The Renin-Angiotensin-Aldosterone System Regulates Sarcoidosis Granulomatous Inflammation.

Rationale: Sarcoidosis is a granulomatous disorder of unclear cause notable for abnormal elevation of blood and tissue ACE1 (angiotensin converting enzyme 1) levels and activity. ACE1 regulates the renin-angiotensin-aldosterone system (RAAS), the terminal product of which is aldosterone, which selectively engages mineralocorticoid receptors to promote inflammation. Objectives: We sought to determine whether the RAAS promotes sarcoidosis granuloma formation and related inflammatory responses. Methods: Using an established ex vivo model, we first determined whether aldosterone was produced by sarcoidosis granulomas and verified the presence of CYP11B2, the enzyme required for its production. We then evaluated the effects of selective inhibitors of ACE1 (captopril), angiotensin type 1 receptor (losartan), and mineralocorticoid receptors (spironolactone, eplerenone) on granuloma formation, reflected by computer image analysis-generated granuloma area, and selected cytokines incriminated in sarcoidosis pathogenesis. Measurements and Main Results: Aldosterone was spontaneously produced by sarcoidosis peripheral blood mononuclear cells, and both intra- and extracellular levels steadily increased during granuloma formation. In parallel, peripheral blood mononuclear cells were shown to express more CYP11B2 during granuloma formation. Significant inhibition of sarcoidosis granulomas and related cytokines (TNFα, IL-1ß, IFNγ, IL-10) was observed in response to pretreatments with captopril, losartan, spironolactone, or eplerenone, comparable to that of prednisone. Conclusions: The RAAS is intact in sarcoidosis granulomas and contributes significantly to early granuloma formation and to related inflammatory mediator responses, with important implications for clinical management.

Molecular analysis of 12 Chinese patients with 11ß-hydroxylase deficiency and in vitro functional study of 20 CYP11B1 missense variants.

Steroid 11ß-hydroxylase deficiency (11ß-OHD) is a rare autosomal recessive disorder caused by pathogenic variants of CYP11B1 gene. This study aimed to perform molecular analysis of a Chinese 11ß-OHD series and in vitro functional study of twenty CYP11B1 missense variants. Twelve Chinese patients with clinical diagnosis of 11ß-OHD were included in the study to analyze their molecular etiology. Genomic DNA of patients was extracted to be sequenced all coding exons and intronic flanking sequences of CYP11B1. Fourteen missense variants found in 12 patients mentioned above along with 6 missense variants previously reported by our team were evaluated functionally. Amino acid substitutions were analyzed with computational program to determine their effects on the three-dimensional structure of CYP11B1 protein. Clinical characteristics and hormone levels at baseline of the 18 patients carrying 18 missense variants aforementioned were recorded to perform genotype-phenotype correlation. A total of 21 rare variants including 9 novel and 12 recurrent ones were identified in 12 patients, out of which 17 were missense, 2 were nonsense, 1 was a splice site variant, and 1 was a deletion-insertion variant. Results of in vitro functional study revealed that 3 out of 20 missense mutants (p.Leu3Pro, p.Gly267Ser, and p.Ala367Ser) had partial enzyme activity and the other 17 had little enzymatic activity. The impairment degree of enzymatic activity in vitro functional study was also reflected in the severity degree of interaction change between the wild-type/mutant-type amino acid and its adjacent amino acids in three-dimensional model. In conclusion, the addition of 9 novel variants expands the spectrum of CYP11B1 pathogenic variants. Our results demonstrate that twenty CYP11B1 variants lead to impaired 11ß-hydroxylase activity in vitro. Visualizing these variants in the three-dimensional model structure of CYP11B1 protein can provide a plausible explanation for the results measured in vitro.

Identification of sulfonylpyrimidines as novel selective aldosterone synthase (CYP11B2) inhibitors.

