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Preclinical and Early Clinical Profile of a Highly Selective and Potent Oral Inhibitor of Aldosterone Synthase (CYP11B2)

Preclinical and Early Clinical Profile of a Highly Selective and Potent Oral Inhibitor of... Aldosterone Synthase Inhibitor Preclinical and Early Clinical Profile of a Highly Selective and Potent Oral Inhibitor of Aldosterone Synthase (CYP11B2) Katrijn Bogman, Dietmar Schwab, Marie-Laure Delporte, Giuseppe Palermo, Kurt Amrein, Susanne Mohr, Maria Cristina De Vera Mudry, Morris J. Brown,* Philippe Ferber* See Editorial Commentary, pp 11–12 Abstract—Primary hyperaldosteronism is a common cause of resistant hypertension. Aldosterone is produced in the adrenal by aldosterone synthase (AS, encoded by the gene CYP11B2). AS shares 93% homology to 11β-hydroxylase (encoded by the gene CYP11B1), responsible for cortisol production. This homology has hitherto impeded the development of a drug, which selectively suppresses aldosterone but not cortisol production, as a new treatment for primary hyperaldosteronism. We now report the development of RO6836191 as a potent (Ki 13 nmol/L) competitive inhibitor of AS, with in vitro selectivity >100- fold over 11β-hydroxylase. In cynomolgus monkeys challenged with synthetic adrenocorticotropic hormone, single doses of RO6836191 inhibited aldosterone synthesis without affecting the adrenocorticotropic hormone–induced rise in cortisol. In −/− repeat-dose toxicity studies in monkeys, RO6836191 reproduced the adrenal changes of the AS mouse: expansion of the −/− zona glomerulosa; increased expression of AS (or disrupted green fluorescent protein gene in the AS mouse); hypertrophy, proliferation, and apoptosis of zona glomerulosa cells. These changes in the monkey were partially reversible and partially preventable by electrolyte supplementation and treatment with an angiotensin-converting enzyme inhibitor. In healthy subjects, single doses of RO6836191, across a 360-fold dose range, reduced plasma and urine aldosterone levels with maximum suppression at a dose of 10 mg, but unchanged cortisol, on adrenocorticotropic hormone challenge, up to 360 mg, and increase in the precursors 11-deoxycorticosterone and 11-deoxycortisol only at or >90 mg. In conclusion, RO6836191 demonstrates that it is possible to suppress aldosterone production completely in humans without affecting cortisol production. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifier: NCT01995383. (Hypertension. 2017;69:189-196. DOI: 10.1161/HYPERTENSIONAHA.116.07716.) Online Data Supplement Key Words: adrenal cortex ■ aldosterone ■ CYP11B2 ■ cytochrome P-450 ■ hyperaldosteronism ■ hydrocortisone ■ zona glomerulosa rimary hyperaldosteronism is recognized as a common cortex, catalyzes the final 3 steps in aldosterone synthesis from Pcause of resistant hypertension and contributes to heart and 11-deoxycorticosterone (11-DOC; sequentially to corticoste- 1–3 kidney diseases. Mineralocorticoid receptor (MR) antago- rone and 18-OH-corticosterone) to aldosterone. Aldosterone nists are currently included among the treatments of choice. synthesis shares several of the same enzymes as that of cortisol However, they are not always well tolerated and induce a synthesis, whose final step (from the precursor 11-deoxycorti- counterregulatory increase in aldosterone production, which sol) is mediated by 11β-hydroxylase, encoded by the CYP11B1 3,4 may limit the efficacy of the MR blockade. Furthermore, gene in the zona fasciculata and zona reticularis (see Figure experimental data suggest that some of the deleterious effects SI in the online-only Data Supplement for a schematic repre- 5,6 8 of aldosterone may occur through nongenomic pathways. sentation of the synthetic pathways). Selective AS inhibition These limitations have led to the exploration of alternative has foundered on the 93% homology between the transcripts approaches to antagonize aldosterone effects by inhibition of of CYP11B2 and CYP11B1 genes. Angiotensin II, potassium, aldosterone synthase (AS, encoded by the gene CYP11B2). and adrenocorticotropic hormone (ACTH) are the main physi- AS, expressed within the zona glomerulosa (ZG) of the adrenal ological agonists that regulate aldosterone secretion. ACTH Received April 28, 2016; first decision May 23, 2016; revision accepted September 13, 2016. From the Clinical Pharmacology (K.B., D.S., M.