Metyrapone Research Articles

Optimal timing of oral metyrapone intake for the suppression of cold-pressor stress-induced cortisol release

BackgroundPharmacological manipulation of cortisol levels is instrumental in elucidating mechanisms underlying acute stress effects and for distinguishing the physiological and behavioral effects of cortisol from those of the adrenergic system. Administration (oral or IV) of hydrocortisone is a direct and efficient method to elevate cortisol, and thus, frequently used in psychobiological stress research. However, lowering of cortisol (i.e. blockade of stress cortisol) requires a more sophisticated approach, such as the administration of the corticostatic compound metyrapone (MET). However, there is insufficient knowledge about the temporal dynamics of MET for the blocking of stress-induced cortisol reactivity. Thus, the present study aimed to build up an experimental protocol suitable to suppress acute behavioral stress-induced cortisol secretion by MET. Methods50 healthy young men were randomly assigned to one of five treatment groups. They received 750 mg oral MET either 30 (n = 9), 45 (n = 11), or 60 (n = 10) minutes before exposure to a combined cold pressor and mental arithmetic test (stress induction), or were subjected to two different control treatments (placebo 60 min before stress (n = 10) or MET 30 min before non-stressful warm-water condition (n = 10)). Salivary cortisol concentration, hemodynamics, and subjective ratings were assessed. ResultsSuppression of cold stress-induced cortisol release was strongest when MET intake was scheduled 30 min prior to stress onset. Cardiovascular stress-responses and subjective ratings remained unaffected by MET. ConclusionIn healthy young males, 750 mg of MET efficiently block cold stress-induced cortisol release when oral administration is scheduled 30 min prior to stress onset. This finding may guide future research in improving timing of suppression of stress-induced cortisol secretion.

Acute stress response of the HPA-axis in children with Prader-Willi syndrome: new insights and consequences for clinical practice.

Prader-Willi syndrome (PWS) is associated with hypothalamic dysfunction. It has been reported that the HPA axis might show a delayed response during acute stress, and it is unknown whether the response of the HPA-axis during acute stress changes with age in children with PWS. To investigate the HPA-axis response during an overnight single-dose metyrapone (MTP) test in children with PWS and to assess if the response changes with age, whether it is delayed and if it changes with repeated testing over time. In addition, we evaluated different cut-off points of ACTH and 11-DOC levels to assess stress-related central adrenal insufficiency (CAI). An overnight single-dose MTP test was performed in 93 children with PWS. Over time, 30 children had a second test and 11 children a third one. Children were divided into age groups (0-2 years, 2-4 years, 4-8 years and > 8 years). Most children did not have their lowest cortisol level at 7.30h, but at 04.00h. Their ACTH and 11-DOC peaks appeared several hours later, suggesting a delayed response. When evaluated according to a subnormal ACTH peak (13-33 pmol/L) more children had an subnormal response compared to evaluation based on a subnormal 11-doc peak (< 200 nmol/L). The percentage of children with a subnormal ACTH response ranged from 22.2 to 70.0% between the age groups, while the percentage of a subnormal 11-DOC response ranged from 7.7 to 20.6%. When using the ACTH peak for diagnosing acute-stress-related CAI, differences between age groups and with repeated testing over time were found, whereas there was no age difference when using the 11-DOC peak. Early morning ACTH or 11-DOC levels are not appropriate to determine acute stress-related CAI in children with PWS, thus multiple measurements throughout the night are needed for an accurate interpretation. Our data suggest a delayed response of the HPA-axis during acute stress. Using the 11-DOC peak for the test interpretation is less age-dependent than the ACTH peak. Repeated testing of the HPA-axis over time is not required, unless clinically indicated.

LC-MS/MS method for simultaneous quantification of osilodrostat and metyrapone in human plasma from patients treated for Cushing’s Syndrome