4-[(5-[2-Methyl-5-(methylsulfonyl)pentan-2-yl]sulfonylpyrimidin-4-yl)amino]benzonitrile 2 was identified as a novel potent aldosterone synthase inhibitor. Compound 2 was found to inhibit human CYP11B2 in the nanomolar range, and showed an aldosterone-lowering effect in a furosemide-treated cynomolgus monkey model. Although human CYP11B2 has the high homology sequence with human CYP11B1, compound 2 showed more than 80 times higher selectivity over human CYP11B1 in vitro.

Cardiovascular risk assessments in patients with cortisol-producing adenoma: impact of clinical features and genetic characteristics.

The causes of adrenal Cushing's syndrome (CS) encompass a wide spectrum of adrenal cortisol proliferations that exhibit clinical and molecular heterogeneity. The aims of our study were to investigate whether clinical and molecular heterogeneity influences endothelial function and metabolic abnormalities in patients with cortisol-producing adenoma (CPA). We retrospectively enrolled 25 patients with CPA and 45 patients with essential hypertension (EH). All CPAs were studied by direct sequencing of PRKACA. Flow-mediated vasodilation (FMD), an index of vascular endothelial function, was significantly lower in CS and subclinical CS (SCS) groups than in the EH group. FMD impairment did not differ significantly between CS and SCS groups. No differences in FMD were seen between PRKACA mutant and wild-type groups. FMD correlated negatively with hemoglobin A1c (HbA1c) in both PRKACA mutant and wild-type groups, as well as in CS and SCS groups. After adrenalectomy, systolic blood pressure (SBP) and HbA1c decreased significantly from baseline in the CS group, and SBP and low-density lipoprotein cholesterol (LDL-C) decreased significantly from baseline in the SCS group. While SBP and LDL-C decreased significantly from baseline in patients with wild-type PRKACA, only HbA1c decreased from baseline in patients harboring PRKACA mutations. Our data showed that patients with CPA have impaired endothelial function compared with EH patients and suggest the need for strict monitoring of atherosclerosis, even in patients with SCS or without PRKACA mutation.

Expression and significance of CEMIP and CYP11B2 in serum in women with foetal growth restriction.

BACKGROUND: Foetal growth restriction (FGR) occurs when a foetus fails to reach its growth potential. This observational study assessed the expression and significance of cell migration-including protein (CEMIP) and aldosterone synthase (CYP11B2) in the serum of pregnant women with FGR. METHODS: 40 singleton FGR-suffered pregnant women, as well as 40 normal singleton pregnant women, were enrolled. The expression of CEMIP and CYP11B2 in serum was detected in early pregnancy. The correlations between parameters were evaluated. The predictive variables for FGR were determined. The diagnostic value of CEMIP and CYP11B2 for FGR was analysed. RESULTS: CEMIP and CYP11B2 mRNA expression in the serum of pregnant women with FGR decreased (both P < 0.001). CEMIP (95%CI: 0.802-0.921, P < 0.001) and CYP11B2 (95%CI: 0.795-0.907, P < 0.001) mRNA expression in serum and soluble fms like tyrosine kinase-1 (sFLT1)/placental growth factor (PlGF) ratio (95%CI: 0.866-0.974, P < 0.001) were independent predictors of FGR, and CEMIP (r = -0.578, P = 0.001) and CYP11B2 (r = -0.602, P < 0.001) mRNA expression in serum were negatively correlated with sFLT1/PlGF ratio. CEMIP (AUC = 0.741) and CYP11B2 (AUC = 0.764) mRNA expression in serum had good diagnostic value for FGR. CONCLUSION: The expression of CEMIP and CYP11B2 is reduced in the serum of pregnant women with FGR and may become new diagnostic markers for FGR.

Foetal growth restriction is the inability of the foetus to reach its growth potential in the uterus due to various factors. This study aimed to investigate the expression and significance of cell migration-including protein and aldosterone synthase in serum of pregnant women with foetal growth restriction. In our study, we found that the expression of cell migration-including protein and aldosterone synthase in serum of pregnant women with foetal growth restriction were decreased. Cell migration-including protein and aldosterone synthase expression was negatively correlated with soluble fms like tyrosine kinase-1/placental growth factor ratio. In addition, the study also found that cell migration-including protein and aldosterone synthase expression in serum had good diagnostic value for foetal growth restriction.

