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

CONTEXT: 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. OBJECTIVE: To compare the effects of osilodrostat, metyrapone, and ketoconazole on adrenal steroidogenesis, and pituitary adenoma cells in vitro. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Dynamics of Adrenocorticotropin after Application of Metyrapone.

Purpose: To investigate the kinetics of adrenocorticotropin (ACTH) following oral metyrapone administration and describe differences between ACTH-deficient and non-ACTH-deficient subjects. Methods: Patients from a tertiary endocrine center at a University Hospital in Munich, Germany, were tested for secondary adrenal insufficiency in a regular patient care setting. Metyrapone (Metopirone, HRA Pharma, France) was administered with a dosage of 40 mg/kg bodyweight at 8 a.m. Consecutive levels of ACTH were determined at 0, 60, 120, 180, and 240 min. Patients were categorized according to their need of glucocorticoid substitution in the follow-up phase. Results: A significant rise in ACTH concentration compared to basal values was found at 60 and 120 min following oral metyrapone administration. ACTH concentrations at 60 and 120 min predicted patients without need for glucocorticoid substitution. ACTH concentrations determined later had no additional benefit. Conclusion: In contrast to previous reports, we found a significant rise in ACTH concentration as soon as one hour after oral metyrapone administration. ACTH values seem to estimate the pituitary corticotrophic function when correlating results to the further clinical course of subjects. Further studies are needed to investigate this finding as a potential basis for a ACTH-based metyrapone short test protocol.

Oral pharmacokinetics and in-vitro metabolism of metyrapone in male rats.

OBJECTIVES: The purpose of this study was to investigate the pharmacokinetics of a single oral administration of metyrapone (MP) and metabolites produced from it in male Wistar rats, and the major tissues and enzymes involved in the production of the MP metabolites. Furthermore, the MP metabolism in human liver subcellular fractions was compared with that in rats. METHODS: High-performance liquid chromatography with ultraviolet detection (HPLC-UV) was used to determine the concentrations of MP and its metabolites in plasma and urine after administration, and the production activity of MP metabolites in subcellular fractions of various tissues. KEY FINDINGS: Plasma concentration of MP was rapidly increased and decreased, and the primary metabolite, metyrapol (MPOL), was immediately produced. The production activity of MPOL was substantially inhibited by an 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) inhibitor in the rat and human liver microsomal and mitochondrial fractions. In the liver cytosolic fraction, the activity was inhibited by a carbonyl reductase inhibitor in the humans but not rats. CONCLUSIONS: In this study, we elucidated the plasma pharmacokinetics of MP and its metabolites in male rats after an oral administration. MPOL is most likely to be produced by 11ß-HSD1 in the male rats and humans.

Pineal¨Cadrenal¨Cimmune system relationship under thermal stress: effect on physiological, endocrine, and non-specific immune response in goats

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The purpose of the investigation was to observe the pineal¨Cadrenal¨Cimmune system relationships and their influence on non-specific immune response in female goats under short-term thermal stress. Six female goats had been exposed to (..)(AU)

Pharmacodynamic and pharmacokinetic characterization of the aldosterone synthase inhibitor FAD286 in two rodent models of hyperaldosteronism: comparison with the 11beta-hydroxylase inhibitor metyrapone.

