Aldosterone synthase inhibitor (BI 690517) therapy for people with diabetes and albuminuric chronic kidney disease: A multicentre, randomized, double-blind, placebo-controlled, Phase I trial.

AIM: This Phase I study evaluated the safety and early efficacy of an aldosterone synthase inhibitor (BI 690517) in people with diabetes and albuminuric chronic kidney disease. METHODS: Double-blind, placebo-controlled study (NCT03165240) at 40 sites across Europe. Eligible participants [estimated glomerular filtration rate ≥20 and <75 ml/min/1.73 m2; urine albumin/creatinine ratio (UACR) ≥200 and <3500 mg/g] were randomized 6:1 to receive once-daily oral BI 690517 3, 10 or 40 mg, or eplerenone 25-50 mg, or placebo, for 28 days. The primary endpoint was the proportion of participants with drug-related adverse events (AEs). Secondary endpoints included changes from baseline in the UACR. RESULTS: Fifty-eight participants were randomized and treated from 27 November 2017 to 16 April 2020 (BI 690517: 3 mg, n = 18; 10 mg, n = 13; 40 mg, n = 14; eplerenone, n = 4; placebo, n = 9) for 28 days. Eight (13.8%) participants experienced drug-related AEs [BI 690517: 3 mg (two of 18); 10 mg (four of 13); 40 mg (two of 14)], most frequently constipation [10 mg (one of 13); 40 mg (one of 14)] and hyperkalaemia [3 mg (one of 18); 10 mg (one of 13)]. Most AEs were mild to moderate; one participant experienced severe hyperkalaemia (serum potassium 6.9 mmol/L; BI 690517 10 mg). UACR responses [≥20% decrease from baseline (first morning void urine) after 28 days] were observed for 80.0% receiving BI 690517 40 mg (eight of 10) versus 37.5% receiving placebo (three of eight). Aldosterone levels were suppressed by BI 690517, but not eplerenone or placebo. CONCLUSIONS: BI 690517 was generally well tolerated, reduced plasma aldosterone and may decrease albuminuria in participants with diabetes and albuminuric chronic kidney disease.

Phase 2 Trial of Baxdrostat for Treatment-Resistant Hypertension.

BACKGROUND: Aldosterone synthase controls the synthesis of aldosterone and has been a pharmacologic target for the treatment of hypertension for several decades. Selective inhibition of aldosterone synthase is essential but difficult to achieve because cortisol synthesis is catalyzed by another enzyme that shares 93% sequence similarity with aldosterone synthase. In preclinical and phase 1 studies, baxdrostat had 100:1 selectivity for enzyme inhibition, and baxdrostat at several dose levels reduced plasma aldosterone levels but not cortisol levels. METHODS: In this multicenter, placebo-controlled trial, we randomly assigned patients who had treatment-resistant hypertension, with blood pressure of 130/80 mm Hg or higher, and who were receiving stable doses of at least three antihypertensive agents, including a diuretic, to receive baxdrostat (0.5 mg, 1 mg, or 2 mg) once daily for 12 weeks or placebo. The primary end point was the change in systolic blood pressure from baseline to week 12 in each baxdrostat group as compared with the placebo group. RESULTS: A total of 248 patients completed the trial. Dose-dependent changes in systolic blood pressure of -20.3 mm Hg, -17.5 mm Hg, -12.1 mm Hg, and -9.4 mm Hg were observed in the 2-mg, 1-mg, 0.5-mg, and placebo groups, respectively. The difference in the change in systolic blood pressure between the 2-mg group and the placebo group was -11.0 mm Hg (95% confidence interval [CI], -16.4 to -5.5; P<0.001), and the difference in this change between the 1-mg group and the placebo group was -8.1 mm Hg (95% CI, -13.5 to -2.8; P = 0.003). No deaths occurred during the trial, no serious adverse events were attributed by the investigators to baxdrostat, and there were no instances of adrenocortical insufficiency. Baxdrostat-related increases in the potassium level to 6.0 mmol per liter or greater occurred in 2 patients, but these increases did not recur after withdrawal and reinitiation of the drug. CONCLUSIONS: Patients with treatment-resistant hypertension who received baxdrostat had dose-related reductions in blood pressure. (Funded by CinCor Pharma; BrigHTN ClinicalTrials.gov number, NCT04519658.).

