Inhibition Of Steroidogenic Enzymes Research Articles

Differences in the spectrum of steroidogenic enzyme inhibition between Osilodrostat and Metyrapone in ACTH-dependent Cushing syndrome patients.

Osilodrostat is a new 11β-hydroxylase inhibitor with a mode of action analogous to Metyrapone. The objective of this study was to compare steroidogenic profiles in patients treated with either Osilodrostat or Metyrapone for adrenocorticotrophic hormone (ACTH)-dependent Cushing's syndrome (CS). Patients followed up at Cochin hospital Endocrinology department between March 2019 and December 2021 for an ACTH-dependent CS, controlled by either Osilodrostat or Metyrapone, were included. A serum profile of five steroids (cortisol, 11-deoxycortisol, 17-hydroxyprogesterone, androstenedione and testosterone) was determined using UPLC- tandem mass spectrometry (UPLC-MS/MS). Nineteen patients treated with Osilodrostat, eight patients treated with Metyrapone and six patients treated with consecutive Metyrapone then Osilodrostat were included. Hypocortisolism (basal cortisol <100 nmol/L) was found in 48% of patients treated with Osilodrostat and 7% of patients treated with Metyrapone. 11-deoxycortisol and androstenedione levels were higher in patients treated with Metyrapone (80.9 (2.2-688.4) and 14.9 (2.5-54.3) nmol/L, respectively) than in patients treated with Osilodrostat (10.3 (0.5-71.9) and 4.0 (0.3-13.3) nmol/L) (P = 0.0009 and P = 0.0005). Testosterone level in women was also higher in Metyrapone group (3.3 (0.93-4.82) nmol/L vs 1.31(0.13-5.09) nmol/L, P = 0.0146). CYP11B1 activity (11-deoxycortisol/cortisol) was not significantly different between the two groups. CYP21A2 activity (17OHprogesterone/11-deoxycortisol) and CYP17A1 activity (17OHprogesterone/androstenedione) were significantly decreased in Osilodrostat group (P < 0.0001). In patients with ACTH-dependent CS, the use of CYP11B1 inhibitors in routine care suggests that Osilodrostat has a less specific effect on the inhibition of steroidogenic enzymes than Metyrapone. This might explain a smaller increase in 11-deoxycortisol and androgen levels in patients treated with Osilodrostat.

Targeted Endocrine Therapy: Design, Synthesis, and Proof-of-Principle of 17β-Hydroxysteroid Dehydrogenase Type 2 Inhibitors in Bone Fracture Healing.

Current therapies of steroid hormone-dependent diseases predominantly alter steroid hormone concentrations (or their actions) in plasma, in target and nontarget tissues alike, rather than in target organs only. Targeted therapy through the inhibition of steroidogenic enzymes may pose an attractive alternative with much less side effects. Here, we describe the design of a nanomolar potent 17β-hydroxysteroid dehydrogenase type 2 (17β-HSD2) inhibitor (compound 15) and successful targeted intracrine therapy in a mouse bone fracture model. Blockade of 17β-HSD2 in bone is thought to increase intracellular estradiol (E2) and testosterone (T), which thereby inhibits bone resorption by osteoclasts and stimulates bone formation by osteoblasts, respectively. Administration of compound 15 in the mouse fracture model strongly increases the mechanical stability of the healing fractured bone because of a larger periosteal callus with newly formed bone without changing the plasma E2 and T concentrations. Steroidogenic 17β-HSD2 inhibition thus enables targeted intracrine therapy.

Regulation of prostate cancer by hormone-responsive G protein-coupled receptors.