Steroidogenesis inhibitors such as metyrapone (MTP) and osilodrostat (ODT) have a key role in the medical treatment of endogenous Cushing’s Syndrome (ECS). Both drugs are characterized by a high inter-individual variability of response and require a dose-titration period to achieve optimal control of cortisol excess. However, PK/PD data remain scarce for both molecules and a pharmacokinetically guided approach could help reaching eucortisolism more rapidly. We aimed to develop and validate a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of ODT and MTP in human plasma. After addition of isotopically labeled internal standard (IS), plasma pretreatment consisted in protein precipitation with acetonitrile including 1% formic acid (v/v). Chromatographic separation was performed on Kinetex® HILIC (4.6 × 50 mm; 2.6 µm) analytical column with an isocratic elution during the 2.0-min run time. The method was linear from 0.5 to 250 ng/mL for ODT and from 2.5 to 1250 ng/mL for MTP. Intra- and inter-assay precisions were < 7.2%, with an accuracy ranging from 95.9% to 114.9%. The IS-normalized matrix effect ranged from 106.0% to 123.0% (ODT) and from 107.0% to 123.0% (MTP) and the range of the IS-normalized extraction recovery was 84.0–101.0% for ODT and 87.0–101.0% for MTP. The LC-MS/MS method was successfully applied in patients’ plasma samples (n = 36), trough concentration of ODT and MTP ranged from 2.7 ng/mL to 8.2 ng/mL and from 10.8 ng/mL to 27.8 ng/mL, respectively. Incurred sample reanalysis exhibits less than 14% difference between the first and the second analysis for both drugs. This accurate and precise method, meeting all validation criteria, can therefore be used for plasma drug monitoring of ODT and MTP within the dose-titration period.

RF21 | PSAT99 Urinary adrenal steroidome modifications by Ketoconazole and Metyrapone alter the urinary free cortisol immunoassay reliability in Cushing Syndrome

Abstract Introduction Twenty-four-hour urinary free cortisol (24h-UFC) is the most used test for follow-up decision-making in patients with Cushing syndrome (CS) under adrenal-directed medical treatment. In CS patients without medical treatment, 24h-UFC determinations by immunoassays (IA) are commonly overestimated because of interference by cross-reacting steroid metabolites. However, it is unknown how induced changes by Ketoconazole (KTZ) or Metyrapone (MTP) on the excretion of urinary steroid metabolites alter the IA cross-reactivity interference of 24h-UFC determinations and what are its clinical implications. Methods 24h-UFC was analyzed by IA and gas chromatography-mass spectrometry (GC-MS) in 193 samples (81 before treatment, 73 during KTZ and 39 during MTP) from 34 CS patients. In addition, 35 urinary steroids were analyzed by GC-MS on each patient before and during treatment. Results Before treatment, 24h-UFC*IA determinations were overestimated by a factor of 1.75: 1 (95% CI 1.60 - 1.94) compared to those by GC-MS. During treatment with KTZ, the cross-reactivity interference of the 24h-UFC results by IA decreased in a dose-dependent manner (p = 0.000). Thus, in patients taking KTZ &amp;gt;400mg/day, 24h-UFC*IA determinations were no longer overestimated, giving similar results to those by GC-MS (factor of 0.98: 1, 95% CI, 0.83–1.20). Baseline 24h-UFC and upper limit normal (ULN) reductions were magnified (&amp;gt;25%) when assessed by IA vs GC-MS leading to higher falsely KTZ efficacy and misleading biochemical classification of 15% of patients. Despite dosage, in patients taking MTP the IA cross-reactivity only decreased 0.55 which resulted in persistence of overestimated 24h-UFC results (factor of 1.33: 1, 95% CI, 1.09 -1.76). When comparing MTP efficacy among IA vs GC-MS results, ULN baseline reductions were &amp;gt;50% different in some patients, while in others, similar reductions were found. Acceptable 24h-UFC method agreement between GC-MS and IA before treatment (R2 = 0.954) declined in patients under KTZ (R2 = 0.632) but not in MTP (R2 = 0.917). Urinary excretion changes of 22 urinary steroid metabolites explained 86% of the 24h-UFC*IA interference. 6β-Hydroxy-cortisol (6β-OH-cortisol) was the metabolite determining most of the 24h-UFC cross-reactivity variability (R2= 48.3%, p = 0.000) followed by 20α-dihydrocortisol (20α-DHF) (R2= 24.4%, p = 0.000). Each treatment induced a unique urinary steroid signature (p &amp;lt;0.001) and larger excretion reductions of 6β-OH-cortisol, 20α-DHF and 18-Hydroxy-cortisol in patients with KTZ elucidated the higher decrement of interference in the 24h-UFC by IA, compared to MTP treated patients (p = 0.001). Conclusion KTZ and MTP alter distinctly the urinary excretion of IA cross-reactive steroid metabolites, thus decreasing the cross-reactive interference of 24h-UFC*IA determinations present before treatment in a unique manner. Consequently, this interference reduction in 24h-UFC*IA leads to loss of method agreement with GC-MS and high risk of overestimating the biochemical impact of KTZ and MTP in controlling CS because of poor reliability of ULN. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Sunday, June 12, 2022 12:36 p.m. - 12:41 p.m.

