Posaconazole-Induced Hypertension Due to Inhibition of 11β-Hydroxylase and 11β-Hydroxysteroid Dehydrogenase 2

We describe two cases of hypertension and hypokalemia due to mineralocorticoid excess caused by posaconazole treatment of coccidioidomycosis and rhinocerebral mucormycosis infections, respectively. Clinical laboratory evaluations, including a comprehensive analysis of blood and urine steroid profiles, revealed low renin and aldosterone and indicated as the underlying mechanism primarily a block of 11β-hydroxylase activity in patient 1, whereas patient 2 displayed weaker 11β-hydroxylase but more pronounced 11β-hydroxysteroid dehydrogenase 2 inhibition. The results show that both previously suggested mechanisms must be considered and emphasize significant interindividual differences in the contribution of each enzyme to the observed mineralocorticoid excess phenotype. The mineralocorticoid symptoms of patient 1 resolved after replacement of posaconazole therapy by isavoconazole, and posaconazole dosage de-escalation ameliorated the effects in patient 2. By providing a thorough analysis of the patients' blood and urine steroid metabolites, this report adds further evidence for two individually pronounced mechanisms of posaconazole-induced hypertension and hypokalemia. The elucidation of the factors responsible for the individual phenotype warrants further research

The Anabolic Androgenic Steroid Fluoxymesterone Inhibits 11β-Hydroxysteroid Dehydrogenase 2-Dependent Glucocorticoid Inactivation

Anabolic androgenic steroids (AAS) are testosterone derivatives used either clinically, in elite sports, or for body shaping with the goal to increase muscle size and strength. Clinically developed compounds and nonclinically tested designer steroids often marketed as food supplements are widely used. Despite the considerable evidence for various adverse effects of AAS use, the underlying molecular mechanisms are insufficiently understood. Here, we investigated whether some AAS, as a result of a lack of target selectivity, might inhibit 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2)-dependent inactivation of glucocorticoids. Using recombinant human 11β-HSD2, we observed inhibitory effects for several AAS. Whereas oxymetholone, oxymesterone, danazol, and testosterone showed medium inhibitory potential, fluoxymesterone was a potent inhibitor of human 11β-HSD2 (half-maximal inhibitory concentration [IC50] of 60-100nM in cell lysates; IC50 of 160nM in intact SW-620, and 530nM in MCF-7 cells). Measurements with rat kidney microsomes and lysates of cells expressing recombinant mouse 11β-HSD2 revealed much weaker inhibition by the AAS tested, indicating that the adverse effects of AAS-dependent 11β-HSD2 inhibition cannot be investigated in rats and mice. Furthermore, we provide evidence that fluoxymesterone is metabolized to 11-oxofluoxymesterone by human 11β-HSD2. Structural modeling revealed similar binding modes for fluoxymesterone and cortisol, supporting a competitive mode of inhibition of 11β-HSD2-dependent cortisol oxidation by this AAS. No direct modulation of mineralocorticoid receptor (MR) function was observed. Thus, 11β-HSD2 inhibition by fluoxymesterone may cause cortisol-induced MR activation, thereby leading to electrolyte disturbances and contributing to the development of hypertension and cardiovascular diseas

Currently available murine Leydig cell lines can be applied to study early steps of steroidogenesis but not testosterone synthesis

Androgen biosynthesis in males occurs to a large extent in testicular Leydig cells. This study focused on the evaluation of three murine Leydig cell lines as potential screening tool to test xenobiotics interfering with gonadal androgen synthesis. The final step of testosterone (T) production in Leydig cells is catalyzed by the enzyme 17β-hydroxysteroid dehydrogenase 3 (17β-hsd3). The endogenous 17β-hsd3 mRNA expression and Δ4-androstene-3,17-dione (AD) to T conversion were determined in the murine cell lines MA-10, BLTK1 and TM3. Additionally, effects of 8-Br-cAMP and forskolin stimulation on steroidogenesis and T production were analyzed. Steroids were quantified in supernatants of cells using liquid chromatography-tandem mass spectrometry. Unstimulated cells incubated with AD produced only very low T but substantial amounts of the inactive androsterone. Stimulated cells produced low amounts of T, moderate amounts of AD, but high amounts of progesterone. Gene expression analyses revealed barely detectable 17β-hsd3 levels, absence of 17β-hsd5 (Akr1c6), but substantial 17β-hsd1 expression in all three cell lines. Thus, MA-10, BLTK1 and TM3 cells are not suitable to study the expression and activity of the gonadal T synthesizing enzyme 17β-hsd3. The low T production reported in stimulated MA-10 cells are likely a result of the expression of 17β-hsd1. This study substantiates that the investigated Leydig cell lines MA-10, BLTK1, and TM3 are not suitable to study gonadal androgen biosynthesis due to altered steroidogenic pathways. Furthermore, this study emphasizes the necessity of mass spectrometry-based steroid quantification in experiments using steroidogenic cells such as Leydig cells