Development of [18F]AldoView as the First Highly Selective Aldosterone Synthase PET Tracer for Imaging of Primary Hyperaldosteronism.

The purpose of this study was to synthesize a fluorine-18 labeled, highly selective aldosterone synthase (hCYP11B2) inhibitor, [18F]AldoView, and to assess its potential for the detection of aldosterone-producing adenomas (APAs) with positron emission tomography in patients with primary hyperaldosteronism (PHA). Using dibenzothiophene sulfonium salt chemistry, [18F]AldoView was obtained in high radiochemical yield in one step from [18F]fluoride. In mice, the tracer showed a favorable pharmacokinetic profile, including rapid distribution and clearance. Imaging in the adrenal tissue from patients with PHA revealed diffuse binding patterns in the adrenal cortex, avid binding in some adenomas, and "hot spots" consistent with aldosterone-producing cell clusters. The binding pattern was in good visual agreement with the antibody staining of hCYP11B2 and distinguished areas with normal and excessive hCYP11B2 expression. Taken together, [18F]AldoView is a promising tracer for the detection of APAs in patients with PHA.

Updates in adrenal steroidogenesis inhibitors for Cushing's syndrome - A practical guide.

Medical therapy is essential in the management of patients with Cushing's syndrome (CS) when curative surgery has failed, surgery is not feasible, when awaiting radiation effect, and in recurrent cases of CS. Steroidogenesis inhibitors have a rapid onset of action and are effective in reducing hypercortisolism, however, adverse effects, including adrenal insufficiency require very close patient monitoring. Osilodrostat is the only steroidogenesis inhibitor to have been assessed in prospective randomized controlled trials and approved for Cushing's disease (CD) by the US Food and Drug Administration and for CS by the European Medical Agency (EMA). Osilodrostat has been shown to be highly effective at maintaining normal urinary free cortisol in patients with CD. Drugs such as metyrapone, ketoconazole (both EMA approved), and etomidate lack prospective evaluation(s). There is, however, considerable clinical experience and retrospective data that show a very wide efficacy range in treating patients with CS. In the absence of head-to-head comparative clinical trials, therapy choice is determined by the specific clinical setting, risk of adverse events, cost, availability, and other factors. In this review practical points to help clinicians who are managing patients with CS being treated with steroidogenesis inhibitors are presented.

Aldosterone synthase inhibitors for cardiovascular diseases: A comprehensive review of preclinical, clinical and in silico data.

Aldosterone, the main mineralocorticoid hormone, plays a fundamental role in maintaining blood pressure (BP)and volume under hypovolemic conditions. However, in numerous diseases, where it is produced in excess, it plays a detrimental role and contributes to cardiovascular events and ultimately to death in a multitude of patients. The seminal observation that the fungicide-derivative fadrozole blunted steroidogenesis has led to develop several agents to inhibit aldosterone synthase (AS, CYP11B2), the mitochondrial NADH-dependent enzyme that is necessary for aldosterone biosynthesis. Aldosterone synthase inhibitors (ASI) have, thereafter, been conceived and investigated in phase I and phase II studies. We herein reviewed the ASIs available so far considering their chemical structure, the related aldosterone synthase binding and pharmacodynamic properties. We also examined the promising results obtained with ASIs that have already been tested in phase II human studies.

The establishment of a novel high-throughput screening system using RNA-guided genome editing to identify chemicals that suppress aldosterone synthase expression.

Aldosterone is synthesized in the adrenal by the aldosterone synthase CYP11B2. Although the control of CYP11B2 expression is important to maintain the mineral homeostasis, its overexpression induced by the depolarization-induced calcium (Ca2+) signaling activation has been reported to increase the synthesis of aldosterone in primary aldosteronism (PA). The drug against PA focused on the suppression of CYP11B2 expression has not yet been developed, since the molecular mechanism of CYP11B2 transcriptional regulation activated via Ca2+ signaling remains unclear. To address the issue, we attempted to reveal the mechanism of the transcriptional regulation of CYP11B2 using chemical screening. We generated a cell line by inserting Nanoluc gene as a reporter into CYP11B2 locus in H295R adrenocortical cells using the CRSPR/Cas9 system, and established the high-throughput screening system using the cell line. We then identified 9 compounds that inhibited the CYP11B2 expression induced by potassium-mediated depolarization from the validated compound library (3399 compounds). Particularly, tacrolimus, an inhibitor of phosphatase calcineurin, strongly suppressed the CYP11B2 expression even at 10 nM. These results suggest that the system is effective in identifying drugs that suppress the depolarization-induced CYP11B2 expression. Our screening system may therefore be a useful tool for the development of novel medicines against PA.

