Behavioral and steroidogenic pharmacology of phenyl ring substituted etomidate analogs in rats.

BACKGROUND: Cushing's syndrome is an endocrine disorder characterized by the overproduction of adrenocortical steroids. Steroidogenesis enzyme inhibitors are the mainstays of pharmacological treatment. Unfortunately, they produce significant side effects. Among the most potent inhibitors is the general anesthetic etomidate whose GABAA receptor-mediated sedative-hypnotic actions restrict use. In this study, we defined the sedative-hypnotic and steroidogenesis inhibiting actions of etomidate and four phenyl-ring substituted etomidate analogs (dimethoxy-etomidate, isopropoxy-etomidate, naphthalene-etomidate, and naphthalene (2)-etomidate) that possess negligible GABAA receptor modulatory activities. METHODS: In the first set of experiments, male Sprague-Dawley rats were assessed for loss of righting reflexes (LoRR) after receiving intravenous boluses of either etomidate (1 mg/kg) or an etomidate analog (40 mg/kg). In the second set of experiments, rats were assessed for LoRR and their abilities to produce adrenocortical and androgenic steroids after receiving 2-h infusions (0.5 mg kg- 1 min- 1) of either etomidate or an etomidate analog. RESULTS: All rats that received etomidate boluses or infusions had LoRR that persisted for minutes or hours, respectively. In contrast, no rat that received an etomidate analog had LoRR. Compared to rats in the vehicle control group, rats that received etomidate analog infusions had plasma corticosterone and aldosterone concentrations that were reduced by 80-84% and 68-94%, respectively. Rats that received etomidate infusions had plasma corticosterone and aldosterone concentrations that were also significantly reduced (by 92 and 96%, respectively). Rats that received etomidate or isopropoxy-etomidate had significant reductions (90 and 57%, respectively) in plasma testosterone concentrations whereas those that received naphthalene-etomidate had significant increases (1400%) in plasma dehydroepiandrosterone concentrations. Neither etomidate nor any etomidate analog significantly affected plasma androstenedione and dihydrotestosterone concentrations. CONCLUSIONS: Our studies demonstrate that the four phenyl-ring substituted etomidate analogs form a novel class of compounds that are devoid of sedative-hypnotic activities and suppress plasma concentrations of adrenocortical steroids but vary in their effects on plasma concentrations of androgenic steroids.

Repurposing steroidogenesis inhibitors for the therapy of neuropsychiatric disorders: Promises and caveats.

Steroids exert a profound influence on behavioral reactivity, by modulating the functions of most neurotransmitters and shaping the impact of stress and sex-related variables on neural processes. This background - as well as the observation that most neuroactive steroids (including sex hormones, glucocorticoids and neurosteroids) are synthetized and metabolized by overlapping enzymatic machineries - points to steroidogenic pathways as a powerful source of targets for neuropsychiatric disorders. Inhibitors of steroidogenic enzymes have been developed and approved for a broad range of genitourinary and endocrine dysfunctions, opening to new opportunities to repurpose these drugs for the treatment of mental problems. In line with this idea, preliminary clinical and preclinical results from our group have shown that inhibitors of key steroidogenic enzymes, such as 5α-reductase and 17,20 desmolase-lyase, may have therapeutic efficacy in specific behavioral disorders associated with dopaminergic hyperfunction. While the lack of specificity of these effects raises potential concerns about endocrine adverse events, these initial findings suggest that steroidogenesis modulators with greater brain specificity may hold significant potential for the development of alternative therapies for psychiatric problems. This article is part of the Special Issue entitled 'Drug Repurposing: old molecules, new ways to fast track drug discovery and development for CNS disorders'.

Abiraterone Acetate for Cushing Syndrome: Study in a Canine Primary Adrenocortical Cell Culture Model.

Abiraterone acetate (AA) is a potent inhibitor of steroidogenic enzyme 17α-hydroxylase/17,20-lyase (CYP17A1). AA is approved for the treatment of prostate cancer but could also be used to treat patients with Cushing syndrome (CS). Similar to humans, canine glucocorticoid synthesis requires CYP17A1, providing a useful animal model. The objective of this study was to preclinically investigate the effect of AA on adrenocortical hormone production, cell viability, and mRNA expression of steroidogenic enzymes in canine primary adrenocortical cell cultures (n = 9) from the adrenal glands of nine healthy dogs. The cells were incubated with AA (0.125 nM to 10 µM) for 72 hours under basal conditions and with 100 nM ACTH(1-24). Adrenocortical hormone concentrations were measured in culture medium using liquid chromatography-mass spectrometry, RNA was isolated from cells for subsequent real-time quantitative PCR analysis, and cell viability was assessed with an alamarBlue™ assay. AA reduced cortisol (IC50, 21.4 ± 4.6 nM) without affecting aldosterone under basal and ACTH-stimulated conditions. AA increased progesterone under basal and ACTH-stimulated conditions but reduced corticosterone under basal conditions, suggesting concurrent inhibition of 21-hydroxylation. AA did not affect the mRNA expression of steroidogenic enzymes and did not inhibit cell viability. In summary, primary canine adrenocortical cell culture is a useful model system for drug testing. For the treatment of CS, AA may to be superior to other steroidogenesis inhibitors due to its low toxicity. For future in vivo studies, dogs with endogenous CS may provide a useful animal model.