Regulation of prostate cancer by androgen and androgen receptor (AR), and blockade of AR signaling by AR antagonists and steroidogenic enzyme inhibitors have been extensively studied. G protein-coupled receptors (GPCRs) are a family of membrane receptors that regulate almost all physiological processes. Nearly 40% of FDA-approved drugs in the market target GPCRs. A variety of GPCRs that mediate reproductive function have been demonstrated to be involved in the regulation of prostate cancer. These GPCRs include gonadotropin-releasing hormone receptor, luteinizing hormone receptor, follicle-stimulating hormone receptor, relaxin receptor, ghrelin receptor, and kisspeptin receptor. We highlight here GPCR regulation of prostate cancer by these GPCRs. Further therapeutic approaches targeting these GPCRs for the treatment of prostate cancer are summarized.

Endocrine Disruptor Compounds (EDC) Effects on Reproduction

A large number of industrial chemical products (paints, flame retardants, pharmaceuticals, pesticides) are suspected or proved to act as endocrine disruptor compounds (EDCs). In the present survey of the literature, the main issue is to consider how to classify individual products as EDC or not. In this perspective, the different mechanisms leading to endocrine disruption are described: either by direct interaction with hormone receptors leading to its stimulation or inhibition, or by effect on endogenous hormone concentration through stimulation or inhibition of its synthesis or its degradation, or its binding to transport binding proteins. The present review takes particularly in consideration the effects of EDCs on the endocrine control of Reproduction in human and animals. Indeed, a number of EDCs can act as anti-androgens, anti-estrogens, and steroidogenic enzyme inhibitors that interfere with steroid action or production and thus can alter reproductive health.

Blockage of androgen and administration of estrogen induce transdifferentiation of testis into ovary.

Induction of sex reversal of XY fish has been restricted to the sex undifferentiated period. In the present study, differentiated XY tilapia were treated with trilostane (TR), metopirone (MN) and glycyrrhetinic acid (GA) (inhibitor of 3β-HSD, Cyp11b2 and 11β-HSD, respectively) alone or in combination with 17β-estradiol (E2) from 30 to 90 dah (days after hatching). At 180 dah, E2 alone resulted in 8.3%, and TR, MN and GA alone resulted in no secondary sex reversal (SSR), whereas TR + E2, MN + E2 and GA + E2 resulted in 88.3, 60.0 and 46.7% of SSR, respectively. This sex reversal could be rescued by simultaneous administration of 11-ketotestosterone (11-KT). Compared with the control XY fish, decreased serum 11-KT and increased E2 level were detected in SSR fish. Immunohistochemistry analyses revealed that Cyp19a1a, Cyp11b2 and Dmrt1 were expressed in the gonads of GA + E2, MN + E2 and TR + E2 SSR XY fish at 90 dah, but only Cyp19a1a was expressed at 180 dah. When the treatment was applied from 60 to 120 dah, TR + E2 resulted in 3.3% of SSR, MN + E2 and GA + E2 resulted in no SSR. These results demonstrated that once 11-KT was synthesized, it could antagonize E2-induced male-to-female SSR, which could be abolished by simultaneous treatment with the inhibitor of steroidogenic enzymes. The upper the enzyme was located in the steroidogenic pathway, the higher SSR rate was achieved when it was inhibited as some of the precursors, such as androstenedione, testosterone and 5α-dihydrotestosterone, could act as androgens. These results highlight the key role of androgen in male sex maintenance.

A novel framework for interpretation of data from the fish short-term reproduction assay (FSTRA) for the detection of endocrine-disrupting chemicals.