Immunohistochemical staining seems mandatory for individualizing and shortening follow-up in unilateral primary aldosteronism.

OBJECTIVE: The clinical significance of immunohistochemistry (IHC) for unilateral primary aldosteronism (PA) has been unclear. Individualized follow-up of PA patients could be in sight. Long-term outcomes of patients, classified based on IHC, need further investigation. We aimed to assess long-term clinical outcomes for unilateral PA, classifying patients based on IHC. DESIGN: A nationwide observational study, with up to 16 years follow-up, executed in 2007-2016 at Landspitali University Hospital, tertiary referral center. Patients were diagnosed and treated in line with the current guidelines. Haematoxylin and eosin (H&E) tissue slides were stained using CYP11B1 and -B2 antibodies. All cases were re-evaluated and classified according to the HISTALDO consensus. Outcomes were assessed using the PASO criteria. PATIENTS: All unilateral PA patients diagnosed in 2007-2016 in Iceland, 26 patients aged 28-73 years, who underwent adrenalectomy, were included. MEASUREMENTS: Aldosterone, renin, and cortisol values, use and dosage of antihypertensives, potassium supplementation, blood pressure and serum potassium pre-intervention and throughout follow-up, and histopathology results post-adrenalectomy. RESULTS: Following IHC, an aldosterone-producing nodule was seen in 12 adrenals, an aldosterone-producing adenoma in 10 and multiple aldosterone-producing micronodules in four. IHC altered histopathology from previous H&E diagnosis in 23% (6/26) of the patients. In total, 81% (21/26) of the patients had partial clinical success. Eight percent (2/26) of the patients needed potassium supplementation during follow-up. In the classical group, the AVS results were more determinative with significantly higher lateralization index (median 10.1 vs. 5.3, p = .04) and more contralateral suppression (median nondominant ratio 0.4 vs. 1.0, p = .03). One out of five patients with complete clinical success at 12 months post-op had severe relapse later, the other four were normotensive without antihypertensives for up to 10 years. CONCLUSIONS: We found IHC mandatory for accurate histopathologic diagnosis of PA. Our results support the importance of contralateral suppression when interpreting AVS results. Also, the study highlights the complicated assessment of clinical outcome and importance of aldosterone and renin measurements during follow-up.

Virtual screening and biological evaluation to identify pharmaceuticals potentially causing hypertension and hypokalemia by inhibiting steroid 11ß-hydroxylase.

Several drugs were found after their market approval to unexpectedly inhibit adrenal 11ß-hydroxylase (CYP11B1)-dependent cortisol synthesis. Known side-effects of CYP11B1 inhibition include hypertension and hypokalemia, due to a feedback activation of adrenal steroidogenesis, leading to supraphysiological concentrations of 11-deoxycortisol and 11-deoxycorticosterone that can activate the mineralocorticoid receptor. This results in potassium excretion and sodium and water retention, ultimately causing hypertension. With the risk known but usually not addressed in preclinical evaluation, this study aimed to identify drugs and drug candidates inhibiting CYP11B1. Two conceptually different virtual screening methods were combined, a pharmacophore based and an induced fit docking approach. Cell-free and cell-based CYP11B1 activity measurements revealed several inhibitors with IC50 values in the nanomolar range. Inhibitors include retinoic acid metabolism blocking agents (RAMBAs), azole antifungals, α2-adrenoceptor ligands, and a farnesyltransferase inhibitor. The active compounds share a nitrogen atom embedded in an aromatic ring system. Structure activity analysis identified the free electron pair of the nitrogen atom as a prerequisite for the drug-enzyme interaction, with its pKa value as an indicator of inhibitory potency. Another important parameter is drug lipophilicity, exemplified by etomidate. Changing its ethyl ester moiety to a more hydrophilic carboxylic acid group dramatically decreased the inhibitory potential, most likely due to less efficient cellular uptake. The presented work successfully combined different in silico and in vitro methods to identify several previously unknown CYP11B1 inhibitors. This workflow facilitates the identification of compounds that inhibit CYP11B1 and therefore pose a risk for inducing hypertension and hypokalemia.

