Inhibition Of Steroidogenesis Research Articles

Medical Management of Cushing's Syndrome: Current and Emerging Treatments.

Endogenous Cushing's syndrome is a chronic disease associated with increased morbidity and mortality if not appropriately treated. Recurrence and/or persistence of hypercortisolemia after surgical treatment, especially for Cushing's disease, are high, and long-term medical treatment is used to decrease cortisol levels and risk of metabolic comorbidities. Medical treatment is also often required while waiting for radiation effects to take place. In some cases, severe or life-threatening hypercortisolism must be urgently and medically treated, via intravenous medications or with combination therapy, before patients can undergo surgery. In the last decade, medical treatment has progressed from a few steroidogenesis inhibitors to three novel drug groups: new inhibitors for steroidogenic enzymes with possibly fewer side effects, pituitary-directed drugs that aim to inhibit the pathophysiological pathways of Cushing's disease, and glucocorticoid receptor antagonists that block cortisol's action. Understanding the pathophysiology of Cushing's syndrome has also led to the identification of potential targets that may decrease adrenocorticotrophic hormone and/or cortisol excess, and/or decrease tumor cell proliferation, and induce senescence or apoptosis. We provide here a review of current and near-future medical options to treat Cushing's syndrome, and discuss updates on clinical trials and the efficacy and safety of novel or in-development drugs, as well as future potential targets.

Osilodrostat Is a Potential Novel Steroidogenesis Inhibitor for the Treatment of Cushing Syndrome: An In Vitro Study.

Metyrapone and ketoconazole, frequently used steroidogenesis inhibitors for treatment of Cushing syndrome, can be associated with side effects and limited efficacy. Osilodrostat is a CYP11B1 and CYP11B2 inhibitor, with unknown effects on other steroidogenic enzymes. To compare the effects of osilodrostat, metyrapone, and ketoconazole on adrenal steroidogenesis, and pituitary adenoma cells in vitro. HAC15 cells, 17 primary human adrenocortical cell cultures, and pituitary adenoma cells were incubated with osilodrostat, metyrapone, or ketoconazole (0.01 to 10 µM). Cortisol and ACTH were measured using chemiluminescence immunoassays, and steroid profiles by liquid chromatography-mass spectrometry. In HAC15 cells, osilodrostat inhibited cortisol production more potently (IC50: 0.035 µM) than metyrapone (0.068 µM; P < 0.0001), and ketoconazole (0.621 µM; P < 0.0001). IC50 values of osilodrostat and metyrapone for basal cortisol production varied with a 25- and 18-fold difference, respectively, with comparable potency. Aldosterone production was inhibited more potently by osilodrostat vs metyrapone and ketoconazole. Osilodrostat and metyrapone treatment resulted in strong inhibition of corticosterone and cortisol, 11-deoxycortisol accumulation, and modest effects on adrenal androgens. No pituitary-directed effects of osilodrostat were observed. Under our study conditions, osilodrostat is a potent cortisol production inhibitor in human adrenocortical cells, comparable with metyrapone. All steroidogenesis inhibitors showed large variability in sensitivity between primary adrenocortical cultures. Osilodrostat might inhibit CYP11B1 and CYP11B2, in some conditions to a lesser extent CYP17A1 activity, and a proximal step in the steroidogenesis. Osilodrostat is a promising treatment option for Cushing syndrome, and in vivo differences with metyrapone are potentially driven by pharmacokinetic differences.