Efficacy and safety of osilodrostat in patients with Cushing's disease (LINC 3): a multicentre phase III study with a double-blind, randomised withdrawal phase.

BACKGROUND: Cushing's disease is a rare endocrine disorder characterised by cortisol overproduction with severe complications. Therapies for cortisol reduction are often necessary. Here we report the outcomes from the pivotal phase III study of osilodrostat (a potent oral inhibitor of cytochrome P450 11B1, mitochondrial [11ß-hydroxylase]; Novartis Pharma AG, Basel, Switzerland) in patients with Cushing's disease. METHODS: LINC 3 was a prospective, multicentre, open-label, phase III study with a double-blind randomised withdrawal period, that comprised four periods. Patients aged 18-75 years, with confirmed persistent or recurrent Cushing's disease (defined as mean 24-h urinary free cortisol [UFC] concentration >1·5 times the upper limit of normal [ULN] and morning plasma adrenocorticotropic hormone above the lower limit of normal) who had previously had pituitary surgery or irradiation, or were newly diagnosed and who refused surgery or were not surgical candidates, were recruited from 66 hospital sites and private clinical practices in 19 countries. In period 1, open-label osilodrostat was initiated in all participants and adjusted every 2 weeks (1-30 mg twice daily; film-coated tablets for oral administration) on the basis of mean 24-h UFC concentration and safety until week 12. In period 2, weeks 13-24, osilodrostat was continued at the therapeutic dose determined during period 1. In period 3, beginning at week 26, participants who had a mean 24-h UFC concentration of less than or equal to the ULN at week 24, without up-titration after week 12, were randomly assigned (1:1), via an interactive-response technology, stratified by osilodrostat dose at week 24 and history of pituitary irradiation, to continue osilodrostat or switch to placebo for 8 weeks. Participants and investigators were masked to treatment assignment. Ineligible participants continued open-label osilodrostat. In period 4, weeks 35-48, all participants were given open-label osilodrostat until core-study end. The primary objective was to compare the efficacy of osilodrostat versus placebo at the end of period 3. The primary endpoint was the proportion of participants who had been randomly assigned to treatment or placebo with a complete response (ie, mean 24-h UFC concentration of ≤ULN) at the end of the randomised withdrawal period (week 34), without up-titration during this period. The key secondary endpoint was the proportion of participants with a complete response at the end of the single-arm, open-label period (ie, period 2, week 24) without up-titration during weeks 13-24. Analysis was by intention-to-treat for all patients who received at least one dose of osilodrostat (full analysis set; key secondary endpoint) or randomised treatment (randomised analysis set; primary endpoint) and safety was assessed in all enrolled patients who received at least one dose of osilodrostat and had at least one post-baseline safety assessment. LINC 3 is registered with ClinicalTrials.gov, NCT02180217, and is now complete. FINDINGS: Between Nov 12, 2014, and March 22, 2017, 202 patients were screened and 137 were enrolled. The median age was 40·0 years (31·0-49·0) and 106 (77%) participants were female. 72 (53%) participants were eligible for randomisation during the withdrawal phase, of whom 36 were assigned to continue osilodrostat and 35 were assigned to placebo; one patient was not randomly assigned due to investigator decision and continued open-label osilodrostat. More patients maintained a complete response with osilodrostat versus with placebo at week 34 (31 [86%] vs ten [29%]; odds ratio 13·7 [95% CI 3·7-53·4]; p<0·0001). At week 24, 72 (53%; 95% CI 43·9-61·1) of 137 patients maintained a complete response without up-titration after week 12. Most common adverse events (ie, occurred in >25% of participants) were nausea (57 [42%]), headache (46 [34%]), fatigue (39 [28%]), and adrenal insufficiency (38 [28%]). Hypocortisolism occurred in 70 (51%) patients and adverse events related to adrenal hormone precursors occurred in 58 (42%) patients. One patient died, unrelated to study drug, after the core study phase. INTERPRETATION: Twice-daily osilodrostat rapidly reduced mean 24-h UFC and sustained this reduction alongside improvements in clinical signs of hypercortisolism; it was also generally well tolerated. Osilodrostat is an effective new treatment option that is approved in Europe for the treatment of endogenous Cushing's syndrome and in the USA for Cushing's disease. FUNDING: Novartis Pharma AG.