Glucocorticoids are differentially synthesized along the murine and human respiratory tree.

BACKGROUND: Synthetic glucocorticoids (GC) are effective in the treatment of inflammatory diseases of the lung. However, long-term use leads to severe side effects. Endogenous GC can be synthesized locally, either de novo from cholesterol in a 11ß-hydroxylase (Cyp11b1)-dependent manner, or by reactivation from 11-dehydrocorticosterone/cortisone by 11ß-hydroxysteroid dehydrogenase 1 (Hsd11b1). We aimed to define the molecular pathways of endogenous GC synthesis along the respiratory tree to provide a basis for understanding how local GC synthesis contributes to tissue homeostasis. METHODS: Expression of steroidogenic enzymes in murine lung epithelium was analyzed by macroscopic and laser capture microdissection, followed by RT-qPCR. Flow cytometry analysis was performed to identify the cellular source of steroidogenic enzymes. Additionally, the induction of steroidogenic enzyme expression in the lung was analyzed after lipopolysaccharide (LPS) injection. mRNA and protein expression of steroidogenic enzymes was confirmed in human lung tissue by RT-qPCR and immunohistochemistry. Furthermore, GC synthesis was examined in ex vivo cultures of fresh tissue from mice and human lobectomy patients. RESULTS: We observed that the murine and human lung tissue differentially expresses synthesis pathway-determining enzymes along the respiratory tree. We detected Hsd11b1 expression in bronchial, alveolar, club and basal epithelial cells, whereas Cyp11b1 expression was detectable only in tracheal epithelial cells of mice. Accordingly, de novo synthesis of bioactive GC occurred in the large conducting airways, whereas reactivation occurred everywhere along the respiratory tree. Strikingly, Cyp11b1 but not Hsd11b1 expression was enhanced in the trachea upon LPS injection in mice. CONCLUSION: We report here the differential synthesis of bioactive GC along the murine and human respiratory tree. Thus, extra-adrenal de novo GC synthesis and reactivation may differentially contribute to the regulation of immunological and inflammatory processes in the lung.

Functional nodules in primary aldosteronism: identification of CXCR4 expression with 68Ga-pentixafor PET/CT.

OBJECTIVES: We analyzed the diagnostic efficiency of 68Ga-pentixafor PET/CT for functional nodules in primary aldosteronism (PA). Furthermore, we compared the correlation of CXCR4 expression with aldosterone synthase (CYP11B2) expression and PET/CT uptake in these patients. METHODS: We prospectively assessed 50 patients diagnosed with PA and 10 patients with non-functional adrenal adenoma (NFA). All patients underwent 68Ga-pentixafor PET/CT before adrenalectomy. Immunohistochemistry (IHC) was performed to detect the protein expression of CYP11B2 and the G-protein-coupled receptor CXCR4. RESULTS: CYP11B2 IHC revealed the presence of 43 functional nodules. Subsequently, 40/43 functional nodules could be detected on 68Ga-pentixafor PET/CT, while negative imaging findings were noted for 11/13 non-functional nodules (sensitivity, 93.0%; specificity, 84.6%). The optimum SUVmax cut-off for the identification of functional nodules was 8.95 (AUC 0.914 [0.828-1.000], p < 0.001). Regarding the size of functional nodules, diagnostic efficiency appeared to be much higher for nodules greater than 1 cm in size (sensitivity, up to 97.3%). Moreover, we examined the relationship between CXCR4 and CYP11B2 expression in 56 lesions. All 43 CYP11B2-positive nodules were CXCR4-positive, but one of the 13 CYP11B2-negative nodules (7.7%) showed false-positive staining for CXCR4. Moreover, the consistency between PET/CT uptake and CXCR4 staining results was 92.9% (52/56). CONCLUSIONS: At least 90% of functional nodules show positive uptake on 68Ga-pentixafor PET/CT, and the detection ability is much better for nodules with a diameter ≥ 1 cm. With its high sensitivity and specificity, 68Ga-pentixafor PET/CT can be considered a promising surgical decision-making tool for patients with PA. KEY POINTS: • 68Ga-pentixafor PET/CT could be a useful tool for the identification of functional adrenal nodules in APAs and even IHAs. • The diagnostic efficiency appears to be much higher for nodules ≥ 1 cm in size. • There is high consistency between the results of 68Ga-pentixafor PET/CT imaging and CXCR4 immunohistochemistry.