MON-461 Drug Repurposing to Explore Novel Treatment for Cushing Disease

Cushing Disease (CD), caused by adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas, leads to excess production of adrenal-derived cortisol. Current medical treatments for CD include adrenal steroidogenesis inhibitors (Ketoconazole, Metyrapone), the somatostatin receptor ligand Pasireotide, and the glucocorticoid receptor antagonist (Korlym). Although Pasireotide is tumor directed, its effect on tumor growth is modest, and it frequently causes hyperglycemia. Additionally, long-term compliance with these drugs is < 30% - either due to escape from control caused by increased tumor-derived ACTH or drug side effects. Clearly there is a large unmet need for improved medical treatments for CD. We hypothesize that an ideal drug would act on the tumor itself, potently and selectively inhibit corticotroph tumor-derived ACTH production to attain sustained eucorticolism and inhibit corticotroph tumor growth. Using our recently developed ACTH quantification assay (ACTH AlphaLISA), we screened an annotated kinase inhibitor library (KIL, n=430) at 10μM, 1μM and 100nM doses. The KIL contains inhibitors of PI3K/AKT/mTOR (n=95), protein tyrosine kinases (n=87), MAPK (n=45), angiogenesis (n=44), cell cycle (n=44), JAK/STAT (n=26), and others (n=89). Out of 430 compounds screened, 6, 20 and 115 compounds exhibited >50% ACTH AlphaLISA inhibition and 36, 105 and 263 compounds exhibited >50% nuclei inhibition in murine corticotroph tumor AtT20 cells at 100nM, 1μM and 10μM respectively. Three out of the 6 compounds that exhibited efficacy >50% at 100nM are currently being evaluated in Phase II clinical trials in a variety of solid tumors. Given the involvement of PI3K/AKT pathway in pituitary tumor pathogenesis and recent findings of aberrant USP8 mutant-associated EGFR activation in corticotroph tumors, we have thus far performed dose-response curves for one of the PI3K inhibitors using two separate assays of ACTH secretion (AlphaLISA and ELISA) and cell proliferation (Hoechst nuclei staining and CellTilter Glow assay). Both demonstrated comparable inhibition rates with an EC50 of 246nM for ACTH inhibition determined by AlphaLISA assay, and LC50 of 18 nM for cytotoxicity. Experiments to elucidate the mode of action of the PI3K inhibitor on ACTH synthesis and/or secretion, demonstrated it inhibited POMC mRNA expression by 40% at 300nM and 87% at 1μM (p<0.001), indicating the drug may block activation of POMC regulatory proteins such as the known PI3K targets AKT and mTOR. Given the already defined drug safety and efficacy profiles of many of these drugs, and their potent action at low doses to inhibit not only ACTH secretion but additionally corticotroph tumor cell proliferation, some may be suitable for drug repurposing studies to develop novel CD therapies.

MON-444 Defining Remission In Cushing Syndrome After Tumor Resection In Cases Of Non-suppressed Postoperative Serum Cortisol Levels

Background: Remission of Cushings syndrome (CS) is generally defined as morning serum cortisol (F) values <5mcg/dL or Urine Free Cortisol < 28 - 56 nmol/d (<10-20/ μg/d) after tumor resection, as normal pituitary corticotropes are suppressed by sustained hypercortisolism (1). In cases where eucortisolemia is achieved by pharmacologic therapy preoperatively, the corticotrope cells can recover their function leading to detectable postoperative cortisol levels not always indicative of unsuccessful tumor resection. Here we describe the use of the 1mg dexamethasone suppression test (DST) and night time serum cortisol as tools to establish criteria for remission in patients where normal pituitary corticotrophs may not have been suppressed. Methods: We retrospectively reviewed 19 patients (pts) with biochemical evidence of ACTH dependent CS who underwent tumor resection. All had measurable serum F measurements within post op day 7 and underwent a 1mg DST to determine if surgery was successful. Remission was defined as serum F value of <1.8 mcg/dl after DST. Recognizing that DST results can be affected by various factors such as concurrent medications or dexamethasone metabolism, in cases where post DST serum F was > 1.8mcg/dl, late night serum F levels were collected at 2330 and 2359. Definition of remission was established as late night F level < 7.5mcg/dl.Results: Of 19 pts, 11 had Ectopic ACTH Syndrome (EAS) and 8 had Cushing’s disease (CD). The median age was 54, 12/19 were female. 15 were deemed to be in surgical remission, 10 having passed the DST with F value <1.8 (range <1 - 5.4mcg/dl). The remaining 5 had night serum F values <7.5mcg/dl (range <1-5.9). 3/10 pts had DST results >2mcg/dl; one had a 2cm adrenal nodule secondary to CD, one had bilateral adrenal hyperplasia, and in the 3rd case a dexamethasone level was not measured. These 3 cases underwent 24 hour urine cortisol measurements, all found within the lower end of the reference range; 5mcg/24hr, 8mcg/24h and 5.2ug/24hr respectively. 12/15 had documented or reported history of cyclic CS and/or were treated with steroidogenesis inhibitors prior to surgery. 21% (4/19) pts were concluded to have persistent hypercortisolemia based on abnormal DST results (F range 4.7-10.8mcg/dl) with 2 also having abnormal night serum F (>20mcg/d). All had CD, and of these 2 had no tumor identified on pathology, one had persistent disease requiring a repeat pituitary surgery with subsequent cure and positive adenoma on pathology, and the 4th had residual disease. Conclusion: In cases of cyclic CS, or where medical management was used prior to surgical resection, normal corticotrophs may not be suppressed, resulting in measurable or normal serum cortisol values post operatively. We suggest the use of the 1 mg DST and/or night time serum cortisol samples to differentiate between persistent hypercortisolism and remission after successful tumor resection.

Physiological and drug-induced changes in blood levels of adrenal steroids and their precursors in cynomolgus monkeys: An application of steroid profiling by LC-MS/MS for evaluation of the adrenal toxicity.