Involvement Of Vascular Aldosterone Synthase In Phosphate-Induced Osteogenic Transformation Of Vascular Smooth Muscle Cells

Vascular calcification resulting from hyperphosphatemia is a major determinant of mortality in chronic kidney disease (CKD). Vascular calcification is driven by aldosterone-sensitive osteogenic transformation of vascular smooth muscle cells (VSMCs). We show that even in absence of exogenous aldosterone, silencing and pharmacological inhibition (spironolactone, eplerenone) of the mineralocorticoid receptor (MR) ameliorated phosphate-induced osteo-/chondrogenic transformation of primary human aortic smooth muscle cells (HAoSMCs). High phosphate concentrations up-regulated aldosterone synthase (CYP11B2) expression in HAoSMCs. Silencing and deficiency of CYP11B2 in VSMCs ameliorated phosphate-induced osteogenic reprogramming and calcification. Phosphate treatment was followed by nuclear export of APEX1, a CYP11B2 transcriptional repressor. APEX1 silencing up-regulated CYP11B2 expression and stimulated osteo-/chondrogenic transformation. APEX1 overexpression blunted the phosphate-induced osteo-/chondrogenic transformation and calcification of HAoSMCs. Cyp11b2 expression was higher in aortic tissue of hyperphosphatemic klotho-hypomorphic (kl/kl) mice than in wild-type mice. In adrenalectomized kl/kl mice, spironolactone treatment still significantly ameliorated aortic osteoinductive reprogramming. Our findings suggest that VSMCs express aldosterone synthase, which is up-regulated by phosphate-induced disruption of APEX1-dependent gene suppression. Vascular CYP11B2 may contribute to stimulation of VSMCs osteo-/chondrogenic transformation during hyperphosphatemia

Absence of hexose-6-phosphate dehydrogenase results in reduced overall glucose consumption but does not prevent 11β-hydroxysteroid dehydrogenase-1-dependent glucocorticoid activation

Hexose-6-phosphate dehydrogenase (H6PD) is thought to be the major source of NADPH within the endoplasmic reticulum (ER), determining 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) reaction direction to convert inert 11-oxo- to potent 11β-hydroxyglucocorticoids. Here, we tested the hypothesis whether H6pd knock-out (KO) in primary murine bone marrow-derived macrophages results in a switch from 11β-HSD1 oxoreduction to dehydrogenation, thereby inactivating glucocorticoids (GC) and affecting macrophage phenotypic activation as well as causing a more aggressive M1 macrophage phenotype. H6pdKO did not lead to major disturbances of macrophage activation state, although a slightly more pronounced M1 phenotype was observed with enhanced proinflammatory cytokine release, an effect explained by the decreased 11β-HSD1-dependent GC activation. Unexpectedly, ablation of H6pd did not switch 11β-HSD1 reaction direction. A moderately decreased 11β-HSD1 oxoreduction activity by 40-50% was observed in H6pdKO M1 macrophages but dehydrogenation activity was undetectable, providing strong evidence for the existence of an alternative source of NADPH in the ER. H6pdKO M1 activated macrophages showed decreased phagocytic activity, most likely a result of the reduced 11β-HSD1-dependent GC activation. Other general macrophage functions reported to be influenced by GC, such as nitrite production and cholesterol efflux, were altered negligibly or not at all. Importantly, assessment of energy metabolism using an extracellular flux analyzer and lactate measurements revealed reduced overall glucose consumption in H6pdKO M1 activated macrophages, an effect that was GC independent. The GC-independent influence of H6PD on energy metabolism and the characterization of the alternative source of NADPH in the ER warrant further investigations. ENZYMES: 11β-HSD1, EC 1.1.1.146; H6PD, EC 1.1.1.47

11β-Hydroxysteroid dehydrogenases control access of 7β,27-dihydroxycholesterol to retinoid-related orphan receptor γ