-L.D.), Biostatistics (G.P.), Discovery (K.A.), Pharmaceutical Sciences (S.M., M.C.D.V.M.), and Translational Medicine, Cardiovascular Diseases (P.F.), Pharma Research and Early Development, Roche Innovation Center Basel, Switzerland; and Clinical Pharmacology, William Harvey Research Institute, Queen Mary University of London, United Kingdom (M.J.B). Current address for P.F.: Novartis Pharma, Basel, Switzerland. *These authors contributed equally to this work. The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA. 116.07716/-/DC1. Correspondence to Katrijn Bogman, F. Hoffmann-La Roche Ltd, Grenzacherstrasse 124, 4070 Basel, Switzerland. E-mail katrijn.bogman@roche.com © 2016 The Authors. Hypertension is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDervis License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made. Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.116.07716 189 190 Hypertension January 2017 inhibitor. In both studies, recovery animals were dosed for 4 weeks stimulates the adrenal glands to release cortisol, and negative and then maintained undosed for a further 4 weeks to assess recovery. feedback of cortisol on ACTH secretion occurs at the hypotha- The studies were performed at contract research organizations lamic and pituitary levels. accredited by the Association for Assessment and Accreditation of The only AS inhibitor to enter phase 2 clinical development Laboratory Animal Care International (AAALAC) study procedures was LCI699. This compound reduced plasma aldosterone in such as necropsy and are in compliance with AAALAC accreditation. Further details are provided in the online-only Data Supplement. healthy subjects and in patients with primary hyperaldosteron- 7,12,13 ism. It decreased blood pressure in patients with primary Histology and Immunohistochemistry hyperaldosteronism, essential hypertension, and resistant Serial sections (nominal thickness of ≈5 µm) were prepared from 13–17 hypertension. However, lack of sufficient selectivity led to formalin-fixed, paraffin-embedded adrenal gland (left) of all animals. cortisol suppression within the clinical efficacious dose range The adrenal gland was stained routinely with hematoxylin-eosin, and consequently apparent termination of the development as well as for Ki-67 (marker for proliferative activity), CYP11B2 (marker for AS), and TUNEL (terminal deoxynucleotidyl transfer- program in hypertension. Indeed, LCI699 is currently under 18,19 ase-mediated deoxyuridine triphosphate nick end labeling; marker development for the treatment of Cushing syndrome. for apoptosis). Further details are provided in the online-only Data RO6836191 was designed as a highly selective and potent Supplement. AS inhibitor, belonging to the series with a novel tetrahy- droisoquinoline structure. We have investigated the selectiv- First-In-Man Study ity, efficacy, and safety of RO6836191 in various preclinical This was an adaptive, single-center, randomized, blinded, single ascend- ing dose, placebo-controlled study to investigate safety, tolerability, test systems, including in vitro cells expressing recombinant pharmacokinetic effects, and pharmacodynamic effects of RO6836191 CYP11B1 and CYP11B2 enzymes. Because there is only 68% in healthy male subjects (unique identifier: NCT01995383) conducted homology between rat and human CYP11B2, preclinical phar- in the Netherlands. RO6836191 was administered as oral solution to macological characterization and chronic toxicity studies were fasted subjects on day 1. Subjects were advised to consume a minimum of 1.5 L fluid daily while in clinic. performed in cynomolgus monkeys. We then conducted a The study was conducted in 2 parts. In part 1, a total of 64 subjects first-in-man single ascending dose study during which we mea- received single ascending oral doses of 1, 3, 10, 30, 90, 180, or 360 mg sured the extent of translation from preclinical into clinical. RO6836191 or matching placebo (the active treatment/placebo ratio was 3/1) under normal-salt diet conditions with ACTH challenge (n=56) or Methods without ACTH challenge (n=8). In part 2, a total of 24 subjects received single oral doses of 1, 3, or 10 mg RO6836191 or matching placebo (the In Vitro Assay of CYP11B2 and CYP11B1 active treatment/placebo ratio was 3/1). Each subject was studied under Inhibition low-salt and normal-salt diet conditions, in a 2-way crossover design The in vitro effect of RO6836191 on CYP11B2 and CYP11B1 proteins with a 2- to 3-week wash out, with no change in treatment assignment. was assessed in human renal leiomyoblastoma cells (ATCC CRL1440) Dosing under low-salt diet conditions required a 3-day in-clinic run-in period in which subjects adhered to a low-salt diet of 50 mmol sodium/d expressing recombinant human or cynomolgus CYP11B1 and and 70 to 100 mmol potassium/d, which was confirmed by urinary so- CYP11B2 enzymes as described for an earlier compound in the series. dium and creatinine assessments. The low-salt diet was continued until Further details are provided in the online-only Data Supplement. 