Neuronal aldosterone elicits a distinct genomic response in pain signaling molecules contributing to inflammatory pain.

BACKGROUND: Recently, mineralocorticoid receptors (MR) were identified in peripheral nociceptive neurons, and their acute antagonism was responsible for immediate and short-lasting (non-genomic) antinociceptive effects. The same neurons were shown to produce the endogenous ligand aldosterone by the enzyme aldosterone synthase. METHODS: Here, we investigate whether endogenous aldosterone contributes to inflammation-induced hyperalgesia via the distinct genomic regulation of specific pain signaling molecules in an animal model of Freund's complete adjuvant (FCA)-induced hindpaw inflammation. RESULTS: Chronic intrathecal application of MR antagonist canrenoate-K (over 4 days) attenuated nociceptive behavior in rats with FCA hindpaw inflammation suggesting a tonic activation of neuronal MR by endogenous aldosterone. Consistently, double immunofluorescence confocal microscopy showed abundant co-localization of MR with several pain signaling molecules such as TRPV1, CGRP, Nav1.8, and trkA whose enhanced expression of mRNA and proteins during inflammation was downregulated following i.t. canrenoate-K. More importantly, inhibition of endogenous aldosterone production in peripheral sensory neurons by continuous intrathecal delivery of a specific aldosterone synthase inhibitor prevented the inflammation-induced enhanced transcriptional expression of TRPV1, CGRP, Nav1.8, and trkA and subsequently attenuated nociceptive behavior. Evidence for such a genomic effect of endogenous aldosterone was supported by the demonstration of an enhanced nuclear translocation of MR in peripheral sensory dorsal root ganglia (DRG) neurons. CONCLUSION: Taken together, chronic inhibition of local production of aldosterone by its processing enzyme aldosterone synthase within peripheral sensory neurons may contribute to long-lasting downregulation of specific pain signaling molecules and may, thus, persistently reduce inflammation-induced hyperalgesia.

Discovery of 3-Pyridyl Isoindolin-1-one Derivatives as Potent, Selective, and Orally Active Aldosterone Synthase (CYP11B2) Inhibitors.

Aldosterone synthase (CYP11B2) inhibitors have been explored in recent years as an alternative therapeutic option to mineralocorticoid receptor (MR) antagonists to reduce elevated aldosterone levels, which are associated with deleterious effects on various organ systems including the heart, vasculature, kidney, and central nervous system (CNS). A benzamide pyridine hit derived from a focused screen was successfully developed into a series of potent and selective 3-pyridyl isoindolin-1-ones CYP11B2 inhibitors. Our systematic structure-activity relationship study enabled us to identify unique structural features that result in high selectivity against the closely homologous cortisol synthase (CYP11B1). We evaluated advanced lead molecules, exemplified by compound 52, in an in vivo cynomolgus monkey acute adrenocorticotropic hormone (ACTH) challenge model and demonstrated a superior 100-fold in vivo selectivity against CYP11B1.

Mitigating risk of aldosterone in diabetic kidney disease.

PURPOSE OF REVIEW: Diabetic kidney disease is a growing problem leading to end-stage kidney disease but also atherosclerotic cardiovascular disease and heart failure. Aldosterone is a key risk factor promoting inflammation and fibrosis causing cardio-renal failure. Current options and challenges with mitigating the risk of aldosterone are reviewed. RECENT FINDINGS: More aggressive renin-angiotensin-aldosterone system (RAAS) blockade can be maintained in individuals with hyperkalemia if new potassium binders are added. Aldosterone synthase inhibitors may lower aldosterone without causing hyperkalemia. Novel nonsteroidal mineralocorticoid receptor antagonists (MRA) are able to lower proteinuria and markers of heart failure, with limited potassium problems and without renal impairment. Ongoing clinical trials are evaluating the safety and potential benefits of nonsteroidal MRAs on progression of renal disease and development of cardiovascular outcomes in type 2 diabetes and kidney disease. SUMMARY: Aldosterone is an important driver of inflammation and fibrosis leading to renal and cardiovascular complications. MRA lower albuminuria but data showing prevention of end-stage kidney disease are lacking. Side effects including hyperkalemia have previously prevented long-term studies in diabetic kidney disease but new treatment strategies with potassium binders, aldosterone synthase inhibitors and nonsteroidal MRA have been developed for clinical testing.