Oxysterols previously were considered intermediates of bile acid and steroid hormone biosynthetic pathways. However, recent research has emphasized the roles of oxysterols in essential physiologic processes and in various diseases. Despite these discoveries, the metabolic pathways leading to the different oxysterols are still largely unknown and the biosynthetic origin of several oxysterols remains unidentified. Earlier studies demonstrated that the glucocorticoid metabolizing enzymes, 11β-hydroxysteroid dehydrogenase (11β-HSD) types 1 and 2, interconvert 7-ketocholesterol (7kC) and 7β-hydroxycholesterol (7βOHC). We examined the role of 11β-HSDs in the enzymatic control of the intracellular availability of 7β,27-dihydroxycholesterol (7β27OHC), a retinoid-related orphan receptor γ (RORγ) ligand. We used microsomal preparations of cells expressing recombinant 11β-HSD1 and 11β-HSD2 to assess whether 7β27OHC and 7-keto,27-hydroxycholesterol (7k27OHC) are substrates of these enzymes. Binding of 7β27OHC and 7k27OHC to 11β-HSDs was studied by molecular modeling. To our knowledge, the stereospecific oxoreduction of 7k27OHC to 7β27OHC by human 11β-HSD1 and the reverse oxidation reaction of 7β27OHC to 7k27OHC by human 11β-HSD2 were demonstrated for the first time. Apparent enzyme affinities of 11β-HSDs for these novel substrates were equal to or higher than those of the glucocorticoids. This is supported by the fact that 7k27OHC and 7β27OHC are potent inhibitors of the 11β-HSD1-dependent oxoreduction of cortisone and the 11β-HSD2-dependent oxidation of cortisol, respectively. Furthermore, molecular docking calculations explained stereospecific enzyme activities. Finally, using an inducible RORγ reporter system, we showed that 11β-HSD1 and 11β-HSD2 controlled RORγ activity. These findings revealed a novel glucocorticoid-independent prereceptor regulation mechanism by 11β-HSDs that warrants further investigation

Acute Effects of 3,4-Methylenedioxymethamphetamine and Methylphenidate on Circulating Steroid Levels in Healthy Subjects

3,4-Methylenedioxymethamphetamine (MDMA, 'ecstasy') and methylphenidate are widely used psychoactive substances. MDMA primarily enhances serotonergic neurotransmission, and methylphenidate increases dopamine but has no serotonergic effects. Both drugs also increase norepinephrine, resulting in sympathomimetic properties. Here we studied the effects of MDMA and methylphenidate on 24-h plasma steroid profiles. Sixteen healthy subjects (eight men, eight women) were treated with single doses of MDMA (125 mg), methylphenidate (60 mg), MDMA + methylphenidate, and placebo on four separate days using a cross-over study design. Cortisol, cortisone, corticosterone, 11-dehydrocorticosterone, aldosterone, 11-deoxycorticosterone, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), androstendione, and testosterone were repeatedly measured up to 24-h using liquid-chromatography tandem mass-spectroscopy. MDMA significantly increased the plasma concentrations of cortisol, corticosterone, 11-dehydrocorticosterone, and 11-deoxycorticosterone and also tended to moderately increase aldosterone levels compared with placebo. MDMA also increased the sum of cortisol + cortisone and the cortisol/cortisone ratio, consistent with an increase in glucocorticoid production. MDMA did not alter the levels of cortisone, DHEA, DHEAS, androstendione, or testosterone. Methylphenidate did not affect any of the steroid concentrations, and it did not change the effects of MDMA on circulating steroids. In summary, the serotonin releaser MDMA has acute effects on circulating steroids. These effects are not observed after stimulation of the dopamine and norepinephrine systems with methylphenidate. The present findings support the view that serotonin rather than dopamine and norepinephrine mediates the acute pharmacologically-induced stimulation of the hypothalamic-pituitary-adrenal axis in the absence of other stressors. © 2014 S. Karger AG, Basel

Effects of lisdexamfetamine on plasma steroid concentrations compared with d-amphetamine in healthy subjects: A randomized, double-blind, placebo-controlled study