CYP11B2 inhibitor dexfadrostat phosphate suppresses the aldosterone-to-renin ratio, an indicator of sodium retention, in healthy volunteers.

AIMS: High aldosterone is a key driver of hypertension and long-term negative sequelae. We evaluated the safety and efficacy of dexfadrostat phosphate (DP13), a novel aldosterone synthase (CYP11B2) inhibitor, in healthy participants. METHODS: This randomized, double-blind, placebo-controlled study was conducted in two parts. In part A, a single-ascending dose escalation, 16 participants received oral DP13 1-16 mg. Part B was a multiple-ascending dose, sequential group study in which 32 participants received oral DP13 4, 8 or 16 mg once daily for 8 days. Safety and tolerability were monitored throughout. An adrenocorticotropic hormone (ACTH) stimulation test at maximal blood drug concentrations defined the dose range for multiple dosing. RESULTS: DP13 was well tolerated at all doses, with no serious adverse events. In part B, all DP13 doses (4, 8 and 16 mg) over 8 days effectively suppressed aldosterone production, increased the urinary sodium/potassium ratio, decreased plasma sodium and increased plasma potassium and renin levels compared with placebo, resulting in potent suppression of the aldosterone-to-renin ratio (ARR). Endocrine counter-regulation resulted in the 4 mg dose no longer sustaining 24-h aldosterone suppression after 8 days of treatment, unlike the 8- and 16 mg doses. There was no evidence of drug-induced adrenal insufficiency (ACTH stress challenge). CONCLUSIONS: In patients with excess aldosterone and ensuing sodium retention driving hypertension, managing sodium balance is critical. A CYP11B2 inhibitor like DP13, whose effectiveness can be monitored by a reduction in ARR, may prove valuable in managing aldosterone-dependent hypertension and primary aldosteronism.

Fluorinated aldosterone synthase (CYP11B2)-inhibitors for differential diagnosis between bilateral and unilateral conditions of primary aldosteronism.

The enzyme aldosterone synthase (CYP11B2) is specifically expressed in aldosterone-producing tissue of the adrenal cortex and is overexpressed in aldosterone-producing adenomas (APA). It therefore represents an ideal target for molecular imaging, particularly for the differential diagnosis between bilateral hyperplasia and unilateral APA in primary aldosteronism. However, the presence of the cortisol-producing enzyme 11ß-hydroxylase (CYP11B1) in the adrenal cortex remains very challenging owing to its high homology to CYP11B2. Within this study, we efficiently synthesized a variety of disubstituted fluorinated pyridines and pyrazines by Suzuki coupling reactions. These compounds were evaluated for their ability to inhibit CYP11B1 and CYP11B2 in transfected Y1 cells and in NCI-h295 cells. Several compounds were found to exhibit excellent affinity (IC50 < 10 nM) to CYP11B2 as well as strong selectivity (up to 125-fold) over CYP11B1. These findings support the further development of an analogous 18F-labelled PET tracer.

Unexpectedly high mutation rate of cyp11b1 compared to cyp21a2 in randomly-selected turkish women: a large screening study.

