium using radioimmunological methods. The addition of testosterone to the culture medium enhanced oestradiol synthesis, suggesting that exogenous testosterone could also be aromatized to oestradiol by ovine Leydig cells in vitro. In the presence of CGS 16949A, the conversion of testosterone to oestradiol was significantly suppressed in a dose-dependent manner. All Leydig cells obtained from testes of mature rams and stained immunohistochemically were positive for aromatase, whereas Leydig cells from immature males were negative. The localization of immunoreactive aromatase appeared to be dependent on the age of the donor ram. It is suggested therefore, that mature Leydig cells in the ram are not only the site for testosterone synthesis, they are also capable of converting androgens into oestrogens.