The novel d-amphetamine prodrug lisdexamfetamine is applied to treat attention-deficit/hyperactivity disorder (ADHD). d-Amphetamine releases dopamine and norepinephrine and stimulates the hypothalamic-pituitary-adrenal (HPA) axis, which may contribute to its reinforcing effects and risk of abuse. However, no data is currently available on the effects of lisdexamfetamine on circulating steroids. This randomized, double-blind, placebo-controlled, cross-over study evaluated the effects of equimolar doses of d-amphetamine (40 mg) and lisdexamfetamine (100 mg) and placebo on circulating steroids in 24 healthy subjects. Plasma steroid and d-amphetamine levels were determined up to 24 h. Delayed increase and peak levels of plasma d-amphetamine concentrations were observed following lisdexamfetamine treatment compared with d-amphetamine administration, however the maximal concentrations and total exposure (area under the curve [AUC]) were similar. Lisdexamfetamine and d-amphetamine significantly enhanced plasma levels of adrenocorticotropic hormone, glucocorticoids (cortisol, cortisone, corticosterone, 11-dehydrocorticosterone, and 11-deoxycortisol), androgens (dehydroepiandrosterone, dehydroepiandrosterone sulfate, and Δ4-androstene-3,17-dione [androstenedione]), and progesterone (only in men) compared with placebo. Steroid concentration-time curves were shifted to later time points due to a non-significantly later onset following lisdexamfetamine administration than after d-amphetamine, however maximal plasma steroid concentrations and AUCs did not differ between the active treatments. None of the active treatments altered plasma levels of the mineralocorticoids aldosterone and 11-deoxycorticosterone or the androgen testosterone compared with placebo. The effects of the amphetamines on glucocorticoid production were similar to those that were previously reported for methylphenidate (60 mg) but weaker than those for the serotonin releaser 3,4-methylenedioxymethamphetamine (MDMA; 125 mg) or direct serotonin receptor agonist lysergic acid diethylamide (LSD; 0.2 mg). Lisdexamfetamine produced comparable HPA axis activation and had similar pharmacokinetics than d-amphetamine, except for a delayed time of onset. Thus, serotonin (MDMA, LSD) may more effectively stimulate the HPA axis than dopamine and norepinephrine (D-amphetamine)

Chemical Tuning Enhances Both Potency Toward Nrf2 and In Vitro Therapeutic Index of Triterpenoids

The transcription factor Nrf2 protects against a number of experimental pathologies, and is a promising therapeutic target. The clinical investigation of a potent Nrf2-inducing agent, the triterpenoid (TP) bardoxolone methyl (BARD), was recently halted due to adverse cardiovascular events in chronic kidney disease patients, although the underlying mechanisms are yet to be resolved. The majority of small molecule Nrf2 inducers are electrophilic and trigger Nrf2 accumulation via the chemical modification of its redox-sensitive repressor Keap1. Therefore, it is pertinent to question whether the therapeutic targeting of Nrf2 could be hindered in many cases by the inherent reactivity of a small molecule inducer toward unintended cellular targets, a key mechanism of drug toxicity. Using H4IIE-ARE8L hepatoma cells, we have examined the relationship between (a) Nrf2 induction potency, (b) toxicity and (c) in vitro therapeutic index (ratio of b:a) for BARD and a number of other small molecule activators of Nrf2. We show that BARD exhibits the highest potency toward Nrf2 and the largest in vitro therapeutic index among compounds that have been investigated clinically (namely BARD, sulforaphane and dimethylfumarate). Through further examination of structurally related TPs, we demonstrate that an increase in potency toward Nrf2 is associated with a relatively smaller increase in toxicity, indicating that medicinal chemistry can be used to enhance the specificity of a compound as an inducer of Nrf2 signaling whilst simultaneously increasing its therapeutic index. These findings will inform the continuing design and development of drugs targeting Nrf

Chemical tuning enhances both potency toward Nrf2 and<em> in vitro</em> therapeutic index of triterpenoids

The transcription factor Nrf2 protects against a number of experimental pathologies, and is a promising therapeutic target. The clinical investigation of a potent Nrf2-inducing agent, the triterpenoid (TP) bardoxolone methyl (BARD), was recently halted due to adverse cardiovascular events in chronic kidney disease patients, although the underlying mechanisms are yet to be resolved. The majority of small molecule Nrf2 inducers are electrophilic and trigger Nrf2 accumulation via the chemical modification of its redox-sensitive repressor Keap1. Therefore, it is pertinent to question whether the therapeutic targeting of Nrf2 could be hindered in many cases by the inherent reactivity of a small molecule inducer toward unintended cellular targets, a key mechanism of drug toxicity. Using H4IIE-ARE8L hepatoma cells, we have examined the relationship between (a) Nrf2 induction potency, (b) toxicity and (c) in vitro therapeutic index (ratio of b:a) for BARD and a number of other small molecule activators of Nrf2. We show that BARD exhibits the highest potency toward Nrf2 and the largest in vitro therapeutic index among compounds that have been investigated clinically (namely BARD, sulforaphane and dimethylfumarate). Through further examination of structurally related TPs, we demonstrate that an increase in potency toward Nrf2 is associated with a relatively smaller increase in toxicity, indicating that medicinal chemistry can be used to enhance the specificity of a compound as an inducer of Nrf2 signaling whilst simultaneously increasing its therapeutic index. These findings will inform the continuing design and development of drugs targeting Nrf2.</p