The adrenal gland is the most common toxicological target of drugs within the endocrine system, and inhibition of adrenal steroidogenesis can be fatal in humans. However, methods to evaluate the adrenal toxicity are limited. The aim of the present study was to verify the usefulness of simultaneous measurement of blood levels of multiple adrenal steroids, including precursors, as a method to evaluate drug effects on adrenal steroidogenesis in cynomolgus monkeys. With this aim, physiological and drug-induced changes in blood levels of adrenal steroids, including cortisol, aldosterone, androgen, and their precursors were examined. First, for physiological changes, intraday and interday changes in blood steroid levels were examined in male and female cynomolgus monkeys. The animals showed circadian changes in steroid levels that are similar to those in humans, while interday changes were relatively small in males. Next, using males, changes in blood steroid levels induced by ketoconazole and metyrapone were examined, which suppress adrenal steroidogenesis via inhibition of CYP enzymes. Consistent with rats and humans, both ketoconazole and metyrapone increased the deoxycorticosterone and deoxycortisol levels, probably via CYP11B1 inhibition, and the increase was observed earlier and with greater dynamic range than the changes in cortisol level. Changes in other steroid levels reflecting the drug mechanisms were also observed. In conclusion, this study showed that in cynomolgus monkeys, simultaneous measurement of blood levels of adrenal steroids, including precursors, can be a valuable method to sensitively evaluate drug effects on adrenal steroidogenesis and to investigate the underlying mechanisms.

Androgens in Cushing's Syndrome.

Although polycystic ovary syndrome (PCOS) is the most common androgen excess disorder, screening for Cushing's Syndrome (CS) should be considered in women with PCOS phenotype, particularly if they are also affected by other disturbances that increase their pretest probability (e.g., osteoporosis/bone fractures). Approximately 70-80% of women with CS present menstrual abnormalities, and PCOS findings are found in 46% of these patients. Diagnostic efforts should strengthen if the clinical picture is severe or of rapid onset in order to ensure the earliest and most appropriate treatment. If the diagnosis of CS is challenging, its differentiation from PCOS is not outdone: isolated PCOS may be associated to hypothalamic-pituitary-adrenal axis disruption, leading to false-positive results in screening tests. Because of this overlap, the diagnosis of CS is initially missed or delayed. Diagnostic utility of serum androgen assessment is controversial, but the widespread use of high-performance liquid chromatography and gas chromatography-mass spectrometry for urinary steroid profiling is showing promising results. According to the role of adrenocorticotropic hormone (ACTH) in adrenal androgen secretion, it is not surprising that the levels of dehydroepiandrosterone, dehydroepiandrosterone-sulfate, and androstenedione (A4) are generally elevated or in the upper normal range in patients with ACTH-dependent CS. Conversely, adrenal androgens are generally low in patients with cortisol-secreting adrenocortical adenoma. However, androgen-secreting adrenal tumors (adenoma and carcinoma) can be also associated with severe hyperandrogenism. Regression of hypercortisolism after treatment causes disappearance of hyperandrogenism. However, signs of androgen excess may be detectable in well-controlled CS as a result of ACTH compensatory response to certain adrenal steroidogenesis inhibitors.

The effects on the endocrine system under hepatotoxicity induction by phenobarbital and di(2-ethylhexyl)phthalate in intact juvenile male rats

Phenobarbital (PB) and Di (2-ethylhexyl) phthalate (DEHP), an anti-epileptic drug and a plasticizer used in flexible polyvinylchloride formulations, respectively, are well-known typical hepatotoxicants. This study investigated the effects of PB (100 mg/kg/day) or DEHP (500 mg/kg/day) on the endocrine system in intact juvenile/peripubertal male rats exposed for 31 days beginning on postnatal day 23. Slight hormone level changes, histopathological changes in thyroid gland or induction of UDP-glucuronosyltransferase in liver were observed in both the PB and DEHP groups. One of the assumed mechanisms inducing thyroid effects is predictable to be secondary changes based on the enhancement in thyroid hormone metabolism via the induction of hepatic microsomal enzymes. No reproductive system-related changes in organ weights, histopathology, and sexual maturation were observed in both groups. Lower testosterone level was observed in the PB group. CYP2B and CYP3A, which are involved in testosterone metabolism, were induced in liver of the PB group. There was no change of 17β-hydroxysteroid dehydrogenase activity in testis of both groups. Lower testosterone level in the PB-treated male rats was attributed to an indirect, hepatotoxicity-associated effect on the reproductive system and not to direct effects on testis such as the antiandrogenic activity and the inhibition of steroidogenesis. These results did not indicate that PB or DEHP exposure affects the endocrine system directly.

Contribution of Adrenal Glands to Intratumor Androgens and Growth of Castration-Resistant Prostate Cancer.