Aldosterone synthase (CYP11B2) inhibitors (ASIs) represent an attractive therapeutic approach for mitigating the untoward effects of aldosterone. We characterized the pharmacokinetic/pharmacodynamic relationships of a prototypical ASI, (+)-(5R)-4-(5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-5-yl]benzonitrile hydrochloride (CGS020286A, FAD286, FAD) and compared these profiles to those of the 11beta-hydroxylase inhibitor metyrapone (MET) in two rodent models of secondary hyperaldosteronism and corticosteronism. In chronically cannulated Sprague-Dawley rats, angiotensin II (ANG II) (300 ng/kg bolus + 100 ng/kg/min infusion) or adrenocorticotropin (100 ng/kg + 30 ng/kg/min) acutely elevated plasma aldosterone concentration (PAC) from approximately 0.26 nM to a sustained level of approximately 2.5 nM for 9 h. Adrenocorticotropin but not ANG II elicited a sustained increase in plasma corticosterone concentration (PCC) from approximately 300 to approximately 1340 nM. After 1 h of Ang II or adrenocorticotropin infusion, FAD (0.01-100 mg/kg p.o.) or MET (0.1-300 mg/kg p.o.) dose- and drug plasma concentration-dependently reduced the elevated PACs over the ensuing 8 h. FAD was approximately 12 times more dose-potent than MET in reducing PAC but of similar or slightly greater potency on a plasma drug concentration basis. Both agents also decreased PCC in the adrenocorticotropin model at relatively higher doses and with similar dose potencies, whereas FAD was 6-fold weaker based on drug exposures. FAD was approximately 50-fold selective for reducing PAC versus PCC, whereas MET was only approximately 3-fold selective. We conclude that FAD is a potent, orally active, and relatively selective ASI in two rat models of hyperaldosteronism. MET is an order of magnitude less selective than FAD but is, nevertheless, more potent as an ASI than as an 11beta-hydroxylase inhibitor.

Transfer of metyrapone and its metabolite, rac-metyrapol, into breast milk.

Metyrapone, an inhibitor of corticosteroid biosynthesis, is used in the diagnosis and treatment of adrenocortical hyperfunction. The authors describe the excretion of metyrapone and its metabolite, rac-metyrapol, in milk from a lactating woman requiring metyrapone treatment (250 mg 4 times daily). At steady state, the average concentrations in milk and absolute and relative infant doses were 11 microg/L, 1.7 microg/kg/d, and 0.02%, respectively, for metyrapone, and 48.5 microg/L, 7.3 microg/kg/d, and 0.08%, respectively, for rac-metyrapol. The findings suggest that maternal metyrapone use during breastfeeding is extremely unlikely to be a significant risk for the breastfed infant.

Effects of the combination of metyrapone and oxazepam on cocaine and food self-administration in rats.

For several years, our laboratory has investigated the role for the HPA axis in cocaine reinforcement. Two classes of drugs that we have studied include corticosterone synthesis inhibitors (e.g., metyrapone) and benzodiazepine receptor agonists (e.g., oxazepam). In the experiments described in this manuscript, we tested the effects of various doses of metyrapone and oxazepam against several doses of self-administered cocaine. Behavioral, endocrine and pharmacokinetic measures of the effects of the combination of metyrapone and oxazepam on cocaine reward are presented. Combinations of metyrapone and oxazepam at doses that produced no observable effects when administered separately significantly reduced cocaine self-administration without affecting food-maintained responding during the same sessions. Changes in pharmacokinetics or endocrine function do not appear to mediate these effects, suggesting a central mechanism of action. Therefore, although these drugs produce their effects through distinct mechanisms, an additive effect on cocaine self-administration is obtained when these drugs are administered together, suggesting that combinations of low doses of metyrapone and oxazepam may be useful in reducing cocaine seeking with a reduced incidence of unwanted side effects and a decreased potential for abuse.

Stereoselective reductive metabolism of metyrapone and inhibitory activity of metyrapone metabolites, metyrapol enantiomers, on steroid 11 beta-hydroxylase in the rat.

Pharmacokinetics of metyrapone and metyrapol enantiomers was studied in the rat to determine the stereoselective reductive metabolism of metyrapone. The HPLC method using a chiral column was developed for the stereoselective analysis of metyrapol enantiomers in rat plasma. The AUC ratio of (-)- and (+)-metyrapol appeared in rat plasma after i.v. administration of metyrapone was about 3:1. The interconversion of (-)- or (+)-metyrapol to its antipode was negligible, and the reverse reaction from metyrapol to metyrapone was insignificant. There were similar kinetic parameters of (-)-metyrapol to those of (+)-metyrapol after i.v. administration of racemic metyrapol. These results indicate metyrapone displays product-stereoselective reductive metabolism in the rat. The inhibition of steroid 11 beta-hydroxylase by metyrapone, racemic metyrapol, (-)-metyrapol or (+)-metyrapol was analyzed in rat adrenal homogenates. Metyrapol was equally as potent as metyrapone in the inhibition of steroid 11 beta-hydroxylase and each enantiomer of metyrapol showed similar inhibitory activity on the rat adrenal steroid 11 beta-hydroxylase. These results indicate there is an insignificant difference in the inhibitory effects on steroid 11 beta-hydroxylase of metyrapol enantiomers, and that the inhibitory effects of metyrapol may be involved in the pharmacological activity of metyrapone in vivo.