48 hours postdose. Adverse events, vital signs, orthostatic vital signs, ECG, standard laboratory safety data (hematology, coagulation, blood In Vivo Monkey Pharmacology Study chemistry, and urinalysis), and body weight were monitored at baseline The effect of RO6836191 in cynomolgus monkeys was assessed as and postdose. The AS inhibition was assessed with different procedures described for an earlier compound in the series. Animals received to stimulate aldosterone release: ACTH challenge (intravenous admin- an oral gavage of vehicle and RO6836191 (0.035, 3, or 30 mg/kg, istration of 0.25 mg Cortrosyn, 1 hour postdose in part 1 only), postural 2 monkeys per dose). At 1 hour postdose, 0.0145 mg/kg Synacthen change (assessed at 6 hours postdose in parts 1 and 2, subjects were re- (ACTH challenge) was given intramuscularly. Serial blood samples quired to be standing or moving for at least 30 minutes before sampling, were collected pre- and postdose for analysis of RO6836191, cortisol, whereas at other timepoints pharmacodynamic samples were taken in aldosterone, and precursors. supine position), and low-salt diet. Serial blood samples (predose and up to maximum 96 hours post- In Vivo Monkey Preclinical Safety Studies dose) were taken for the assessment of plasma aldosterone, cortisol, and their precursors, plasma renin activity (PRA), plasma ACTH, and A 4-week high-dose study and 4-week mechanistic study were per- plasma electrolytes. Serial blood samples were taken before and up formed. The high-dose study explored doses from 0 to 40 mg/kg and to 4 hours post-ACTH challenge for the measurement of cortisol and included a treatment-free period to assess the reversibility, persis- precursors. Urine (collected from day −1 up to 48 hours postdose) tence, or delayed occurrence of any changes noted at 40 mg/kg. At 40 was analyzed for electrolytes, aldosterone, tetrahydroaldosterone, mg/kg, clinical signs of dehydration (skin tenting, urine drinking, and and cortisol. All pharmacodynamic assessments and procedures increase water consumption) after 16 days of dosing necessitated a were performed at baseline (day −1, time-matched with day 1 assess- suspension of dosing for 3 days. Animals received free access to elec- ments) and at multiple time points postdose. Serial blood samples for trolyte solution bottles from day 17 onward, which resulted in rapid RO6836191 pharmacokinetic analysis were collected up to 96 hours reversal of dehydration status and allowed redosing of the animals postdose, and a sample was taken on day 9 and at the follow-up visit. until end of the 4-week treatment period. The study was approved by the local independent ethics committees A 4-week mechanistic study explored doses from 0 to 1 mg/ and health authorities and conducted according to the provisions of the kg, testing whether adrenal changes in the high-dose study were Declaration of Helsinki. Written informed consent was obtained from entirely because of salt depletion and consequent activation of the each study participant before conducting any protocol-related proce- renin–angiotensin system, and investigating the reversibility of any dures. Further details are provided in the online-only Data Supplement. remaining changes in the adrenal ZG. Animals received either ve- hicle alone or RO6836191 0.15 or 1 mg/kg, with electrolyte supple- Pharmacokinetic and Statistical Analysis mentation and with or without angiotensin-converting enzyme (ACE) inhibitor (lisinopril 0.5 mg/kg). An additional group received 1 mg/ Results are reported as mean and standard deviation. Estimates of kg RO6836191 alone without electrolyte supplementation or ACE ANOVA models are reported together with 90% confidence interval (CI). Bogman et al An Oral Selective Inhibitor of Aldosterone Synthase 191 Percentage change from baseline was calculated as follows: (PD Table 1. In Vitro Potency of RO6836191 as Inhibitor of treated − PD baseline) × 100/PD baseline, where PD refers to a phar- CYP11B1 and CYP11B2 in Human Renal Leiomyoblastoma macodynamic marker concentration at a specific time point or over a Cells Expressing Recombinant Human and Cynomolgus specific time interval. Monkey Enzymes Pharmacokinetic parameters for RO6836191 and area under the plasma concentration versus time curve (AUC) for the pharmacody- Monkey Ki, Human Ki, namic markers were estimated by noncompartmental methods, and Enzyme Substrate nmol/L nmol/L exploratory pharmacokinetic and pharmacodynamic (PKPD) analy- Aldosterone 11-DOC 4±2.2 13±2.2 sis was performed using WinNonlin software (Phoenix version 6.2; synthase Pharsight Corporation, Mountain View, CA). (CYP11B2) Plasma concentrations for aldosterone and precursors that were be- low limit of quantification were set to the lower limit of quantification 11β-Hydroxylase 11-deoxycortisol 3150±1410 1310±533 value to allow further