Tumor androgens in castration-resistant prostate cancer (CRPC) reflect de novo intratumoral synthesis or adrenal androgens. We used C.B.-17 SCID mice in which we observed adrenal CYP17A activity to isolate the impact of adrenal steroids on CRPC tumors in vivo. We evaluated tumor growth and androgens in LuCaP35CR and LuCaP96CR xenografts in response to adrenalectomy (ADX). We assessed protein expression of key steroidogenic enzymes in 185 CRPC metastases from 42 patients. Adrenal glands of intact and castrated mice expressed CYP17A. Serum DHEA, androstenedione (AED), and testosterone (T) in castrated mice became undetectable after ADX (all P < 0.05). ADX prolonged median survival (days) in both CRPC models (33 vs. 179; 25 vs. 301) and suppressed tumor steroids versus castration alone (T 0.64 pg/mg vs. 0.03 pg/mg; DHT 2.3 pg/mg vs. 0.23 pg/mg; and T 0.81 pg/mg vs. 0.03 pg/mg, DHT 1.3 pg/mg vs. 0.04 pg/mg; all P ≤ 0.001). A subset of tumors recurred with increased steroid levels, and/or induction of androgen receptor (AR), truncated AR variants, and glucocorticoid receptor (GR). Metastases from 19 of 35 patients with AR positive tumors concurrently expressed enzymes for adrenal androgen utilization and nine expressed enzymes for de novo steroidogenesis (HSD3B1, CYP17A, AKR1C3, and HSD17B3). Mice are appropriate for evaluating adrenal impact of steroidogenesis inhibitors. A subset of ADX-resistant CRPC tumors demonstrate de novo androgen synthesis. Tumor growth and androgens were suppressed more strongly by surgical ADX than prior studies using abiraterone, suggesting reduction in adrenally-derived androgens beyond that achieved by abiraterone may have clinical benefit. Proof-of-concept studies with agents capable of achieving true "nonsurgical ADX" are warranted.

Successful Use of Fludrocortisone in a Child with Refractory Cerebral Salt Wasting Syndrome: A Case Report and Review of Literature

Hyponatremia post-neurosurgical intervention can be dangerous and potentially life-threatening. Two of its most common causes are cerebral salt wasting (CSW) and syndrome of inappropriate anti-diuretic hormone release (SIADH). CSW is proposed to be secondary not only to the elevated levels of circulating atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) but inhibition of steroidogenesis in the zona glomerulosa of the adrenal cortex, thus resulting in mineralocorticoid deficiency. We present a two-year-old male who had developed acute hyponatremia secondary to CSW on post-operative day two after a sub-total resection of a low-grade juvenile pilocytic astrocytoma (WHO grade I). Fludrocortisone was successfully used to manage the refractory hyponatremia and alleviated the need to use very large amounts of oral sodium supplementation.

Adjuvant and Neoadjuvant Therapy, Treatment for Advanced Disease, and Genetic Considerations for Adrenocortical Carcinoma: An Update from the SSO Endocrine and Head and Neck Disease Site Working Group.

This is the second of a two-part review on adrenocortical carcinoma (ACC) management. While margin-negative resection provides the only potential cure for ACC, recurrence rates remain high. Furthermore, many patients present with locally advanced, unresectable tumors and/or diffuse metastases. As a result, selecting patients for adjuvant therapy and understanding systemic therapy options for advanced ACC is important. Herein, we detail the current literature supporting the use of adjuvant mitotane therapy, consideration of adjuvant radiation therapy, and utility of cytotoxic chemotherapy in patients with advanced disease. Ongoing investigation into molecular targeted agents, immunotherapy, and inhibitors of steroidogenesis for the treatment of ACC are also highlighted. Lastly, the importance of genetic counseling in patients with ACC is addressed as up to 10% of patients will have an identifiable hereditary syndrome.

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.

New Insights in Cushing Disease Treatment With Focus on a Derivative of Vitamin A.

Cushing’s disease (CD) is an endocrine disorder originated by a corticotroph tumor. It is linked with high mortality and morbidity due to chronic hypercortisolism. Treatment goals are to control cortisol excess and achieve long-term remission, therefore, reducing both complications and patient’s mortality. First-line of treatment for CD is pituitary’s surgery. However, 30% of patients who undergo surgery experience recurrence in long-term follow-up. Persistent or recurrent CD demands second-line treatments, such as pituitary radiotherapy, adrenal surgery, and/or pharmacological therapy. The latter plays a key role in cortisol excess control. Its targets are inhibition of adrenocorticotropic hormone (ACTH) production, inhibition of adrenal steroidogenesis, or antagonism of cortisol action at its peripheral receptor. Retinoic acid (RA) is a metabolic product of vitamin A (retinol) and has been studied for its antiproliferative effects on corticotroph tumor cells. It has been shown that this drug regulates the expression of pro-opiomelanocortin (POMC), ACTH secretion, and tumor growth in corticotroph tumor mouse cell lines and in the nude mice experimental model, via inhibition of POMC transcription. It has been shown to result in tumor reduction, normalization of cortisol levels and clinical improvement in dogs treated with RA for 6 months. The orphan nuclear receptor COUP-TFI is expressed in normal corticotroph cells, but not in corticotroph tumoral cells, and inhibits RA pathways. A first clinical human study demonstrated clinical and biochemical effectiveness in 5/7 patients treated with RA for a period of up to 12 months. In a recent second clinical trial, 25% of 16 patients achieved eucortisolemia, and all achieved a cortisol reduction after 6- to 12-month treatment. The goal of this review is to discuss in the context of the available and future pharmacological treatments of CD, RA mechanisms of action on corticotroph tumor cells, and future perspectives, focusing on potential clinical implementation.