Ketoconazole- and Metyrapone-Induced Reductions on Urinary Steroid Metabolites Alter the Urinary Free Cortisol Immunoassay Reliability in Cushing Syndrome.

IntroductionTwenty-four-hour urinary free cortisol (24h-UFC) is the most used test for follow-up decision-making in patients with Cushing syndrome (CS) under medical treatment. However, 24h-UFC determinations by immunoassays (IA) are commonly overestimated because of steroid metabolites’ cross-reaction. It is still uncertain how ketoconazole (KTZ)- and metyrapone (MTP)-induced changes on the urinary steroid metabolites can alter the 24h-UFC*IA determinations’ reliability.Methods24h-UFC was analyzed by IA and gas chromatography-mass spectrometry (GC-MS) in 193 samples (81 before treatment, 73 during KTZ, and 39 during MTP) from 34 CS patients. In addition, urinary steroidome was analyzed by GC-MS on each patient before and during treatment.ResultsBefore treatment, 24h-UFC*IA determinations were overestimated by a factor of 1.75 (95% CI 1.60–1.94) compared to those by GC-MS. However, during KTZ treatment, 24h-UFC*IA results were similar (0.98:1) to those by GC-MS (95% CI, 0.83–1.20). In patients taking MTP, IA bias only decreased 0.55, resulting in persistence of an overestimation factor of 1.33:1 (95% CI, 1.09–1.76). High method agreement between GC-MS and IA before treatment (R2 = 0.954) declined in patients under KTZ (R2 = 0.632) but not in MTP (R2 = 0.917). Upper limit normal (ULN) reductions in patients taking KTZ were 27% larger when using 24h-UFC*IA compared to 24h-UFC*GC-MS, which resulted in higher false efficacy and misleading biochemical classification of 15% of patients. Urinary excretion changes of 22 urinary steroid metabolites explained 86% of the 24h-UFC*IA interference. Larger urinary excretion reductions of 6β-hydroxy-cortisol, 20α-dihydrocortisol, and 18-hydroxy-cortisol in patients with KTZ elucidated the higher 24h-UFC*IA bias decrement compared to MTP-treated patients.ConclusionKTZ and MTP alter the urinary excretion of IA cross-reactive steroid metabolites, thus decreasing the cross-reactive interference of 24h-UFC*IA determinations present before treatment. Consequently, this interference reduction in 24h-UFC*IA leads to loss of method agreement with GC-MS and high risk of overestimating the biochemical impact of KTZ and MTP in controlling CS because of poor reliability of reference ranges and ULN.

Inverse autonomic stress reactivity in depressed patients with and without prior history of depression

IntroductionThere is a considerable association between major depressive disorder (MDD) and cardiovascular disease, most possibly relying on abnormalities in the autonomic nervous system (ANS)-related cardiac reactivity, although the exact underlying pathophysiological pathway is unclear.ObjectivesThis study tends to shed some additional light on this background by investigating ANS reactivity in MDD with respect to previous depression history through an objective stress challenge paradigm.MethodsThe study assessed the effects of an overnight hypothalamus-pituitary-adrenal (HPA) axis stimulation with metyrapone (MET) on baseline ANS activity through linear and non-linear heart rate variability (HRV) measures in the morning of two continuous days in a group of 14 physically healthy, antidepressant-free patients with clinical, non-psychotic MDD, to investigate differences in autonomic reactivity with respect to prior MDD history.ResultsThe main findings of this study include statistically significant time x group interactions with respect to several HRV measures, suggesting substantial differences on autonomic reactivity between patients with and without depression history. Hereby, recurrent-episode MDD patients showed lower vagal activity, while first-episode MDD patients increased PNS activity after HPA axis stimulation.ConclusionsThese findings indicate that HPA axis stimulation in MDD patients leads to inverse vagal response according to MDD history. We suggest that chronic stress system overactivation, as found in MDD, might lead to a progressive inversion of the original stress response through HPA axis and ANS divergence over the course of a recurrent illness. HRV could, thus, represent a significant biomarker in MDD with temporal sensitivity.