calculations. (CYP11B1) GraphPad Prism 6, version 6.04 (GraphPad Software, Inc, La Jolla, CA) was used for creation of figures and for calculation of in vitro Ki values. To calculate the inhibition constant (Ki) value, the amount Selectivity ratio 800 100 produced was plotted versus the substrate concentration for every in- Cells were incubated in the presence of 11-deoxycorticosterone (11-DOC) hibitor concentration tested; the resulting data set was then analyzed or 11-deoxycortisol as substrates for CYP11B2 and CYP11B1, respectively. Ki using the mixed model equation that is part of the statistic program values are means of replicates ±SD (n=5 and 8–13 for CYP11B2 and CYP11B1, GraphPad Prism. respectively). Selectivity ratio is defined as the ratio of Ki for CYP11B1 versus For the monkey study, individual percentage change from vehicle Ki for CYP11B2. in analytes in plasma at 2 hours postdose (or 1 hour after Synacthen application) was calculated. To test the effect of dose on plasma aldosterone, cortisol, and their present in vehicle-treated monkeys was absent or indistinct precursors in the clinical study, an ANOVA model was applied on re- in RO6836191-treated animals. Furthermore, both apoptosis sults from part 1 on the change from baseline for the log-transformed and proliferation of ZG cells were increased in dosed animals AUC. Baseline AUC (as a continuous variable) and dose (as a cat- compared with vehicle controls. After a 4-week treatment- egorical variable) were included as fixed effects in the model. The 90% CIs were derived for the percentage change from baseline on free period, there was a trend toward reversibility of increased AUC for each dose and for the placebo-adjusted percentage change CYP11B2 expression, while apoptosis and proliferation of from baseline on AUC for each active dose of RO6836191. ZG cells persisted (see Figure 1A). An exploratory pharmacokinetic and pharmacodynamic analysis Changes in clinical chemistry and urinalysis were consistent using data from the clinical study was applied on percentage change with the predicted natriuretic action of RO6836191, notably PRA from baseline in plasma aldosterone, 11-DOC, and 11-deoxycortisol concentration versus the corresponding RO6836191 plasma concen- was increased in both sexes at 1 and 7 mg/kg. At these dose lev- tration (details of the analysis are in Table SIII in the online-only Data els, aldosterone levels were clearly decreased without changing Supplement). cortisol. At the highest dose of 40 mg/kg, there was no change in renin, most probably because high-dose animals received rescue Analytical Methods electrolyte supplementation. At this dose, a decrease in cortisol Details are provided in the online-only Data Supplement. was observed in addition to the expected decrease in aldoste- rone, indicating a loss of selectivity against 11β-hydroxylase Results (CYP11B1). All values returned to control group values by the Estimation of In Vitro Selectivity of RO6836191 end of the 4-week treatment-free period (see Figure SIIIA). Cortisol or aldosterone production by human renal leiomyo- In the mechanistic study, supplementation with electro- blastoma cells expressing recombinant human or monkey lytes ameliorated the histopathologic findings in the adrenal CYP11B1 or CYP11B2 was measured in the presence of the gland (see Figure 1B), which had occurred in the previous respective substrate for these enzymes. The in vitro Ki values study at doses of 1 mg/kg and higher, namely, resulting in of RO6836191 in these cell lines showed that RO6836191 is only minimally reduced ZG expansion, CYP11B2 expression, a potent, selective, and competitive inhibitor of AS (Table 1 and cell proliferation. In addition, coadministration of an ACE and Figure SII). Its inhibition of AS was 800- and 100-fold inhibitor in monkeys treated with 1 mg/kg RO6836191 with more potent than that of 11β-hydroxylase (CYP11B1) in the electrolytes conferred further protection, notably with the monkey and human cell lines, respectively. retention of the transitional zone. At 0.15 mg/kg, no expan- sion of the ZG was observed. PRA was increased only for Histopathologic Effect of RO6836191 on Adrenal doses >0.15 mg/kg. PRA was still high when electrolyte sup- ZG in Monkey plementation was present, but almost comparable with control The ZG of the adrenal gland was the main affected tissue, values in the group receiving electrolyte supplementation and and changes occurred at the lowest dose tested, that is, 1 mg/ ACE inhibitor treatment. kg, which was then used as the high dose in the mechanis- Measurements of aldosterone and cortisol showed the tic study. Results of the semiquantitative analysis from both expected decrease and no change, respectively, at all doses. studies are in Table SI. There was drug-related expansion of The expected decrease of aldosterone was also observed in the ZG, hypertrophy, and increased AS (CYP11B2) expres- the group treated with ACE inhibitor alone (see Figure SIIIB). sion of ZG cells at 1 or 7 mg/kg. At 40 mg/kg RO6836191, Table SII shows that the efficacy and selectivity of the 0.15 the width of the ZG and CYP11B2 expression was reduced and 1 mg/kg doses are consistent with the RO6836191 expo- compared with the lower doses. The typically unstained tran- sures being equal or above the Ki for CYP11B2 and below Ki sitional zone at the junction of the ZG and zona fasciculata for CYP11B1. 