Spontaneous adrenocorticotropic hormone (ACTH) normalisation due to tumour regression induced by metyrapone in a patient with ectopic ACTH syndrome: case report and literature review

BackgroundEctopic adrenocorticotropic hormone (ACTH) syndrome (EAS) is caused by tumours releasing ACTH. Ectopic ACTH-producing tumour regression is rarely induced using steroidogenesis inhibitors. We presented a case of EAS in which ACTH production by a lung tumour was reduced by metyrapone (MTP) and also reviewed previous cases of ectopic ACTH production suppressed via steroidogenesis inhibition.Case presentationA 71-year-old female with general fatigue, central obesity and impaired glucose tolerance was diagnosed with Cushing’s syndrome due to elevated ACTH (192.9 pg/mL; normal range, 7.2–63.3 pg/mL), cortisol (73.1 μg/dL; 6.4–21.0 μg/dL) and 24-h urinary free cortisol (UFC) (6160 μg/day; 11.2–80.3 μg/day) levels. Chest computed tomography identified a solid 26.6 × 22.9 × 30.0 mm tumour with a cavity in the upper lobe of the left lung. There was no adrenal gland enlargement. Tumour markers were not significantly elevated; ACTH levels were not suppressed by 8-mg dexamethasone. A corticotropin-releasing hormone stimulation test revealed blunted ACTH response (basal ACTH, 204.6 pg/mL; highest ACTH level during the 120-min stimulation test, 214.0 pg/mL). She was diagnosed with EAS due to a lung lesion. MTP treatment was started to reduce cortisol production. ACTH levels and cortisol and UFC levels were normalised and the ACTH-producing lung tumour was ablated after MTP treatment. In several reported cases, plasma ACTH levels reduced during steroidogenesis inhibitor treatment for EAS. Among the 10 patients, three cases of pheochromocytoma, one of thymic carcinoid and one of islet cell carcinoma were reported. In four cases, the tumour was not detected. In our case, the pathology of the lung tumour was unknown because of lack of tumour cells in biopsy. The patients were treated with ketoconazole (KTZ) and/or MTP and exhibited ACTH and cortisol/UFC suppression, but tumour regression was observed only in our case.ConclusionMTP and/or KTZ may reduce ACTH and cortisol production. The tumour spontaneously regressed after MTP treatment, indicating that MTP may reduce the tumour size without surgery. The mechanisms of therapeutic effects of steroidogenesis inhibitors and prognosis of spontaneous remission should be elucidated further via molecular biology studies.

The in vitro modulation of steroidogenesis by inflammatory cytokines and insulin in TM3 Leydig cells

BackgroundCytokines and hormones, including insulin, are known to modulate the hypothalamic-pituitary-testes axis and steroidogenesis, both centrally and peripherally. In the context of chronic inflammation and hyperinsulinaemia mediating male hypogonadism associated with obesity, metabolic syndrome and type 2 diabetes mellitus, these mechanisms are poorly understood and the impact of cytokines and insulin on Leydig cell steroidogenesis has not been fully elicited. This study aimed to further investigate the in vitro impact of TNFα, IL1ß, IL6, IL8 and insulin on Leydig cell function and steroidogenesis.MethodshCG-stimulated TM3 Leydig cells were exposed to various concentrations of TNFα, IL1ß, IL6, IL8 (100 ng/ml, 10 ng/ml, 1 ng/ml and 0.1 ng/ml) and insulin (10 ng/ml, 1 ng/ml, 0.1 ng/ml and 0.01 ng/ml) in optimal cell culture conditions over 48 h. Cell viability (XTT) and testosterone and progesterone concentrations (ELISA) were assessed using standardised laboratory techniques.ResultsTNFα significantly decreased cell viability and progesterone and testosterone concentrations in a dose-dependent relationship. IL1ß and IL6 had a subtle but significant negative effect on cell viability and testosterone concentrations, with a marked significant decrease in progesterone concentration at all concentrations investigated. IL8 showed an increase in cell viability, with no significant effect on testosterone concentrations alongside a significant decrease in progesterone concentrations. Insulin significantly increased cell viability and testosterone concentrations in a dose dependent relationship, but interestingly significantly decreased progesterone concentrations.ConclusionsThe inflammatory cytokines TNFα, IL1β and IL6 cause a dose dependent decline in steroidogenesis in TM3 Leydig cells. These results suggest that chronic inflammation may downregulate steroidogenesis in males via direct modulation of Leydig cell function. However, IL8 may stimulate TM3 Leydig cell growth. Insulin is associated with a dose-dependent increase in testosterone synthesis, with a significant decline in progesterone synthesis. With the phenomenon of insulin resistance, the literature is unclear on the potential role of hyperinsulinaemia in steroidogenesis. Further studies are warranted in order to fully elicit the molecular mechanisms and interactions of these molecules on male steroidogenesis.