Histamine H4 receptor as a novel therapeutic target for the treatment of Leydig-cell tumours in prepubertal boys.

Leydig-cell tumours (LCTs) are rare endocrine tumours of the testicular interstitium, with recent increased incidence. Symptoms include precocious puberty in children; and erectile dysfunction, infertility and/or gynaecomastia, in adults. So far, scientific evidence points to aromatase (CYP19) overexpression and excessive oestrogen and insulin-like growth factor (IGF) -1 production as responsible for Leydig-cell tumourigenesis. LCTs are usually benign; however, malignant LCTs respond poorly to chemo/radiotherapy, highlighting the need to identify novel targets for treatment. Herein, we investigated the potential role of the histamine receptor H4 (HRH4) as a therapeutic target for LCTs using R2C rat Leydig tumour cells, a well-documented in vitro model for Leydigioma. Also, we studied for the first time the expression of CYP19, IGF-1R, oestrogen receptor (ER) α, ERß, androgen receptor (AR) and HRH4 in human prepubertal LCTs versus normal prepubertal testes (NPTs). HRH4 agonist treatment inhibited steroidogenesis and proliferation in R2C cells and also negatively affected their pro-angiogenic capacity in vitro and in vivo, as assessed by evaluating the proliferative activity of human umbilical vein endothelial cells and by means of the quail chorioallantoic membrane assay, respectively. Moreover, E2 and IGF-1 inhibited HRH4 mRNA and protein levels. In human prepubertal LCTs, CYP19, IGF-1R, ERα and ERß were overexpressed compared with NPTs. In contrast, HRH4 staining was weak in LCTs, but moderate/strong and confined to the interstitium in NPTs. Importantly, HRH4 was absent or barely detectable in seminiferous tubules or germ cells. Overall, our results point to HRH4 as a novel therapeutic target in LCTs.

The optimal treatment of metastatic hormone-naive prostate cancer: abiraterone acetate or docetaxel?

AIM: To determine whether abiraterone acetate or docetaxel should be regarded as the current standard of care for metastatic hormone-naive prostate cancer (mHNPC). METHODS & MATERIALS: A network meta-analysis (NMA) using the frequentist approach and generalized pairwise modeling was computed. RESULTS: The results of this NMA favored abiraterone acetate over docetaxel-based regimens (hazard ratio: 0.79; 95% CI: 0.64-0.99) in patients with mHNPC. The results also suggest a reconsideration of the role of prednisone in view of the absence of a survival benefit (hazard ratio: 0.98; 95% CI: 0.59-1.65) with its use. CONCLUSION: Despite the paucity of direct comparative evidence, the results of this NMA favor the use of abiraterone acetate in the first-line treatment of mHNPC.

2-Phenylimidazo[1,2-a]pyridine-containing ligands of the 18-kDa translocator protein (TSPO) behave as agonists and antagonists of steroidogenesis in a mouse leydig tumor cell line.

Ligands of 18-kDa translocator protein (TSPO) are known for their ability to potently and dose-dependently stimulate steroid biosynthesis in steroidogenic cells. In this study, we investigated a number of 2-phenyl-imidazo[1,2-a]pyridine acetamide derivatives, analogs of alpidem, for their ability to bind TSPO and to affect steroidogenesis in a mouse Leydig tumor cell line. We observed that not only some compounds behaved as agonists, stimulating steroidogenesis (e.g., 3 and 4) with EC50 values (15.9 and 6.99µM) close to that determined for FGIN-1-27 used as positive control (7.24µM), but two compounds, namely 5 and 6, which on the other hand are the most lipophilic ones in the investigated series, behaved as antagonists, by significantly inhibiting steroid production at concentrations at least twenty times lower than the cytotoxic ones. To our surprise, the newly synthesized compound 3, which is a strict analog of alpidem bearing at the para position of the 2-phenyl group a methoxy group instead of chlorine, achieved a ten-fold stimulation of the steroid production (for comparison FGIN-1-27 achieved 1.6-fold stimulation). Within the limits of the examined property space, some unprecedented SARs were unveiled, which can help in understanding the key molecular factors underlying the transition from agonism to antagonism in the steroidogenesis process. Besides the substitution pattern and the physicochemical features (mainly hydrogen bonding potential) of the substituents at the positions C(6) and C(8) of the imidazo[1,2-a]pyridine nucleus, and at the para position of the 2-phenyl group, the structure-activity relationship analysis suggested lipophilicity, whose increase seems to be generally related to steroidogenesis inhibition, and steric hindrance, which appeared as a stimulation-limiting factor, as two main properties to control in the design or optimization of novel imidazo[1,2-a]pyridine-based TSPO ligands endowed with potential in modulating the steroidogenesis process.