PURPOSE: Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders resulting from enzyme deficiencies associated with steroidogenesis. The clinical presentation of non-classic CAH (NCAH) in females is often indistinguishable from other hyperandrogenic disorders like polycystic ovary syndrome (PCOS). The data on the prevalence of NCAH in unselected women in the literature is scanty. The research aimed to evaluate the prevalence of NCAH, carrier frequencies, and the correlation between clinical symptoms and genotype in Turkish women. METHODS: The study group comprised two hundred and seventy randomly-selected unrelated asymptomatic women of reproductive age (18-45). Subjects were recruited from female blood donors. All volunteers underwent clinical examination and hormone measurements. The protein-encoding exons and exon-intron boundaries of the CYP21A2, CYP11B1, HSD3ß2 and CYP21A2 promoter were sequenced by direct DNA sequencing. RESULTS: After genotyping, seven (2.2%) individuals were diagnosed with NCAH. The heterozygous carrier frequencies of CYP21A2, CYP21A2 promoter, CYP11B1, and HSD3ß2 genes with 34, 34, 41, and 1 pathologic mutation were determined at 12.6%, 12.6%, 15.2%, and 0.37% of volunteers, respectively. Gene-conversion (GC) frequencies between CYP21A2/CYP21A1P and CYP11B1/CYP11B2 were determined as 10.4% and 14.8%, respectively. CONCLUSION: Despite GC-derived higher mutation frequency determined in the CYP11B1 gene, the reason for the low frequency of NCAH due to 11OHD compared to 21OHD might be that gene-conversion arises with active CYP11B2 rather than an inactive pseudogene. HSD3ß1 exhibits high homology with HSD3ß2 located on the same chromosome; remarkably, it demonstrates low heterozygosity and no GC, most probably the outcome of a tissue-specific expression pattern.

Pathology and gene mutations of aldosterone-producing lesions.

The human adrenal cortex secretes aldosterone and cortisol as major corticosteroids. For their production, CYP11B2 and CYP11B1 catalyze the last steps in the syntheses of aldosterone and cortisol, respectively. In our previous study, CYP11B2 was the first successfully purified from rat adrenals and human clinical samples and then was proved to be aldosterone synthase. We demonstrated the immunohistochemistry for CYP11B2 of both rats and humans and applied it clinically to visualize the functional histology of aldosterone-producing adenoma (APA) causing primary aldosteronism (PA). We discovered aldosterone-producing cell clusters (APCCs) and possible APCC-to-APA transitional lesions (pAATLs) and further visualized aldosterone-producing lesions for rare forms of PA including familial hyperaldosteronism type 3 and novel non-familial juvenile PA. Here we review the history of our research on aldosterone-producing lesions.

Pathogenicity of Congenital Adrenal Hyperplasia Induced by the p.P377L Mutation of CYP11B1.

CYP11B1 encodes an 11ß-hydroxylase that is involved in the catalysis of adrenal glucocorticoids and the production of cortisol. Mutations in CYP11B1 can result in congenital adrenal hyperplasia. We discovered a proband with a CYP11B1 gene mutation. Gene sequencing revealed a homozygous missense mutation of c.1130C > T in the 7th exon of the CYP11B1 gene that resulted in the change from Pro377 to leucine in the encoded protein. Based on the proband's clinical symptoms and the prognosis according to the database, this mutation may be harmful. However, the pathogenicity has not yet been reported. Thus, we created an expression vector for the mutation in vitro, transfected cells, observed the changes in gene expression, and determined its pathogenicity. To determine the pathogenicity of the CYP11B1 p.P377L mutation site through in vitro verification. The eukaryotic expression vector of the CYP11B1 mutation site was constructed in vitro, and the success of the construct was confirmed by sequencing. Fluorescence microscopy was used to determine the transfection effectiveness, GFP fluorescent tag labeling was used to detect changes in protein localization, and qRT‒PCR and Western blotting were used to detect CYP11B1 mRNA and protein expression. Sequencing revealed that the proband harbored a homozygous missense mutation of CYP11B1 (p.P377L). The expression of the protein decreased but the localization did not change when cells were transfected with the CYP11B1 mutation vector compared to the wild-type vector. The p.P377L mutation of CYP11B1 could affect protein expression and enzymatic activity and may be pathogenic.

Pertinence between risk of preeclampsia and the renin-angiotensin-aldosterone system (RAAS) gene polymorphisms: an updated meta-analysis based on 73 studies.