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

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

Identification of the fungicide epoxiconazole by virtual screening and biological assessment as inhibitor of human 11ß-hydroxylase and aldosterone synthase.

Humans are constantly exposed to a multitude of environmental chemicals that may disturb endocrine functions. It is crucial to identify such chemicals and uncover their mode-of-action to avoid adverse health effects. 11ß-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) catalyze the formation of cortisol and aldosterone, respectively, in the adrenal cortex. Disruption of their synthesis by exogenous chemicals can contribute to cardio-metabolic diseases, chronic kidney disease, osteoporosis, and immune-related disorders. This study applied in silico screening and in vitro evaluation for the discovery of xenobiotics inhibiting CYP11B1 and CYP11B2. Several databases comprising environmentally relevant pollutants, chemicals in body care products, food additives and drugs were virtually screened using CYP11B1 and CYP11B2 pharmacophore models. A first round of biological testing used hamster cells overexpressing human CYP11B1 or CYP11B2 to analyze 25 selected virtual hits. Three compounds inhibited CYP11B1 and CYP11B2 with IC50 values below 3 µM. The most potent inhibitor was epoxiconazole (IC50 value of 623 nM for CYP11B1 and 113 nM for CYP11B2, respectively); flurprimidol and ancymidol were moderate inhibitors. In a second round, these three compounds were tested in human adrenal H295R cells endogenously expressing CYP11B1 and CYP11B2, confirming the potent inhibition by epoxiconazole and the more moderate effects by flurprimidol and ancymidol. Thus, the in silico screening, prioritization of chemicals for initial biological tests and use of H295R cells to provide initial mechanistic information is a promising strategy to identify potential endocrine disruptors inhibiting corticosteroid synthesis. A critical assessment of human exposure levels and in vivo evaluation of potential corticosteroid disrupting effects by epoxiconazole is required.

Adipocyte Mineralocorticoid Receptor.

Mineralocorticoid receptor (MR) has been recently identified in adipose tissue, where its excessive activation contributes to several metabolic derangements often observed in obesity and metabolic syndrome. Recent findings support the existence of a bidirectional cross-talk between adipose tissue and adrenal glands, contributing to obesity-related hyperaldosteronism and subsequent adipocyte MR excessive activation. In this regard, MR pharmacological blockade has led to prevention of weight gain and metabolic benefits in murine models of genetic or diet-induced obesity. However, there is still a lack of knowledge on the potential metabolic effects of MR antagonists in clinical settings. Hence, larger clinical studies are deemed necessary to clarify the role of MR antagonism in obesity and metabolic syndrome in humans.

A steady state system for in vitro evaluation of steroidogenic pathway dynamics: Application for CYP11B1, CYP11B2 and CYP17 inhibitors.

Disorders featuring dysregulated adrenal steroidogenesis, such as primary aldosteronism, can benefit from targeted therapies. The aldosterone and cortisol producing enzymes, aldosterone synthase (CYP11B2) and 11-beta-hydroxylase (CYP11B1), share 93% homology requiring selective drugs for pharmacological treatment. Herein, we introduce an effective in vitro assay for evaluation of steroidogenic enzyme kinetics based on intracellular flux calculations. H295RA cells were cultured in chambers under constant medium flow. Four hourly samples were collected (control samples), followed by collections over an additional four hours after treatment with fadrozole (10 nM), metyrapone (10 µM), SI_191 (5 nM), a novel CYP11B2 inhibitor or SI_254 (100 nM), a newly synthesized 17-alpha-hydroxylase/17,20-lyase inhibitor. Mass spectrometric measurements of multiple steroids combined with linear system computational modeling facilitated calculation of intracellular fluxes and changes in rate constants at different steroidogenic pathway steps, enabling selectivity of drugs for those steps to be evaluated. While treatment with fadrozole, metyrapone and SI_191 all reduced fluxes of aldosterone, corticosterone and cortisol production, treatment with SI_254 led to increased flux through the mineralocorticoid pathway and reduced production of steroids downstream of 17-alpha-hydroxylase/17,20-lyase. Drug-induced decreases in rate constants revealed higher selectivity of SI_191 compared to other drugs for CYP11B2 over CYP11B1, this reflecting additional inhibitory actions of SI_191 on catalytic steps of CYP11B2 downstream from the initial 11-beta-hydroxlase step. By culturing cells under perfusion the described system provides a realistic model for simple and rapid calculations of intracellular fluxes and changes in rate constants, thereby offering a robust procedure for investigating drug or other effects at specific steps of steroidogenesis.