Coordinated Action of Corticotropin-Releasing Hormone and Cortisol Shapes the Acute Stress-Induced Behavioural Response in Zebrafish

Introduction: The stress response mediated by the hypothalamus-pituitary-adrenal (HPA) axis activation is highly conserved in vertebrates. Hyperactivity is one such established acute stress response, and corticotropin-releasing hormone (CRH), the primary step in HPA activation, signalling has been implicated in this stressor-mediated behaviour. However, whether CRH mediates the acute behavioural effects either alone or in conjunction with glucocorticoids (GCs) are far from clear. We hypothesized that the CRH receptor 1 (CRHR1)-mediated rise in GCs post-stress is necessary for the initiation and maintenance of the acute stress-related behaviour. Methods: We first generated zebrafish (Danio rerio) with a mutation in the CRHR1 gene (CRHR1-KO) to assess the function of CRH. The behavioural readout utilized for this study was the locomotor activity of larval zebrafish in response to an acute light exposure, a protocol that freezes the larvae in response to the light stimulus. To test whether cortisol signalling is involved in the stress-mediated hyperactivity, we treated wildtype fish with metyrapone (MET), an inhibitor of 11β-hydroxylase, to suppress cortisol production. The temporal role for cortisol signalling in the stress-related hyperactivity was tested using the glucocorticoid receptor knockout (GRKO) and mineralocorticoid receptor knockout (MRKO) zebrafish mutants. Results: CRHR1-KO larvae did not increase cortisol, the principal GC in teleosts, post-stress, confirming a functional knockout. An acute stress resulted in the hyperactivity of the larvae in light at 15, 60, and 240 min post-stress, and this was absent in CRHR1-KO larvae. Addition of MET effectively blocked the attendant rise in cortisol post-stress; however, the stress-mediated hyperactivity was inhibited only at 60 and 240 min but not at 15 min post-stress. Addition of human CRH peptide caused hyperactivity at 15 min, and this response was also abolished in the CRHR1-KO mutants. The stress-induced hyperactivity was absent in the MRKO fish, while GRKO mutants showed transient effects. Conclusions: The results suggest that the stress-induced hyperactivity is induced by the CRH/CRHR1 system, while the temporal activation of cortisol production and the associated GR/MR signalling is essential for prolonging the stressor-induced hyperactivity. This study underscores the importance of systems-level analysis to assess stress responsivity.

Pesticide tolerance induced by a generalized stress response in wood frogs (Rana sylvatica).

Increasing evidence suggests that phenotypic plasticity can play a critical role in ecotoxicology. More specifically, induced pesticide tolerance, in which populations exposed to a contaminant show increased tolerance to the contaminants later, has been documented in multiple taxa. However, the physiological mechanisms of induced tolerance remain unclear. We hypothesized that induced pesticide tolerance is the result of a generalized stress response based on previous studies showing that both natural stressors and anthropogenic stressors can induce tolerance to pesticides. We tested this hypothesis by first exposing larval wood frogs (Rana sylvatica) to either an anthropogenic stressor (sublethal carbaryl concentration), a natural stressor (cues from a caged predator), or a simulated stressor via exogenous exposure to the stress hormone corticosterone (125 nM). We also included treatments that inhibited corticosterone synthesis with the compound metyrapone (MTP). We then exposed the larvae to a lethal carbaryl treatment to assess time to death. We found that prior exposure to 125 nM of exogenous CORT and predator cues induced tolerance to a lethal concentration of carbaryl through a slight delay in time to death. Pre-exposure to sublethal carbaryl, as well as MTP alone or in combination with predator cues, did not induce tolerance to the lethal carbaryl concentration relative to the ethanol vehicle control treatment. Our study provides evidence that pesticide tolerance can be induced by a generalized stress response both in the presence and absence (exogenous CORT) of specific cues and highlights the importance of considering physiological ecology and environmental context in ecotoxicology.