The aetiological mechanism of preeclampsia (PE) is unclear exactly, so we attempted to investigate the association between susceptibility to preeclampsia and renin-angiotensin-aldosterone system (RAAS) gene polymorphisms to explore the aetiology in terms of genetics. A systematic search was performed in electronic databases to identify relevant studies. Eventually 73 studies were enrolled, odds ratios were generated by 5 genetic models. In overall analysis, significant associations were detected for AGT M235T, AT1R A1166C and CYP11B2 C344T whereas negative correlation was shown for AGT T174M. As stratified by race and geography, AGT 235T allele and AT1R 1166C allele increased preeclampsia risk and AGT T174M was justified uncorrelated with preeclampsia. Our meta-analysis illustrated that AGT 235T allele and AT1R 1166C allele increased and CYP11B2 344T allele decreased preeclampsia risk while AGT T174M polymorphism did not change preeclampsia risk. Hence, pregnant women carrying high-risk genotypes need strengthened management to prevent and early identification of preeclampsia.

Structural and functional insights into aldosterone synthase interaction with its redox partner protein adrenodoxin.

Aldosterone is the major mineralocorticoid in the human body controlling blood pressure and salt homeostasis. Overproduction of aldosterone leads to primary aldosteronism, which is the most common form of secondary hypertension with limited treatment options. Production of aldosterone by cytochrome P450 11B2 (CYP11B2, aldosterone synthase) requires two reduction events with the electrons delivered by the iron/sulfur protein adrenodoxin. Very limited information is available about the structural and functional basis of adrenodoxin/CYP11B2 interaction, which impedes the development of new treatment options for primary aldosteronism. A systematic study was carried out to determine if adrenodoxin interaction with CYP11B2 might also have an allosteric component in addition to electron transfer. Indeed, local increases in adrenodoxin concentration promote binding of the substrate 11-deoxycorticosterone and the inhibitor osilodrostat (LCI699) in the active site-over 17 Å away-as well as enhance the inhibitory effect of this latter drug. The CYP11B2 structure in complex with adrenodoxin identified specific residues at the protein-protein interface interacting via five salt bridges and four hydrogen bonds. Comparisons with cholesterol-metabolizing CYP11A1 and cortisol-producing CYP11B1, which also bind adrenodoxin, revealed substantial structural differences in these regions. The structural and functional differences between different P450 interactions with adrenodoxin may provide valuable clues for an orthogonal treatment approach for primary aldosteronism by specifically targeting the interaction between CYP11B2 and adrenodoxin.

Expression of CYP11B1 and CYP11B2 in adrenal adenoma correlates with clinical characteristics of primary aldosteronism.

OBJECTIVE: Primary aldosteronism (PA) shows histological heterogeneity and clinical variability, including the coexistence of hypercortisolemia. Immunohistochemical analyses of steroidogenic enzymes in adrenal tissues have provided new insights into the pathogenesis of PA. However, a comprehensive analysis of the association between enzyme expression and clinical characteristics of PA has rarely been conducted. We aimed to investigate the correlation between clinical characteristics and steroidogenic enzyme expression in PA. DESIGN: A retrospective case-control study. PATIENTS: Consecutive patients who underwent unilateral adrenalectomy for PA (n = 180). Patients with adrenal Cushing's syndrome (CS) (n = 29) and nonfunctioning adenoma (n = 6) as comparator groups. MEASUREMENTS: A tissue microarray of adrenal adenomas was constructed and immunostained for CYP11B1, CYP11B2 and CYP17A1. The expression of the three enzymes was compared between PA and other adrenal diseases and between PA with and without mild autonomous cortisol excess (MACE). RESULTS: Adrenal adenomas in PA showed lower CYP11B1, higher CYP11B2 and lower CYP17A1 expression than those in adrenal CS (p < .001). Nonfunctioning adenomas showed low expression of the three enzymes. PA with MACE showed higher CYP11B1 expression than PA without MACE. CYP11B1 expression was positively correlated with the severity of hypercortisolemia, and CYP11B2 was positively correlated with that of hyperaldosteronism. The expression of CYP11B1 and CYP11B2 had a negative correlation. Patients with absent clinical improvement after adrenalectomy had lower CYP11B2 expression than those with complete success. CONCLUSIONS: Variable expression of steroidogenic enzymes in adrenal adenoma underlies the clinical heterogeneity of PA and is associated with treatment outcomes.