Identification and Characterization of a Selective Human Carbonyl Reductase 1 Substrate.

During drug discovery efforts targeting inhibition of cytochrome P450 11B2 (CYP11B2)-mediated production of aldosterone as a therapeutic approach for the treatment of chronic kidney disease and hypertension, (S)-6-(5-fluoro-4-(1-hydroxyethyl)pyridin-3-yl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxamide (1) was identified as a potent and selective inhibitor of CYP11B2. Preclinical studies characterized 1 as low clearance in both in vitro test systems and in vivo in preclinical species. Despite low metabolic conversion, an active ketone metabolite (2) was identified from in vitro metabolite-identification studies. Due to the inhibitory activity of 2 against CYP11B2 as well as the potential for it to undergo reductive metabolism back to 1, the formation and elimination of 2 were characterized and are the focus of this manuscript. A series of in vitro investigations determined that 1 was slowly oxidized to 2 by cytochrome P450s 2D6, 3A4, and 3A5, followed by stereoselective reduction back to 1 and not its enantiomer (3). Importantly, reduction of 2 was mediated by an NADPH-dependent cytosolic enzyme. Studies with human cytosolic fractions from multiple tissues, selective inhibitors, and recombinantly expressed enzymes indicated that carbonyl reductase 1 (CBR1) is responsible for this transformation in humans. Carbonyl reduction is emerging as an important pathway for endogenous and xenobiotic metabolism. With a lack of selective substrates and inhibitors to enable characterization of the involvement of CBR1, 2 could be a useful probe to assess CBR1 activity in vitro in both subcellular fractions and in cell-based systems.

Discovery of Novel Pyrazole-Based Selective Aldosterone Synthase (CYP11B2) Inhibitors: A New Template to Coordinate the Heme-Iron Motif of CYP11B2.

It is necessary for aldosterone synthase (CYP11B2) inhibitors to have both high potency and high selectivity over 11ß-hydroxylase (CYP11B1), a critical enzyme for cortisol synthesis. Previous studies have reported a number of CYP11B2 inhibitors, most of which have an imidazole or pyridine ring to coordinate the heme-iron motif of CYP11B2; however, highly selective inhibitors of human CYP11B2 are still needed. To expand the selectivity in humans, we explored alternative templates and found that pyrazoles were suitable templates for CYP11B2 inhibitors. Investigation of pyrazoles, especially N-alkyl pyrazoles, as a new template to coordinate the heme-iron motif led to a potent and highly selective CYP11B2 inhibitor 28 with an aldosterone-lowering effect at 1 mg/kg dosing in cynomolgus monkeys.

Future pharmacological therapy in hypertension.

PURPOSE OF REVIEW: Hypertension (HTN) is a widespread and growing disease, with medication intolerance and side-effect present among many. To address these obstacles novel pharmacotherapy is an active area of drug development. This review seeks to explore future drug therapy for HTN in the preclinical and clinical arenas. RECENT FINDINGS: The future of pharmacological therapy in HTN consists of revisiting old pathways to find new targets and exploring wholly new approaches to provide additional avenues of treatment. In this review, we discuss the current status of the most recent drug therapy in HTN. New developments in well trod areas include novel mineralocorticoid antagonists, aldosterone synthase inhibitors, aminopeptidase-A inhibitors, natriuretic peptide receptor agonists, or the counter-regulatory angiotensin converting enzyme 2/angiotensin (Ang) (1-7)/Mas receptor axis. Neprilysin inhibitors popularized for heart failure may also still hold HTN potential. Finally, we examine unique systems in development never before used in HTN such as Na/H exchange inhibitors, vasoactive intestinal peptide agonists, and dopamine beta hydroxylase inhibitors. SUMMARY: A concise review of future directions of HTN pharmacotherapy.