The effect of estrogen synthesis inhibition on hippocampal memory.

17-Beta-estradiol (E2) facilitates long term-potentiation (LTP) and increases spine synapse density in hippocampal neurons of ovariectomized rodents. Consistent with these beneficial effects on the cellular level, E2 improves hippocampus-dependent memory. A prominent approach to study E2 effects in rodents is the inhibition of its synthesis by letrozole, which reduces LTPs and spine synapse density. In the current longitudinal functional magnetic resonance imaging (fMRI) study, we translated this approach to humans and compared the impact of E2 synthesis inhibition on memory performance and hippocampal activity in post-menopausal women taking letrozole (n = 21) to controls (n = 24). In particular, we employed various behavioral memory paradigms that allow the disentanglement of hippocampus-dependent and -independent memory. Consistent with the literature on rodents, E2 synthesis inhibition specifically impaired hippocampus-dependent memory, however, this did not apply to the same degree to all of the employed paradigms. On the neuronal level, E2 depletion tended to decrease hippocampal activity during encoding, whereas it increased activity in the anterior cingulate and the dorsolateral prefrontal cortex. We thus infer that the inhibition of E2 synthesis specifically impairs hippocampal functioning in humans, whereas the increased prefrontal activity presumably reflects a compensatory mechanism, which is already known from studies on cognitive aging and Alzheimer's disease.

Therapy of endocrine disease: steroidogenesis enzyme inhibitors in Cushing's syndrome.

Steroidogenesis enzyme inhibitors are the mainstay of medical therapy in Cushing's syndrome (CS). Ketoconazole (KTZ) and metyrapone are the most commonly used agents. Although there is considerable experience of their use in individual specialist centres, these drugs have not been rigorously tested in prospective clinical trials. Clinicians face uncertainties and concerns with respect to the safety profile of these agents, and best means to monitor effect. We review steroidogenesis inhibitors in the management of CS, including older agents (KTZ, metyrapone, etomidate and mitotane) and those currently under development (LCI699, non-racemic KTZ), and offer a practical approach for their use in clinical practice.

Long-term effects of traumatic stress on subsequent contextual fear conditioning in rats.

Exposure to stressful events affects subsequent sensitivity to fear. We investigated the long-term effects of a traumatic experience on subsequent contextual fear conditioning and anxiety-like behaviors in rats (Experiment 1). In addition, we tested whether the administration of the glucocorticoid synthesis inhibitor metyrapone (MET) attenuated the sensitization of fear induced by traumatic stress (Experiment 2). Male rats were subjected to a multiple stress (MS) session, which consisted of 4 foot shocks (1mA, 1s) and forced swimming for 20min, followed by exposure to a situational reminder 7days after the MS session. MET (25 or 100mg/kg, intraperitoneal) was administered 30min before MS. The contextual fear conditioning was performed 14days after MS. MS enhanced the conditioned fear response for at least 14days after the conditioning, and pretreatment with MET did not affect the enhancement of conditioned fear. These results suggest that glucocorticoid secretion triggered by MS is not involved in regulating the long-term stress-induced sensitization of fear.

Metyrapone blocks maternal food restriction-induced changes in female rat offspring lung development.

Maternal food restriction (MFR) during pregnancy affects pulmonary surfactant production in the intrauterine growth-restricted (IUGR) offspring through unknown mechanisms. Since pulmonary surfactant production is regulated by maternal and fetal corticosteroid levels, both known to be increased in IUGR pregnancies, we hypothesized that metyrapone (MTP), a glucocorticoid synthesis inhibitor, would block the effects of MFR on surfactant production in the offspring. Three groups of pregnant rat dams were used (1) control dams fed ad libitum; (2) MFR (50% reduction in calories) from days 10 to 22 of gestation; and (3) MFR + MTP in drinking water (0.5 mg/mL), days 11 to 22 of gestation. At 5 months, the MFR offspring weighed significantly more, had reduced alveolar number, increased septal thickness, and decreased surfactant protein and phospholipid synthesis. These MFR-induced effects were normalized by the antiglucocorticoid MTP, suggesting that the stress of MFR causes hypercorticoidism, altering lung structure and function in adulthood.