The fish short-term reproduction assay (FSTRA) is a key component of the US Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP), which uses a weight-of-evidence analysis based on data from several assays to identify the potential for chemicals to act as agonists or antagonists of the estrogen or androgen receptors (ER and AR), or inhibitors of steroidogenic enzymes. The FSTRA considers a variety of mechanistic and apical responses in 21-d exposures with the fathead minnow (Pimephales promelas), including plasma steroid and vitellogenin (VTG; egg yolk protein) concentrations, secondary sex characteristics, gonad size and histopathology, and egg production. Although the FSTRA initially was described several years ago, recent data generation associated with implementation of the EDSP highlighted the need for more formal guidance regarding evaluation of information from the assay. The authors describe a framework for interpretation of FSTRA data relative to perturbation of endocrine pathways of concern to the EDSP. The framework considers end points individually and as suites of physiologically related responses relative to pathway identification. Sometimes changes in single end points can be highly diagnostic (e.g., induction of VTG in males by ER agonists, production of male secondary sex characteristics in females by AR agonists); in other instances, however, multiple, related end points are needed to reliably assess pathway perturbation (e.g., AR antagonism, steroid synthesis inhibition). In addition to describing an interpretive framework, the authors demonstrate its practical utility using publicly available FSTRA data for a wide range of known and hypothesized endocrine-disrupting chemicals. Environ Toxicol Chem 2014;33:2529-2540. Published 2014 Wiley Periodicals Inc., on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Steroid synthesis by Taenia crassiceps WFU cysticerci is regulated by enzyme inhibitors

Cysticerci and tapeworms from Taenia crassiceps WFU, ORF and Taenia solium synthesize sex-steroid hormones in vitro. Corticosteroids increase the 17β-estradiol synthesis by T. crassiceps cysticerci. T. crassiceps WFU cysticerci synthesize corticosteroids, mainly 11-deoxycorticosterone (DOC). The aim of this work was to investigate whether classical steroidogenic inhibitors modify the capacity of T. crassiceps WFU cysticerci to synthesize corticosteroids and sex steroid hormones. For this purpose, T. crassiceps WFU cysticerci were obtained from the abdominal cavity of mice, pre-cultured for 24h in DMEM+antibiotics/antimycotics and cultured in the presence of tritiated progesterone (3H-P4), androstendione (3H-A4), or dehydroepiandrosterone (3H-DHEA) plus different doses of the corresponding inhibitors, for different periods. Blanks with the culture media adding the tritiated precursors were simultaneously incubated. At the end of the incubation period, parasites were separated and media extracted with ether. The resulting steroids were separated by thin layer chromatography (TLC). Data were expressed as percent transformation of the tritiated precursors. Results showed that after 2h of exposure of the cysticerci to 100μM formestane, the 3H-17β-estradiol synthesis from tritiated androstenedione was significantly inhibited. The incubation of cysticerci in the presence of 3H-DHEA and danazol (100nM) resulted in 3H-androstenediol accumulation and a significant reduction of the 17β-estradiol synthesis. The cysticerci 3H-DOC synthesis was significantly inhibited when the parasites were cultured in the presence of different ketoconazole dosis. The drug treatments did not affect parasite’s viability. The results of this study showed that corticosteroid and sex steroid synthesis in T. crassiceps WFU cysticerci can be modified by steroidogenic enzyme inhibitors. As was shown previously by our laboratory and others, parasite survival and development depends on sex steroids, therefore the inhibition of their synthesis is a good starting point exploited in situations where the inhibition of steroidogenesis could help to control the infection for the development of new treatments, or replacement of the usual therapy in resistant parasite infections. We raise the possibility that these drug actions may be beneficially.

Abstract 3467: Identification of androgen receptor splice variants, ESR1, CYP17, and CYP19 in human breast cancer.