Inverse autonomic stress reactivity in depressed patients with and without prior history of depression

BackgroundThere is a considerable association between major depressive disorder (MDD) and cardiovascular disease, most possibly relying on abnormalities in the autonomic nervous system (ANS)-related cardiac reactivity, although the exact underlying pathophysiological pathway is unclear. This study tends to shed some additional light on this background by investigating ANS reactivity in MDD with respect to previous depression history through an objective stress challenge paradigm. MethodsThe study assessed the effects of an overnight hypothalamus-pituitary-adrenal (HPA) axis stimulation with metyrapone (MET) on baseline ANS activity through linear and non-linear heart rate variability (HRV) measures in the morning of two continuous days in a group of 14 physically healthy, antidepressant-free patients with clinical, non-psychotic MDD, to investigate differences in autonomic reactivity with respect to prior MDD history. ResultsThe main findings of this study include statistically significant time × group interactions with respect to several HRV measures, suggesting substantial differences on autonomic reactivity between patients with and without depression history. Hereby, recurrent-episode MDD patients showed lower vagal activity, while first-episode MDD patients increased PNS activity after HPA axis stimulation. ConclusionsThese findings indicate that HPA axis stimulation in MDD patients leads to inverse vagal response according to MDD history. We suggest that chronic stress system overactivation, as found in MDD, might lead to a progressive inversion of the original stress response through HPA axis and ANS divergence over the course of a recurrent illness. HRV could, thus, represent a significant biomarker in MDD with temporal sensitivity.

Affinity Effects on the Release of Non-Conventional Antifibrotics from Polymer Depots.

For many chronic fibrotic conditions, there is a need for local, sustained antifibrotic drug delivery. A recent trend in the pharmaceutical industry is the repurposing of approved drugs. This paper investigates drugs that are classically used for anthelmintic activity (pyrvinium pamoate (PYR)), inhibition of adrenal steroidgenesis (metyrapone (MTP)), bactericidal effect (rifampicin (RIF), and treating iron/aluminum toxicity (deferoxamine mesylate (DFOA)), but are also under investigation for their potential positive effect in wound healing. In this role, they have not previously been tested in a localized delivery system suitable for obtaining the release for the weeks-to-months timecourse needed for wound resolution. Herein, two cyclodextrin-based polymer systems, disks and microparticles, are demonstrated to provide the long-term release of all four tested non-conventional wound-healing drugs for up to 30 days. Higher drug affinity binding, as determined from PyRx binding simulations and surface plasmon resonance in vitro, corresponded with extended release amounts, while drug molecular weight and solubility correlated with the improved drug loading efficiency of cyclodextrin polymers. These results, combined, demonstrate that leveraging affinity interactions, in combination with drug choice, can extend the sustained release of drugs with an alternative, complimentary action to resolve wound-healing and reduce fibrotic processes.

Acute hypercortisolemia inhibits innate immune parameters in Piaractus mesopotamicus experimentally infected with Aeromonas hydrophila

Cortisol is a key modulator of the immune response in fish, and considered an essential molecule for adaptation of animals to stress and overcoming the stressor. Antagonist effects, pro and anti-inflammatory have been associated to acute increases in cortisol levels, however knowledge about the mechanisms of this regulation in tropical teleosts is scarce. The effects of pharmacological inhibition of cortisol release with metyrapone (MTP) on innate immune response were investigated for the freshwater teleost, Piaractus mesopotamicus, using Aeromonas hydrophila to induce an inflammatory-immune response. Pacus were randomly distributed into four experimental groups. T1: basal control, without any previous handling. T2: without MTP treatment, inoculated via swim bladder with lethal concentration 50%-96 h (LC50- 96h) of A. hydrophila. T3: supplemented with 30mgkg−1 MTP in the diet for five days, injected on the sixth day with 50mgkg−1 live weight (LW) MTP and immediately inoculated with the bacteria. T4: supplemented with MTP, without bacterial inoculation. Blood samples were taken at 1, 3, 6 and 9 h post infection (hpi), for assessment of cortisolemia, glycemia, hematological and innate immunity parameters. The results revealed that high cortisol concentrations, during the initial phase of A. hydrophila infection, inhibited the immune-inflammatory response. Fish treated with MTP and inoculated with the bacterium showed significant higher total blood leukocytes and lymphocytes numbers, blood phagocyte respiratory activity and plasma lytic activity when compare to infected fish that did not receive MTP. It was concluded that the inhibition of cortisol release favored the immune-inflammatory response during the initial phase of bacterial infection.

Prenatal metyrapone treatment modulates neonatal cerebrovascular structure, function, and vulnerability to mild hypoxic-ischemic injury.