Dihydrobenzisoxazole-4-one compounds are novel selective inhibitors of aldosterone synthase (CYP11B2) with in vivo activity.

6,7-Dihydro-5H-2,1-benzisoxazol-4-one analogs are potent inhibitors of aldosterone synthase (CYP11B2) with selectivity over the highly homologous enzyme cortisol synthase (CYP11B1). These compounds are unique among inhibitors of CYP11B2 in their lack of a strong-heme binding group such as a pyridine or imidazole. Poor metabolic stability in hepatocyte incubations was found to proceed via a reduction of the isoxazole ring. While the enzyme responsible for the reductive metabolism remains unknown, the rate of metabolism could be attenuated by the addition of polar functionality. The in vitro CYP11B2 potency and selectivity were confirmed in vivo in a cynomolgus monkey model by the inhibition of ACTH stimulated aldosterone production without impacting plasma cortisol concentrations.

LY3045697: Results from two randomized clinical trials of a novel inhibitor of aldosterone synthase.

INTRODUCTION: LY3045697 is a potent and selective aldosterone synthase (CYP11B2) inhibitor that was developed as a safer alternative to mineralocorticoid receptor antagonists. Effects of LY3045697 on aldosterone and cortisol synthesis, as well as potassium ion homeostasis, were evaluated in two clinical studies in healthy subjects. MATERIALS AND METHODS: Two incomplete, placebo-controlled crossover-design clinical studies examined safety, pharmacodynamics, and pharmacokinetics under single and repeated dose conditions in healthy subjects. Pharmacodynamics was assessed following oral potassium challenge and intravenous adrenocorticotropic hormone procedures with spironolactone 25 mg/d as an active comparator. RESULTS: A total of 51 subjects participated in the two studies, which included 38 males and 13 females (of non-childbearing potential), from 18-65 years old. LY3045697 caused rapid dose and concentration-dependent unstimulated plasma aldosterone concentration reduction seen as early as 4 h after the first dose at dose levels as low as 1 mg, and reaching near complete suppression at high doses. The potency (IC50) decreased significantly upon multiple dosing. After eight days of dosing, post-adrenocorticotropic hormone challenge plasma aldosterone concentration increase was dose-dependently blunted by LY3045697 with high potency with a dose as low as 0.1 mg resulting in substantial effect, and with an overall IC50 of 0.38 ng/ml. Minor reductions in cortisol were observed only at the top dose of 300 mg. LY3045697 is generally safe and tolerated, and exhibits linear pharmacokinetics. CONCLUSIONS: LY3045697 is a potent and highly selective aldosterone synthase inhibitor with selectivity for CYP11B2, offering a substantial potential advantage over previous aldosterone synthase inhibitors evaluated in the clinic.

Impact of Aldosterone Synthase Inhibitor FAD286 on Steroid Hormone Profile in Human Adrenocortical Cells.

Inhibition of aldosterone synthase (CYP11B2) is an alternative treatment option to mineralocorticoid receptor antagonism to prevent harmful aldosterone effects. FAD286 is the best characterized aldosterone synthase inhibitor. However, to date, no study has used sensitive liquid chromatography-tandem mass spectrometry to characterize in detail the effect of FAD286 on the secreted steroid hormone profile of adrenocortical cells. Basal aldosterone production in NCI-H295R cells was detectable and 9-fold elevated after stimulation with angiotensin II. FAD286 inhibited this increase, showing a maximal effect at 10 nmol/l. Higher concentrations of FAD286 did not further reduce aldosterone concentrations, but showed a parallel reduction in corticosterone, cortisol and cortisone levels, reflecting additional inhibition of steroid-11ß-hydroxylase (CYP11B1). Pregnenolone, progesterone and 17-OH-progesterone levels remained unaffected. In conclusion, the aldosterone synthase inhibitor FAD286 lowers angiotensin II-induced aldosterone concentrations in adrenocortical cells but the relative lack of selectivity over CYP11B1 is evident at higher FAD286 concentrations.