Use of ketoconazole in the treatment and control of clinical and hematological changes in canine hyperadrenocorticism: Case Report

Hyperadrenocorticism (HAC), also known as Cushing's Syndrome, is a disease manifested by a series of clinical changes and multisystem abnormalities resulting from increased chronic exposure to glucocorticoids secreted by the adrenal glands or excessive administration of this hormone. Two different categories of medications are currently used in the treatment of HAC: inhibitors of steroidogenesis and modulators of ACTH secretion. Among the drugs inhibiting steroidogenesis the most indicated in practice by veterinarians is trilostane. However, due to its high cost, there is a need to study drugs that may have a systemic action in the control of clinical and hematological alterations in CAH. Thus, the present study aims to evaluate the use of ketoconazole as the drug of choice in the treatment and control of clinical and hematological changes of canine hyperadrenocorticism. At 8-year-old Poodle dog with diagnosis of hyperadrenocorticism was attended at the Metropolitan Veterinary Polytechnic of Caucaia - HVM. The guardian reported that the dog was 4 months without using Trilostan because it did not have financial conditions to maintain the treatment, which significantly worsened the clinical signs previously presented: polydipsia, polyuria, polyphagia, hyperpigmented skin with lesions, symmetrical alopecia, telangiectasia, pendular abdomen, muscle weakness in the pelvic limbs. Dyspnea was visually detected and cardiac auscultation detected tachycardia. Laboratory, ultrasonographic and cardiac evaluation (electrocardiogram and echocardiogram) were requested. Due to changes in the results of the tests, the veterinarian suggested the use of ketoconazole at the initial dose of 5mg / kg / VO / BID for 7 days and silymarin 180mg / VO / SID.com for purpose of controlling the patient's clinical symptoms and the observed changes in the examinations carried out. After this period, there was a significant improvement in the hematological and biochemical alterations of the patient, however the animal clinically presented worsening, presenting frequent vomiting and inappetence. In view of the above, it can be concluded that the use of ketoconazole in the treatment of patients with canine CAH should be used only as an adjuvant treatment, since despite the significant improvement of the hematological and biochemical changes related to the endocrine disorder does not improve clinical symptoms.

Triiodothyronine stimulates VEGF expression and secretion via steroids and HIF-1α in murine Leydig cells

ABSTRACTLeydig cells are the principal steroidogenic cells of the testis. Leydig cells also secrete a number of growth factors including vascular endothelial growth factor (VEGF) which has been shown to regulate both testicular steroidogenesis and spermatogenesis. The thyroid hormone, T3, is known to stimulate steroidogenesis in Leydig cells. T3 has also been shown to stimulate VEGF production in a variety of cell lines. However, studies regarding the effect of T3 on VEGF synthesis and secretion by the Leydig cells were lacking. Therefore, we investigated the effect of T3 on VEGF synthesis and secretion in a mouse Leydig tumour cell line, MLTC-1. The effect of T3 was compared with that of LH/cAMP and hypoxia, two known stimulators of Leydig cell functions. The cells were treated with T3, 8-Br-cAMP (a cAMP analogue), or CoCl2 (a hypoxia mimetic) and VEGF secreted in the cell supernatant was measured using ELISA. The mRNA levels of VEGF were measured by quantitative RT-PCR. In the MLTC-1 cells, T3, 8-Br-cAMP, and CoCl2 stimulated VEGF mRNA levels and the protein secretion. T3 also increased steroid secretion as well as HIF-1α protein levels, two well-established upstream regulators of VEGF. Inhibitors of steroidogenesis as well as HIF-1α resulted in inhibition of T3-stimulated VEGF secretion by the MLTC-1 cells. This suggested a mediatory role of steroids and HIF-1α protein in T3-stimulated VEGF secretion by MLTC-1 cells. The mediation by steroids and HIF-1α were independent of each other.Abbreviations: 8-Br-cAMP: 8-bromo – 3ʹ, 5ʹ cyclic adenosine monophosphate; CoCl2: cobalt chloride; HIF-1α: hypoxia inducible factor −1α; LH: luteinizing hormone; T3: 3, 5, 3ʹ-L-triiodothyronine; VEGF: vascular endothelial growth factor

Dimethoxy-etomidate: A Nonhypnotic Etomidate Analog that Potently Inhibits Steroidogenesis.