Abstract Many advances have been made in the diagnosis and treatment of breast cancer in recent years, but it still remains one of the leading causes of death in women. It is well known that the growth of breast cells is regulated by the interactions of different steroid hormones, in particular androgens and estrogens that are related due to their connected metabolic pathways. A number of novel inhibitors of steroidogenic enzymes have been developed that target pre-receptor events_those pertaining to the production, transport, and conversion of steroid ligands. Critical pre-receptor steps include the conversion of pregnenolone-like steroids into androgens, mediated largely by the 17α-hydroxylase/lyase (CYP17) enzyme complex, and the conversion of testosterone and androstenedione to estradiol and estrone, mediated by aromatase (CYP19). Both conversions are implicated in the emergence of tumor resistance and thus are targets for intervention. Androgen Receptor (AR) is the sex hormone receptor most frequently found in both primary and secondary breast tumors, which is indicitative of the importance of AR in regulating the growth of breast cancer cells. It is estimated that 90% of human genes undergo alternative splicing and AR is no exception. Alternative splicing of the AR could culminate in a receptor that is capable of translocation, or can bind DNA without ligand, leading current AR therapies to be less efficacious. Using TaqMan qRT-PCR we examined 213 female breast-cancer FFPET samples, 80 ER- PR- Her2- samples, 68 ER- PR- Her2+ samples, and 64 ER+ PR+ Her2- samples, as well as 8 breast-cancer cell lines for the presence of ESR1, CYP17, CYP19, full length AR and AR splice variants ARV1, ARV3/V7, ARV567, and Delta3AR. ARV3/V7 and Delta3AR were the most prevalent variants in the ER+ PR+ Her2- and ER- PR- Her2+ sample sets, with &amp;gt;85% of these samples showing expression of either or both of these variants. On the other hand, ARV1, ARV3/V7, and ARV567 were the most prevalent variants in the ER- PR- Her2- sample set, with &amp;gt;90% of these samples showing expression of one or a combination of these variants. Lower expression values of most of the AR variants were observed in higher grade ER+ PR+ Her2- and ER- PR- Her2+ samples as compared to the lower grade samples. CYP19 was highly prevalent in all sample sets with &amp;gt;75% of all samples showing expression, while CYP17 expression was observed in &amp;lt;30% of all the samples tested. Our findings show the relatively high expression of AR variants and CYP19 in breast cancer tissues, which may indicate their role in regulating the growth of these tumors. Hence increased expression of AR splice variants in breast cancer tumors may be an important biomarker of resistance and targets for AR related therapy. Citation Format: Gabriela Martinez, Dana Gaffney, Katherine Bell, Suso Platero, Deborah Ricci, Weimin Li, Jayaprakash D. Karkera. Identification of androgen receptor splice variants, ESR1, CYP17, and CYP19 in human breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3467. doi:10.1158/1538-7445.AM2013-3467

Selective antitumour activity and ERα molecular docking studies of newly synthesizedd-homo fused steroidal tetrazoles

Here we report a convenient “click” synthesis for D-homo fused steroidal tetrazoles 11–14 from androstane and estratriene 16,17-seco-16-nitrile-17-mesyloxy derivatives 5–8, via intramolecular 1,3-dipolar cycloaddition from in situ generated 16,17-seco-17-azido-16-nitriles 5a–8a. Products were validated by 1H/13C-NMR, IR, HRMS, and structurally characterized by X-ray crystallography and computational methods. Compounds were evaluated as potential anti-proliferative agents against a panel of human cancer cell lines. D-Homo fused steroidal tetrazoles 13 and 14 appear to have specific, selective anti-proliferative effects against estrogen receptor positive (ER+) breast adenocarcinoma cells, which correlate with binding energies calculated from molecular docking to the estrogen receptor α-ligand binding domain (ERα-LBD). Moreover, molecular docking suggests that D-ring fused steroidal tetrazoles 13 and 14 could bind to ERα-LBD in a manner similar to known anti-estrogenic compounds. Addition of a D-homo fused tetrazole group appears to structurally mimic the hydrogen bonding potential of β-estradiol, suggesting their general utility in designing novel steroidal tetrazole derivatives as anti-estrogens or inhibitors of steroidogenic enzymes.