This study explored the hypothesis that late gestational reduction of corticosteroids transforms the cerebrovasculature and modulates postnatal vulnerability to mild hypoxic-ischemic (HI) injury. Four groups of Sprague-Dawley neonates were studied: 1) Sham-Control, 2) Sham-MET, 3) HI-Control, and 4) HI-MET. Metyrapone (MET), a corticosteroid synthesis inhibitor, was administered via drinking water from gestational day 11 to term. In Shams, MET administration 1) decreased reactivity of the hypothalamic-pituitary-adrenal (HPA) axis to surgical trauma in postnatal day 9 (P9) pups by 37%, 2) promoted cerebrovascular contractile differentiation in middle cerebral arteries (MCAs), 3) decreased compliance ≤46% and increased depolarization-induced calcium mobilization in MCAs by 28%, 4) mildly increased hemispheric cerebral edema by 5%, decreased neuronal degeneration by 66%, and increased astroglial and microglial activation by 10- and 4-fold, respectively, and 5) increased righting reflex times by 29%. Regarding HI, metyrapone-induced fetal transformation 1) diminished reactivity of the HPA axis to HI-induced stress in P9/P10 pups, 2) enhanced HI-induced contractile dedifferentiation in MCAs, 3) lessened the effects of HI on MCA compliance and calcium mobilization, 4) decreased HI-induced neuronal injury but unmasked regional HI-induced depression of microglial activation, and 5) attenuated the negative effects of HI on open-field exploration but enhanced the detrimental effects of HI on negative geotaxis responses by 79%. Overall, corticosteroids during gestation appear essential for normal cerebrovascular development and glial quiescence but induce persistent changes that in neonates manifest beneficially as preservation of postischemic contractile differentiation but detrimentally as worsened ischemic cerebrovascular compliance, increased ischemic neuronal injury, and compromised neurobehavior.

SAT-446 No Central Adrenal Insufficiency Found in Adults with Prader-Willi Syndrome Tested by Multiple-Dose Metyrapone Test

Introduction Individuals with Prader-Willi syndrome (PWS) have hypothalamic dysfunction, with deficiencies of several hypothalamic-pituitary axes. Prevalence of central hypogonadism, hypothyroidism and growth hormone deficiency are increased in comparison with non-PWS individuals. Central adrenal insufficiency (CAI) has also been reported in PWS. Several studies, using different testing modalities, have reported strikingly differing prevalences of CAI in PWS, ranging from 0% to 60%. It is speculated that CAI may be responsible, in part, for high mortality (3%) in patients with PWS. If CAI is present, timely diagnosis and treatment is needed in order to prevent avoidable mortality. Due to the lack of consensus, there are no guidelines or recommendations on the appropriate evaluation and management of CAI in the adult PWS population. Many adults patients with PWS receive standard hydrocortisone (HC) stress dose during physical and/or psychological stress. Frequent administration of HC increases the risk of obesity, hypertension, osteoporosis and diabetes, already a major problem in adults with PWS. It is therefore of utmost importance to assess the prevalence of CAI in order to prevent overtreatment with HC. Methods We screened medical histories of all patients for symptoms of CAI. We performed multiple dose metyrapone (MTP) test in 45 adults with genetically confirmed PWS. At day one, oral MTP 750 mg (Laboratoire HRA Pharma, Paris, France) was administered orally six times (every 4 hours, starting from 8h00). At 0800 h on day two, blood was drawn for determining 11-deoxycortisol (11-DOC) levels. At both days, blood was drawn after 8 hours fasting. Levels of 11-DOC greater than 7.6 g/dL (230 nmol/L) were classified as adrenal sufficiency. Results Mean age of participants was 30.9. Seventeen were using GH treatment since childhood. Male/female ratio was 28/17. Revision of medical histories revealed that a substantial part of patients had undergone operations or had infections without receiving HC stress dose, without any negative consequences. All 45 patients had 11-DOC greater than 7.6 g/dL during MTP test and therefore CAI was excluded in all patients. MTP test was tolerated well by all individuals. Conclusion Central adrenal insufficiency was absent in 45 adults with Prader-Willi syndrome assessed by a multiple dose metyrapone test. This indicates that CAI is rare, or even absent, in adults with PWS. Based on these results, we recommend to perform MTP test instead of routinely prescribing HC stress dose in adults with PWS.

Metyrapone treatment in Cushing's syndrome: a real-life study.