Cushing's syndrome is characterized by the overproduction of adrenocortical steroids. Steroidogenesis inhibitors are mainstays of medical therapy for Cushing's syndrome; unfortunately, adverse side effects and treatment failures are common with currently available drugs. The general anesthetic induction agent etomidate is among the most potent inhibitors of adrenocortical steroidogenesis. However, its use as a treatment of Cushing's syndrome is complicated by its sedative-hypnotic activity and ability to produce myoclonus, central nervous system actions thought to be mediated by the GABAA receptor. Here, we describe the pharmacology of the novel etomidate analog (R)-ethyl 1-(1-(3,5-dimethoxyphenyl)ethyl)-1H-imidazole-5-carboxylate (dimethoxy-etomidate). In contrast to etomidate, dimethoxy-etomidate minimally enhanced GABA-evoked GABAA receptor-mediated currents even at a near-saturating aqueous concentration. In Sprague-Dawley rats, dimethoxy-etomidate's potency for producing loss of righting reflexes-an animal model of sedation/hypnosis-was 2 orders of magnitude lower than that of etomidate, and it did not produce myoclonus. However, similar to etomidate, dimethoxy-etomidate potently suppressed adrenocortical steroid synthesis primarily by inhibiting 11β-hydroxylase. [3H]etomidate binding to rat adrenocortical membranes was inhibited by dimethoxy-etomidate in a biphasic manner with IC50 values of 8.2 and 3970 nM, whereas that by etomidate was monophasic with an IC50 of 22 nM. Our results demonstrate that, similar to etomidate, dimethoxy-etomidate potently and dose-dependently suppresses adrenocortical steroid synthesis by inhibiting 11β-hydroxylase. However, it is essentially devoid of etomidate's GABAA receptor positive modulatory and sedative-hypnotic activities and produces no myoclonus, providing proof of concept for the design of etomidate analogs without important central nervous system actions for the pharmacologic treatment of Cushing's syndrome.

Increased 27-hydroxycholesterol production during luteolysis may mediate the progressive decline in progesterone secretion.

Does 27-hydroxycholesterol (27OH) actively facilitate the progression of luteolysis? There is increased mRNA expression of the enzyme that produces 27OH during luteolysis in vivo in rhesus macaques and sheep, and 27OH reduces progesterone secretion from human luteinized granulosa cells. There is an increase in mRNA expression of liver x receptor (LXR) and a decrease in sterol regulatory element binding protein 2 (SREBP2) target genes during spontaneous luteolysis in primates, which could result in reduced cholesterol availability for steroidogenesis. Concentrations of 27OH are also increased in primate corpora lutea (CL) during luteolysis, and 27OH is a dual LXR agonist and SREBP2 inhibitor. This was an in vitro study using primary human luteinized granulosa cells in a control versus treatment(s) design. Analyses of CL from sheep undergoing induced or spontaneous luteolysis were also performed, along with database mining of microarray data from rhesus macaque CL. Primary luteinizing granulosa cells were obtained from 37 women aged 24-44 who were undergoing oocyte donation or IVF for male factor or idiopathic infertility, and cells were further luteinized in vitro using human chorionic gonadotropin. Three approaches to test the effect of 27OH produced via CYP27A1 (cytochrome p450, family 27, subfamily A, polypeptide 1) on luteinized granulosa cells were used: (i) direct 27OH supplementation, (ii) induction of endogenous CYP27A1 activity via pharmacologic inhibition of steroidogenesis, and (iii) siRNA-mediated knockdown to directly inhibit CYP27A1 as well as cholesterol transport into the mitochondria via the steroidogenic acute regulatory protein (STAR). Endpoints included: progesterone (P4) secretion into culture media determined by enzyme immunoassay, cholesterol efflux and uptake assays using fluorescent lipid analogs, and mRNA expression determined via semi-quantitative real-time PCR (QPCR). An additional experiment involved QPCR analysis of 40 CL collected from ewes undergoing induced or spontaneous luteolysis, as well as database mining of microarray data generated from 16 rhesus macaque CL collected during spontaneous luteolysis and 13 macaque CL collected during a luteinizing hormone ablation and replacement protocol. The mRNA expression of CYP27A1 was significantly increased during luteolysis in rhesus macaques and sheep in vivo, and CYP27A1 transcription was suppressed by luteinizing hormone and hCG. There was a significant decrease in hCG-stimulated P4 secretion from human luteinized granulosa cells caused by 27OH treatment, and a significant increase in basal and hCG-stimulated P4 synthesis when endogenous 27OH production was inhibited via CYP27A1 knockdown, indicating that 27OH inhibits steroidogenesis. Pharmacologic inhibition of steroidogenesis by aminoglutethimide significantly induced LXR and inhibited SREBP2 target gene mRNA expression, indicating that increased oxysterol production occurs when steroidogenesis is suppressed. Inhibiting cholesterol delivery into the mitochondria via knockdown of STAR resulted in reduced SREBP2 target gene mRNA expression, indicating that STAR function is necessary to maintain SREBP2-mediated transcription. The effects of 27OH treatment on markers of LXR and SREBP2 activity were moderate, and knockdown of CYP27A1 did not prevent aminoglutethimide-induced changes in LXR and SREBP2 target gene mRNA expression. These observations indicate that 27OH inhibits P4 secretion partially via mechanisms separate from its role as an LXR agonist and SREBP2 inhibitor, and also demonstrate that other oxysterols are involved in modulating LXR and SREBP2-mediated transcription when steroidogenesis is suppressed. None. Luteinized granulosa cells may differ from luteal cells, and the effect on luteal function in vivo was not directly tested. The mechanisms that cause the initial rise in CYP27A1 mRNA expression during luteolysis are also not clear. The factors causing luteolysis in primates have not yet been determined. This study provides functional evidence of a novel mechanism via increased 27OH synthesis during luteolysis, which subsequently represses progesterone secretion. Increased 27OH may also facilitate the progression of luteolysis in domestic animal species. The authors have nothing to disclose. Support was provided by the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) of the National Institutes of Health (NIH), award number R00HD067678 to R.L.B.