Exposure to leachate from municipal battery recycling site: implication as key inhibitor of steroidogenic enzymes and risk factor of prostate damage in rats

Few or no studies have measured the effect of short- and long-term exposure to industrial leachate. Mature male Wistar strain albino rats (175-220 g) underwent sub-chronic exposure to leachate from the Elewi Odo municipal battery recycling site (EOMABRL) via oral administration for a period of 60 days at different doses (20%, 40%, 60%, 80%, and 100%) per kilogram of body weight to evaluate the toxic effects of the leachate on male reproductive function using steroidogenic enzymes and biomarkers of prostate damage. Control groups were treated equally but were given distilled water instead of the leachate. After the treatment periods, results showed that the treatment induced systemic toxicity at the doses tested by causing a significant (p<0.05) loss in absolute body weight and decline in growth rate. There was a marked significant decrease (p<0.05) in testicular activities of Δ(5)-3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase. Conversely, the activity of prostatic acid phosphatase, a key marker enzyme for prostrate damage was significantly (p<0.05) elevated in the treated rats. Similarly, the administration of EOMABRL significantly (p<0.05) exacerbated the activity of total acid phosphatase with concomitant increase in the activity of prostatic alkaline phosphatase. These findings conclude that exposure to leachate from a battery recycling site induces sub-chronic testicular toxicity by inhibiting key steroidogenic enzymes and activating key markers linked with prostate damage/cancer in rats.

Steroidal and gonadal effects on neural cell proliferation in vitro in an adult songbird

Neurogenesis in the adult songbird brain occurs along the ventricular zone (VZ), a specialized cell layer surrounding the lateral ventricles. To examine the acute effects of sex steroids on VZ cell proliferation, male and female adult zebra finch brain slices containing the VZ were exposed to 5-bromo-2′-deoxyuridine-5′-monophosphate (BrdU) in vitro. Slices from one hemisphere served as the control, while contralateral slices were treated with steroids, steroidogenic enzyme inhibitors or gonadal tissue itself. There were no significant effects on VZ cell proliferation in either sexes by acute exposure to 17β-estradiol (E2), dihydrotestosterone (DHT), a cocktail of four sex steroids, and inhibitors of sex steroid synthesis (aminoglutethimide, ketoconazole, and fadrozole), or by activation of a mitochondrial cholesterol transporter. By contrast, dehydroepiandrosterone (DHEA) suppressed VZ cell proliferation in males, but not females, replicating previous observations involving treatments with corticosterone and RU-486. This suggests that DHEA suppresses proliferation in males via a glucocorticoid receptor-related mechanism. These results suggest that neurosteroidogenesis per se has little effect on acute VZ cell proliferation. Co-incubation with an ovary of female, but not male, slices significantly increased VZ cell proliferation; testicular tissue had no impact on proliferation in males or females. This suggests a role for a non-steroidal ovarian factor on adult female VZ cell proliferation. We also have evidence that previously reported sex-differences in BrdU-labeling along the adult VZ (males>females) result from a more rapid loss of cells in females. Sex differences in steroid action and cell death along the VZ may contribute to the maintenance of the sexually dimorphic song system.

A validated and rapid high-performance liquid chromatography method for the quantification of conversion of radio-labelled sex steroids

The 17β-hydroxysteroid dehydrogenase enzymes modify the availability of potent sex steroids and have thus attracted interest in the study of several steroid-dependent pathologies including breast, endometrial and prostate cancers. An increased awareness of the importance of steroidogenic enzymes has brought forth a demand for efficient assays to study the effects of individual enzymes on steroid levels. Methods used for assessing steroid conversion are often laborious and frequently involve hazardous sample preparation steps. We developed and validated an optimised simple method for sample preparation of sex steroids using protein precipitation by the addition of zinc sulphate/sodium hydroxide. The interconversion of radio-labelled oestrogens and androgens was quantified using high-performance liquid chromatography separation of oestrone, oestradiol, androstenedione and testosterone followed by online radiometric flow scintillation analysis. The method, which can be applied for assessing, e.g., the efficacy of inhibitors of steroidogenic enzymes, was successfully used for evaluating oestrogenic interconversion in breast cancer cell lines MCF7 and T-47D.