Medical treatment is increasingly used in patients with Cushing's syndrome (CS). Metyrapone (MET) is an inhibitor of 11β-hydroxylase: retrospective studies reported a decrease of cortisol secretion in 50% of cases. We evaluated the effectiveness of MET in an observational study, considering the normalization of urinary-free cortisol (UFC) and late-night salivary cortisol (LNSC) levels. We enrolled 31 patients with CS, treated with MET for at least 1 month (16 for primary treatment and 15 after surgical failure). A planned dose-titration regimen considering baseline UFC levels was adopted; MET dose was uptitrated until UFC normalization, surgery, or side effect occurrence. UFC and LNSC levels were routinely measured by liquid chromatography-tandem mass spectrometry. Patients were treated with a median dose of 1000 mg for 9 months. UFC and LNSC decreased quickly after the first month of treatment (-67 and -57% from baseline), with sustained UFC normalization up to 12 and 24 months (in 13 and 6 patients, respectively). UFC and LNSC normalized later (after 3-6 months) in patients with severe hypercortisolism (>5-fold baseline UFC). Regarding the last visit, 70 and 37% of patients normalized UFC and LNSC, respectively. Body weight reduction (-4 kg) was observed after UFC normalization. Severe side effects were not reported, half of the female patients complained of hirsutism, and blood pressure was not increased. MET therapy is a rapid-onset, long-term effective, and safe medical treatment in CS patients, achieving UFC normalization (in 70% of patients) more than cortisol rhythm recovery (in 37% of subjects).

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.

Usefulness of Simultaneous Measurement of Plasma Steroids, Including Precursors, for the Evaluation of Drug Effects on Adrenal Steroidogenesis in Rats.

The aim of this study was to evaluate the usefulness of simultaneous measurement of plasma steroids, including precursors, for the evaluation of drug effects on adrenal steroidogenesis in vivo. Plasma concentrations of corticosterone and its precursors were examined in rats dosed with compounds that affect adrenal steroidogenesis via different modes of action as well as the relationships of the changes with blood chemistry and adrenal histopathology. Male rats were dosed with tricresyl phosphate, aminoglutethimide, trilostane (TRL), metyrapone (MET), ketoconazole (KET), or mifepristone for 7 days. In the TRL, MET, and KET groups, precursor levels were markedly increased, while there were no significant changes in the corticosterone level, suggesting that the precursors are more sensitive biomarkers to detect the effect on adrenal steroidogenesis. Also, the precursors with increased levels were those that are normally metabolized by the inhibited enzymes, reflecting the modes of action of the compounds. In addition, different patterns of changes were observed in blood chemistry and histopathology, supporting the mechanism suggested by the steroid changes. These results show that simultaneous measurement of plasma steroids, including precursors, can be a valuable method to sensitively evaluate drug effects on adrenal steroidogenesis and to investigate the underlying mechanisms.

A polymorphism in the CYP17A1 gene influences the therapeutic response to steroidogenesis inhibitors in Cushing's syndrome.

Steroidogenesis inhibitors, such as ketoconazole (KTZ) and metyrapone (MTP), are used to lower hypercortisolism in patients with Cushing's syndrome (CS). Cortisol normalization is not reached in all patients taking these medications. To test the hypothesis that variants in genes affecting steroidogenesis contribute to different responses to KTZ and/or MTP in patients with CS. Fifty-four CS patients (46 women; mean [±SD] age, 39.7±12.7; 83% with Cushing's disease [CD] and 17% with an adrenal adenoma) preoperatively treated with KTZ (20%), MTP (37%) or a combination of both (43%). Thirty-nine of these (72%) were described in a previous study investigating the outcome of preoperative treatment with KTZ or MTP in CS patients. Following single-nucleotide polymorphisms (SNPs) were analysed: rs6410 (CYP11B1 gene), rs1799998 and rs4546 (CYP11B2 gene), and rs6163 (CYP17A1 gene). The associations between SNPs and cortisol levels at the end of medical treatment were evaluated. Normalization of urinary free cortisol (UFC) was achieved in 50% of patients after 5months of treatment. Patients carrying the CC genotype of SNP rs6163 were more likely to be controlled than AC/AA (OR 0.25 [95%CI, 0.075-0.88]; P=.031). When only patients reaching eucortisolism after medical treatment were analysed, median interquartile range (IQR) duration of treatment was shorter in patients carrying the CC genotype of SNP rs6163 as compared to AA/AC carriers (4 [4.57]months vs 5.2 [6.1]months; P=.026). A polymorphism in the CYP17A1 gene was associated with the response to steroidogenesis inhibitors in CS. Genetic differences in the steroidogenic enzymes might account for inter-individual variations in the responsiveness to adrenal-blocking agents.