Adrenal androgens versus cortisol for primary aldosteronism subtype determination in adrenal venous sampling.

OBJECTIVE: We examined if measurement of adrenal androgens adds to subtype diagnostics of primary aldosteronism (PA) under cosyntropin-stimulated adrenal venous sampling (AVS). DESIGN: A prospective pre-specified secondary endpoint analysis of 49 patients with confirmed PA, of whom 29 underwent unilateral adrenalectomy with long-term follow-up. METHODS: Concentrations of androstenedione, dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEAS) were measured during AVS in addition to aldosterone and cortisol. Subjects with lateralisation index (LI) of ≥4 were treated with unilateral adrenalectomy, and the immunohistochemical subtype was determined with CYP11B2 and CYP11B1 stains. The performance of adrenal androgens was evaluated by receiver operating characteristics (ROC) curve analyses in adrenalectomy and medical therapy groups. RESULTS: During AVS, the correlations between cortisol and androstenedione, DHEA and DHEAS for LI and selectivity index (SI) were highly significant. The right and left side SIs for androstenedione and DHEA were higher (p < .001) than for cortisol. In ROC analysis, the optimal LI cut-off values for androstenedione, DHEA and DHEAS were 4.2, 4.5 and 4.6, respectively. The performance of these LIs for adrenal androgens did not differ from that of cortisol. CONCLUSIONS: Under cosyntropin-stimulated AVS, the measurement of androstenedione and DHEA did not improve the cannulation selectivity. The performance of cortisol and adrenal androgens are confirmatory but not superior to cortisol-based results in lateralisation diagnostics of PA.

The discovery of BMS-737 as a potent, CYP17 lyase-selective inhibitor for the treatment of castration-resistant prostate cancer.

We report herein, the discovery of BMS-737 (compound 33) as a potent, non-steroidal, reversible small molecule inhibitor demonstrating 11-fold selectivity for CYP17 lyase over CYP17 hydroxylase, as well as a clean xenobiotic CYP profile for the treatment of castration-resistant prostate cancer (CRPC). Extensive SAR studies on the initial lead 1 at three different regions of the molecule resulted in the identification of BMS-737, which demonstrated a robust 83% lowering of testosterone without any significant perturbation of the mineralocorticoid and glucocorticoid levels in cynomologous monkeys in a 1-day PK/PD study.

Investigation of combined effects of propyl paraben and methyl paraben on the hypothalamic-pituitary-adrenal axis in male rats.

The aim of this study was to investigate the endocrine-disrupting effects of methyl paraben (MeP) and propyl paraben (PrP) mixture on the hypothalamic-pituitary-adrenal axis (HPA). In this study, six experimental groups were designated. These groups included three control groups (control, corn oil control, and positive control (50 mg/kg/day BPA)) and three dose groups (10, 100, and 500 mg/kg/day MeP+PrP). MeP with PrP were mixed in a 1:1 ratio and administered to the 42-day-old male rats by oral gavage for 30 days. At the end of the experiment, adrenocorticotropic hormone (ACTH), corticosterone and aldosterone hormones were analyzed in serum. Effects of MeP+PrP on the adrenal glands were investigated by immunohistochemical staining of 11ß hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) enzymes involved in the synthesis steps of corticosterone and aldosterone. Also, pituitary and adrenal glands were examined histopathologically. In the histopathological findings, cortical nodule, congestion, and edema were found in the tissues. In the pituitary gland, cytokeratin rings were detected in all MeP+PrP dose groups, supporting the increase of corticosterone and ACTH. Serum corticosterone, aldosterone, and ACTH hormone levels were increased in the 100 mg/kg/day MeP+PrP and BPA groups. Results obtained from immunohistochemical staining showed that increased staining parallelled increased corticosterone and aldosterone hormone levels. In summary, the results showed that exposure to the MeP+PrP mixture caused a significant increase in ACTH and corticosterone. Also, the MeP+PrP mixture caused a significant increase of CYP11B1 and CYP11B2. MeP+PrP exposure disrupts the normal HPA axis.