Development of relevant assay system to identify steroidogenic enzyme inhibitors

Steroidogenesis in the adrenals, testes, and ovaries plays an important roles in hormonal homeostasis in vivo and is mediated by various enzymes, such as acid cholesterol esterase (CEase), neutral CEase, CYP11A, 3β-hydroxysteroid dehydrogenase (HSD), CYP21, CYP11B, CYP17, CYP19 and 17β-HSD. Compounds that are potent inhibitors of one or more steroidogenic enzymes can thus cause serious endocrine toxicity so that relevant assay systems for detecting such enzyme inhibition are useful for safety assessment. Methods already reported involve incubation of radiolabeled steroid precursors with enzyme sources followed by separation of products by TLC or other methods. However, it is unclear whether the systems in use are kinetically-optimized (i.e., reactions performed in the linear range in terms of both reaction time and substrate concentrations), so that they might not be applicable for appropriate inhibition studies. The purpose of this present study was to establish kinetically-optimized methods for rat adrenal, testis, and ovary tissues with radio-HPLC. After development of HPLC methods for separation of precursor substrates and derived metabolites, we investigated the time course and substrate concentration dependence of individual reactions, using radiolabeled substrates and enzyme sources (mitochondria-lysosomal, microsomal, and cytosolic fractions from rat adrenal, testis and ovary tissues). Linearity of metabolism was confirmed in terms of both reaction time and substrate concentrations, and we conclude that this approach can be utilized to assess the inhibition profiles of compounds impacting on steroidogenic enzyme activity.

Specific estradiol biosynthetic pathway in choriocarcinoma (JEG-3) cell line

Estradiol (E2) plays a crucial role in all reproduction processes. In the placenta, it is well recognized that E2 is synthesized from fetal dehydroepiandrosterone sulfate (DHEAS). However, there is some controversy about the biosynthetic pathway involved, some authors suggest that E2 is produced by aromatization of testosterone (T), while others suggest that E2 is produced by the conversion of estrone (E1) into E2 by type 1 17β-HSD, subsequent to the aromatization of 4-androstenedione (4-dione) into E1. In the present report, using the precursor [ 14C]DHEA, inhibitors of steroidogenic enzymes (chemical inhibitors and siRNA) and a choriocarcinoma (JEG-3) cell line that expresses all the enzymes necessary to transform DHEA into E2, we could determine the sequential steps and the specific steroidogenic enzymes involved in the transformation of DHEA into E2. Quantification of mRNA expression levels using real-time PCR, strongly suggests that type 1 3β-hydroxysteroid dehydrogenase (3β-HSD1), aromatase and type 1 17β-HSD (17β-HSD1) that are highly expressed in JEG-3 cells are the enzymes responsible for the transformation of DHEA into E2. Analysis of the intermediates produced in the absence and presence of 3β-HSD, aromatase and 17β-HSD1 inhibitors permits to determine the following sequential steps: DHEA is transformed into 4-dione by 3β-HSD1, then 4-dione is aromatized into E1 by aromatase and E1 is finally transformed into E2 by 17β-HSD1. Our data are clearly in favor of the pathway in which the step of aromatization precedes the step of reduction by 17β-HSD.

Castration-Resistant Prostate Cancer: Locking Up the Molecular Escape Routes

The understanding of the key role that androgens play on the normal and pathological physiology of the prostate guided the development of different therapies for the treatment of locally advanced or metastatic prostate cancer (PCa). These so-called androgen deprivation therapies include surgical or chemical castration, achieved by the administration of gonadotropin-releasing hormone analogs; inhibition of steroidogenic enzymes; and finally, blocking of the binding of androgens to their receptor (AR) by the use of antiandrogens. Despite an excellent initial response, in approximately 2 to 3 years, most of these patients will succumb to the castration resistant form of the disease. Remarkably, even in the presence of castration levels of circulating androgens, these tumors are still dependent on a functional AR, and several molecular mechanisms have been proposed to explain this phenomenon. These include: (1) gene amplification and increased expression of the AR mRNA and protein, (2) selection of mutations in the AR that confer broader ligand specificity, (3) changes in the ratios or expression between the AR and its coregulators, (4) increased expression of steroidogenic enzymes, and (5) up-regulation of cross-talk signal transduction pathways that can activate the AR in a ligand-independent manner. We will summarize how these molecular hypotheses are being tested in the clinic by the latest therapeutic modalities.