Biotransformation and detoxification of insecticidal metyrapone analogues by carbonyl reduction in the human liver.

1. The carbonyl reduction of insecticidal metyrapone analogues to their hydroxyl metabolites by human liver microsomes and cytosol was examined. Metabolite quantification was performed by means of hplc determination and inhibition experiments, using specific carbonyl reductase inhibitors, were conducted. 2. The cytotoxicity of the ketones and their hydroxy metabolites was assessed with the MTT test, using Chang liver cells. 3. It was found that the alcohol derivatives are the major metabolite, both in microsomes and cytosol. The microsomal reductive metabolism, considered to be mediated by 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD) (EC 1.1.1.146), was more extensive than the cytosolic carbonyl reduction. In each case, this metabolism was inhibited significantly by equimolar concentrations of the microsomal 11 beta-HSD inhibitor glycyrrhetinic acid and the cytosolic carbonyl reductase inhibitor quercitrin, respectively. 4. The parent ketones were more cytotoxic than their alcohol metabolites. 5. These results demonstrate that the metyrapone analogues are extensively metabolized by human liver microsomes, presumably by 11 beta-HSD, to the less cytotoxic and readily excretable alcohols. 6. Since the metyrapone analogues can inhibit ecdysone 20-monooxygenase (EC 1.14.99.22), our results indicate potential application of these compounds as insecticides, which would be safer for humans, due to their reductive detoxification, mainly by the hepatic microsomal 11 beta-HSD, to the less toxic hydroxy metabolites.

Characterization of the in vivo inhibition of rat hepatic microsomal aldehyde dehydrogenase activity by metyrapone.

Microsomal aldehyde dehydrogenase (mALDH; EC 1.2.1.3) has been proposed to catalyze the oxidation of various aldehydic products of lipid peroxidation, but the regulation of the enzyme has not been characterized. Metyrapone administration (100 mg/kg, i.p.) produced a rapid decline in the rates of mALDH-catalyzed decanal dehydrogenation; other xenobiotics were generally without effect. Thus, a 22% decrease in activity was detected 2 hr following metyrapone administration, and 52% of the activity remained at 6 hr. The decrease in microsomal decanal dehydrogenation was also dose-dependent with 70, 43, and 12% of the control activity remaining following pretreatment with 25, 100, and 250 mg/kg metyrapone, respectively. This disease in microsomal decanal dehydrogenase activity occurred without a change in mALDH immunoreactive protein, and metyrapone did not inhibit the activity in vitro. The kinetic analysis revealed similar decreases in the maximal reaction velocities (Vmax) for both decanal and NAD in the metyrapone-treated group (200 +/- 10 and 190 +/- 20 nmol NADH produced/min/mg protein, respectively) compared with the untreated group (330 +/- 10 and 350 +/- 20 nmol NADH produced/min/mg protein, respectively), but the Michaelis constants (Km) were unchanged. These data are consistent with the in vivo inactivation of a portion of the mALDH enzyme. A possible consequence of the in vivo inhibition of this enzyme by metyrapone could be the accumulation of toxic aldehydes in the vicinity of the microsomal membrane following lipid peroxidation.

Urinary metabolic profile in rat of 1-(2-methoxyphenyl)-2-methyl-2-(3-pyridyl)-1-propanone: a potential radioligand for functional diagnosis of adrenal pathology.