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.

Postnatal treatment with metyrapone attenuates the effects of diet-induced obesity in female rats exposed to early-life stress.

Experimental studies in rodents have shown that females are more susceptible to exhibiting fat expansion and metabolic disease compared with males in several models of fetal programming. This study tested the hypothesis that female rat pups exposed to maternal separation (MatSep), a model of early-life stress, display an exacerbated response to diet-induced obesity compared with male rats. Also, we tested whether the postnatal treatment with metyrapone (MTP), a corticosterone synthase inhibitor, would attenuate this phenotype. MatSep was performed in WKY offspring by separation from the dam (3 h/day, postnatal days 2-14). Upon weaning, male and female rats were placed on a normal (ND; 18% kcal fat) or high-fat diet (HFD; 60% kcal fat). Nondisturbed littermates served as controls. In male rats, no diet-induced differences in body weight (BW), glucose tolerance, and fat tissue weight and morphology were found between MatSep and control male rats. However, female MatSep rats displayed increased BW gain, fat pad weights, and glucose intolerance compared with control rats (P < 0.05). Also, HFD increased plasma corticosterone (196 ± 51 vs. 79 ± 18 pg/ml, P < 0.05) and leptin levels (1.8 ± 0.4 vs. 1.3 ± 0.1 ng/ml, P < 0.05) in female MatSep compared with control rats, whereas insulin and adiponectin levels were similar between groups. Female control and MatSep offspring were treated with MTP (50 µg/g ip) 30 min before the daily separation. MTP treatment significantly attenuated diet-induced obesity risk factors, including elevated adiposity, hyperleptinemia, and glucose intolerance. These findings show that exposure to stress hormones during early life could be a key event to enhance diet-induced obesity and metabolic disease in female rats. Thus, pharmacological and/or behavioral inflection of the stress levels is a potential therapeutic approach for prevention of early life stress-enhanced obesity and metabolic disease.

Abstract 078: Postnatal Treatment with Metyrapone Attenuates the Effects of Early Life Stress (ELS) on Diet-induced Obesity in Female Rats

Clinical studies have shown a positive correlation between ELS and the development of cardiometabolic disease, particularly affecting women. We previously reported that male rats exposed to Maternal Separation (MatSep), a model of ELS in rodents, do not develop exaggerated diet-induced obesity. Thus, this study tested the hypothesis that MatSep exacerbates the response to an obesogenic diet in female rats. Also, we tested whether the postnatal treatment with metyrapone (MTP), a corticosterone synthase inhibitor, would attenuate this phenotype. MatSep was performed in WKY offspring during 3 hours/day from postnatal days 2-14. Non-disturbed littermates were used as controls. Female rat offspring were untreated or treated with MTP (50 mg/kg, i.p.), 30 minutes prior the daily separation. Upon weaning, rats were placed on regular chow (ND, 18% kcal fat) or HFD (60% kcal fat) for 12 weeks. Despite no differences in food intake (metabolism cages) and blood pressure (DSI radiotelemetry) MatSep exaggerated body weight gain and fat pad weights (p&lt;0.05) in response to HFD. Also, MatSep increased plasma corticosterone (189±48 vs. 79±18 pg/ml, p&lt;0.05) and leptin (2.1±0.4 vs. 1.5±0.4 ng/ml, p&lt;0.05) levels compared to control while insulin and adiponectin levels were similar between groups. Oral glucose tolerance test was impaired in MatSep rats showing a greater AUC compared to control rats (p&lt;0.05). Importantly, MTP-treated female MatSep rats showed significantly attenuated diet-induced obesity, glucose intolerance, plasma corticosterone and leptin levels. Histological analysis revealed that MTP treatment ameliorated the adipocyte size in visceral fat from MatSep rats as well (p&lt;0.05). Gene expression in liver indicated that glucose 6 phospatase, but not other gluconeogenic enzymes, were increased in obese MatSep rats, whereas the MTP treatment abrogated this effect. The visceral fat gene expression showed lower levels of insulin receptor in MatSep rats, but no differences in other genes related to the glucose disposal. Overall, these data reveal that female MatSep rats display a greater susceptibility for the synergistic effect between obesogenic diet and ELS compared to male rats, and this effect may be linked to early life exposure to stress hormones.