Design and validation of specific inhibitors of 17 -hydroxysteroid dehydrogenases for therapeutic application in breast and prostate cancer, and in endometriosis

17beta-Hydroxysteroid dehydrogenases (17beta-HSDs) are enzymes that are responsible for reduction or oxidation of hormones, fatty acids and bile acids in vivo, regulating the amount of the active form that is available to bind to its cognate receptor. All require NAD(P)(H) for activity. Fifteen 17beta-HSDs have been identified to date, and with one exception, 17beta-HSD type 5 (17beta-HSD5), an aldo-keto reductase, they are all short-chain dehydrogenases/reductases, although overall homology between the enzymes is low. Although named as 17beta-HSDs, reflecting the major redox activity at the 17beta-position of the steroid, the activities of these 15 enzymes vary, with several of the 17beta-HSDs able to reduce and/or oxidise multiple substrates at various positions. These activities are involved in the progression of a number of diseases, including those related to steroid metabolism. Despite the success of inhibitors of steroidogenic enzymes in the clinic, such as those of aromatase and steroid sulphatase, the development of inhibitors of 17beta-HSDs is at a relatively early stage, as at present none have yet reached clinical trials. However, many groups are now working on inhibitors specific for several of these enzymes for the treatment of steroid-dependent diseases, including breast and prostate cancer, and endometriosis, with demonstrable efficacy in in vivo disease models. In this review, the recent advances in the validation of these enzymes as targets for the treatment of these diseases, with emphasis on 17beta-HSD1, 3 and 5, the development of specific inhibitors, the models used for their evaluation, and their progress towards the clinic will be discussed.

Olanzapine counteracts stress-induced anxiety-like behavior in rats

Atypical antipsychotics, such as olanzapine, have been reported to display anxiolytic properties as shown in several preclinical and clinical studies. Furthermore, several experimental evidences have shown that olanzapine reduces fear and anxiety in activated anxiety-like behavior test such as Geller-Seifter test, ultrasonic vocalization test and stress-induced EtOH consumption. Here, we hypothesized that the anxiolytic action of olanzapine might be due to via an indirect activation of the gamma-amino butyric acid (GABA)-ergic system through 3alpha-hydroxy-5alpha-pregnan-20-one [allopregnanolone (ALLO)], a potent neuroactive steroid that positively modulates the benzodiazepine-gamma-aminobutyric acid type A (GABA(A))/benzodiazepine receptors complex. To address this question, we used a preclinical animal test to screen for novel anxiolytic compounds - the elevated plus-maze (EPM) - in basal condition and after 45 min restrain stress after acute or repeated (21 days) administration of olanzapine (0.5mg/kg, i.p.). In this condition, we therefore study the effect of the 5-alpha-reductase inhibitor finasteride (FIN) (50mg/kg) after co-administration with olanzapine. FIN is an inhibitor of steroidogenic enzymes which acts by inhibiting type II 5-alpha reductase, the enzyme that converts into 5-alpha-reduced metabolites like the GABA(A) positive neuroactive steroid ALLO. Results showed an anxiolytic effect of the acute, but not of the chronic, treatment with olanzapine only in stressed rats. This anxiolytic effect was counteracted by the co-administration with FIN. These evidences suggest that the anxiolytic effects of olanzapine might be due to possible action of olanzapine on steroid function via activation of GABA system.