1. The metabolism of 1-(2-methoxyphenyl)-2-methyl-2-(3-pyridyl)-1-propanone (2-MPMP) was studied in the male Sprague-Dawley rat after 50 mg/kg, i.v. dose. 2. Organic solvent extracts of urine samples were directly analysed by reversed-phase gradient hplc. The identified metabolites were also isolated by preparative tlc, and analyzed by direct probe mass spectrometry. In the case of conjugated metabolites, the urine samples were deconjugated by enzyme hydrolysis prior to extraction. The structures of metabolites were confirmed by comparison of their chromatographic behaviours, UV spectra, and mass spectra with those of authentic standards. 3. The metabolites identified in the 0-24-h urine samples were 2-hydroxyphenyl-metyrapone (2-OHPMP) and 2-hydroyphenylmetyrapone N-oxide (2-OHPMP-NO), which were present predominantly as their glucuronide and/or sulphate conjugates. 4. 2-MPMP and four of its metabolites present in the 0-24-h urine samples were quantified by a reversed-phase hplc method. The mean total urinary excretion was 75.4% of the administered dose. The major metabolites present in the urine were conjugates of 2-OHPMP-NO (54.4%) and of 2-OHPMP (18.6%). The excretion of the unchanged drug, unconjugated 2-OHPMP and 2-OHPMP-NO accounted for 1.1, 1.1 and 0.2% of the dose respectively.

High carbonyl reductase activity in adrenal gland and ovary emphasizes its role in carbonyl compound detoxication.

Carbonyl reduction has been studied in liver, kidney, adrenal gland and ovary of female Wistar and Sprague-Dawley rats as well as of female NMRI mice by using metyrapone as a substrate and by means of direct HPLC analysis of the reduced alcohol metabolite metyrapol. Carbonyl reducing activities were found in all tissues examined so far, with that in rat ovary and adrenal gland cytosol exceeding the liver cytosolic specific activity severalfold: 15-fold and 12-fold in the Wistar strain; 12-fold and 7-fold in the Sprague-Dawley strain, respectively. In general, Wistar rat enzyme activities were about four times higher than those of Sprague-Dawley rats in all fractions, which indicates an interesting genetic difference between the two rat strains. Due to the sensitivity towards the diagnostic inhibitor quercitrin, carbonyl reductase (EC 1.1.1.184) seems to be mainly responsible for metyrapone reduction in rat and mouse adrenal gland and ovary cytosol. However, sensitivity towards dicoumarol in microsomal fractions of mouse tissues points to the involvement of further carbonyl reducing enzymes. Western blot experiments revealed immunological differences between metyrapone reductase from liver microsomes and respective enzymes of all other tissues. In conclusion, the difference in tissue and intracellular distribution suggests that several enzymes are involved in carbonyl reduction of metyrapone and the intracellular multiplicity of the enzymes may have some relation to their significance in carbonyl compound detoxification. These results support the hypothesis that carbonyl reductases, besides their participation in the metabolism of physiologically occurring substances, provide the enzymatic basis for the detoxification of xenobiotic carbonyl compounds in adrenal gland and ovary which have escaped their metabolic conversion by the liver.

The urinary metabolic profile of metyrapone in the rat. Identification of two novel isomeric metyrapol N-oxide metabolites.

This study was designed to fully characterize the urinary metabolic profile of metyrapone following a 50 mg/kg ip dose to male Sprague-Dawley rats. Preliminary examination of alkaline dichloromethane extracts of urine by TLC and HPLC showed the absence of the intact drug, but the presence of moderate amounts of the two isomeric metyrapone mono-N-oxides, together with small amounts of metyrapol and the alpha-pyridone metabolite. These compounds have previously been reported as in vitro metabolites. Large amounts of two new metabolites of metyrapone were also observed and were conclusively identified as the isomeric metyrapol N-oxides by a combination of low- and high-resolution mass spectrometry. Quantitation by HPLC showed that metyrapol and the two metyrapone mono-N-oxides accounted for 4 and 10% of the dose, respectively. The two metyrapol mono-N-oxides accounted for more than 75% of the dose. These are novel metabolites not previously reported either as in vivo or in vitro metabolites.