Different therapeutic options in patients with Cushing's syndrome due to bilateral macronodular adrenal hyperplasia.

Bilateral macronodular adrenal hyperplasia (BMAH) is a relatively rare cause of Cushing's Syndrome (CS). In recent years, growing evidence has shown that steroidogenesis is regulated by aberrant G-protein-coupled receptors (GPCRs) expression and their ligands, in a significant proportion of patients with BMAH. The screening of patients with overt or subclinical CS demonstrates the frequent expression of several GPCRs that opened the option to potential therapeutic applications. Thus, several studies have demonstrated that targeting the involved receptor with specific antagonists may result in a more or less effective control of cortisol excess. Bilateral adrenalectomy has traditionally been considered the treatment of choice for BMAH. However, unilateral adrenalectomy has been recently proposed as an alternative in selective patients to avoid the long-term necessity of gluco/mineralocorticoid replacement. Adrenal steroidogenesis inhibitors remain a valid option when medical treatment is needed due to high surgical risk.

Effects of Ketoconazole on the Pharmacokinetics of Mifepristone, a Competitive Glucocorticoid Receptor Antagonist, in Healthy Men.

IntroductionMifepristone, a competitive glucocorticoid receptor antagonist approved for Cushing syndrome, and ketoconazole, an antifungal and steroidogenesis inhibitor, are both inhibitors of and substrates for cytochrome P450 (CYP3A4). This study evaluated the pharmacokinetic effects of concomitant ketoconazole, a strong CYP3A4 inhibitor, on mifepristone.MethodsIn an open-label, two-period, single-center study, healthy adult men received mifepristone 600 mg orally daily for 12 days (period 1) followed by mifepristone 600 mg daily plus ketoconazole 200 mg orally twice daily for 5 days (period 2). Serial pharmacokinetic blood samples were collected predose and over 24 h postdose on days 12 (period 1) and 17 (period 2). A cross-study comparison (using data on file) further examined whether systemic exposure to mifepristone plus ketoconazole exceeded the exposure following mifepristone 1200 mg orally administered for 7 days.ResultsSixteen subjects were enrolled and 14 completed the study. Concomitant administration with ketoconazole increased the systemic exposure to mifepristone, based on geometric least squares mean ratios, by 28% for C max and 38% for AUC0–24. This increase was 85% and 87% of the exposure observed following mifepristone’s highest label dose of 1200 mg/day for C max and AUC0–24, respectively. Adverse events (AEs) were reported in 56.3% (9/16) of subjects during administration of mifepristone alone and in 57.1% (8/14) during combination with ketoconazole. No serious AEs were reported.ConclusionSystemic exposure to mifepristone increased following multiple doses of mifepristone 600 mg daily plus ketoconazole 200 mg twice daily. Little to no increase in AEs occurred. Dose adjustment of mifepristone may be needed when given with ketoconazole.FundingCorcept Therapeutics.