Involvement of neurosteroids in the anxiolytic-like effects of AC-5216 in mice

AC-5216, a ligand for the translocator protein (18 kDa) (TSPO), previously called the peripheral benzodiazepine receptor (PBR), produces anxiolytic-like effects mediated by TSPO in animal models of anxiety. Since stimulation of TSPO is considered to promote the synthesis of neurosteroids, we investigated the possible role of endogenous neurosteroids that positively act on the GABA A receptor in the anxiolytic-like effects of AC-5216. In our experiments, the effects of trilostane and finasteride, two inhibitors of steroidogenic enzymes, and picrotoxin, a GABA A receptor-gated Cl − channel blocker, on the anxiolytic-like effects of AC-5216 were examined in the social interaction test in mice. Also, the anxiolytic-like effects of allopregnanolone and progesterone were examined. The anxiolytic-like effects of AC-5216 (0.1 mg/kg, p.o.) were inhibited by trilostane (10–30 mg/kg, s.c.), finasteride (10–30 mg/kg, s.c.), and picrotoxin (0.03–0.3 mg/kg, s.c.), while those of diazepam (0.1 mg/kg, p.o.) were inhibited by picrotoxin only. The anxiolytic-like effects of progesterone (1–3 mg/kg, s.c.) were inhibited by finasteride (3–30 mg/kg) and picrotoxin (0.1–0.3 mg/kg), although those of allopregnanolone (10 mg/kg, s.c.) were inhibited by picrotoxin only. These results demonstrate that the anxiolytic-like effects of AC-5216 are due to newly synthesized neurosteroids that enhance GABA A receptor function.

UPDATE ON THE MEDICAL TREATMENT OF ENDOMETRIOSIS

The treatment of women with endometriosis can be a challenge. Therapeutic strategies must be tailored to the individual symptoms, age, and desire for fertility. Medical therapy continues to be based on endocrine treatment, such as oral contraceptives, progestins, danazol, and GnRH agonists. Unfortunately, recurrence rates are high after discontinuation of therapy. Recent clinical research on GnRH analogues plus add-back therapy has produced favorable results. Long-term treatment of patients using this approach has successfully reduced pain while minimizing symptoms of hypoestrogenism and adverse metabolic effects, such as loss of bone mineral density. Currently, GnRH analogues given with add-back therapy seems to be the most effective long-term approach to the treatment of symptomatic endometriosis. In the future, other modalities, such as medicated vaginal rings, inhibitors of steroidogenic enzymes, and GnRH antagonists, will most likely be options.

Developmental Synergism of Steroidal Estrogens in Sex Determination

Gonadal sex in the red-eared slider turtle, Trachemys scripta, is determined by incubation temperature during embryonic development. Evidence suggests that temperature determines sex by influencing steroid hormone metabolism and/or sensitivity: steroidogenic enzyme inhibitors or exogenous sex steroid hormones and their man-made analogs override (or enhance) temperature effects on sex determination. Specifically, nonaromatizable androgens and aromatase inhibitors induce testis differentiation at female-producing temperatures, whereas aromatizable androgens and estrogens induce ovary differentiation at male-producing temperatures. Moreover, natural estrogens and temperature synergize to produce more females than would be expected if estrogens and temperature had purely additive effects on sex determination. In this study, we use sex reversal of turtle embryos incubated at a male-producing temperature to examine synergism among steroidal estrogens: estrone, 17ss-estradiol, and estriol. A low dose of 17ss-estradiol (200 ng) showed significant synergism when administered with a single low dose of estriol (10 ng). Likewise, a single low dose of estrone (250 ng) had a synergistic effect when combined with the same low dose of estriol (10 ng). We conclude that the weak natural estrogens estrone and 17ss-estradiol synergize with a low dose of the more potent estriol to reverse gonadal sex during the critical period of sexual differentiation. These results suggest that weak environmental estrogens may also synergize with stronger natural estrogens.