192 Hypertension January 2017 but absence of change in cortisol levels post-ACTH chal- lenge across the 1000-fold dose range of RO6836191, was consistent with the selectivity ratio predicted from in vitro experiments. The observed aldosterone reduction at the low- est dose and the related free drug exposure are in line with the in vitro Ki value for AS. By contrast, the rise in 11-DOC and 11-deoxycortisol was consistent with the free drug exposure at the highest dose being comparable with the in vitro Ki value for 11β-hydroxylase (see Table 2). RO6836191 plasma expo- sure data are provided in Table SII. Proof of Mechanism in Healthy Subjects Safety, Tolerability, and Pharmacokinetics RO6836191 was well tolerated at all doses tested. The most Figure 1. Histopathologic changes in the cynomolgus monkey frequently reported adverse events were headache, nasophar- zona glomerulosa in the high-dose (A) and mechanistic (B) safety studies. A, Histopathology of representative monkeys in the high- yngitis, asthenia, and diarrhea, which were also reported in dose study. Animals were administered RO6836191 at 0, 1, 7, or the placebo group and which were all of mild intensity. There 40 mg/kg for 4 weeks and recovery after 40 mg/kg. Staining with was no dose-related increase in incidence or in the severity of hematoxylin-eosin (upper); immunostaining with human anti- reported adverse events. There were no other apparent treat- CYP11B2 antibody to visualize the zona glomerulosa (lower). Scale bar=100 µm. Dotted lines delineate the demarcation ment-related trends or any clinically significant findings in between the zona glomerulosa from the underlying zonal any other safety assessments. Mean time-matched blood pres- fasciculata. B, Histopathology of representative monkeys in the sure and pulse rate changes from baseline up to day 3 did not mechanistic study. Animals were administered RO6836191 at 1 mg/kg for 4 weeks, with or without electrolytes (E) or angiotensin- show a trend with time, dose, or diet, and no difference from converting enzyme inhibitor lisinopril 0.5 mg/kg (A), recovery after placebo was observed. treatment with 1 mg/kg with electrolytes (E*), control monkeys RO6836191 was rapidly absorbed reaching maximum were administered electrolytes alone. Staining with hematoxylin- plasma concentrations between 0.5 and 2 hours postdose. eosin (scale bar=100 µm; upper), double-sided arrows delineate width of the zona glomerulosa (ZG), single-sided arrows indicate Exposure of RO6836191 increased dose-proportionally. Mean the transitional zone of the ZG. Immunostaining with human half-life was 29 hours, and ≈10% of the dose was recovered anti-CYP11B2 antibody to visualize the zona glomerulosa (scale unchanged in the urine (RO6836191 plasma exposure data are bar=50 µm; lower). provided in Table SII). Pharmacological Characterization in Cynomolgus Pharmacological Characterization in Healthy Subjects Monkeys With ACTH Challenge and Comparison With Monkey Data The effect of a single oral dose RO6836191 (0–30 mg/kg) The time course of plasma aldosterone and cortisol in healthy on aldosterone, cortisol, and precursors after an ACTH chal- subjects after single ascending doses of RO6836191 or pla- lenge is shown in Figure 2. RO6836191 blunted aldosterone cebo with ACTH challenge is shown in Figure 3A and 3B. production at all doses tested compared with vehicle, with a The dose-dependent change from baseline in plasma expo- 70% to 90% decrease in aldosterone from the 0.035 mg/kg sure (AUC ) to aldosterone, cortisol, and their precursors is 0-24 to the 30 mg/kg dose, respectively. The precursors 11-DOC shown in Figure 3C and 3D. In the absence of drug, the ACTH and 11-deoxycortisol were increased only by high doses (up challenge induced a plasma aldosterone and cortisol surge to 600% at 30 mg/kg), but cortisol and corticosterone levels peaking ≈0.5 and 2 hours after the challenge, respectively. were not different from vehicle. The reduction in aldosterone, The postural change induced an immediate surge in plasma Figure 2. Dose-dependent effect of RO6836191 on plasma aldosterone, cortisol, and precursors in cynomolgus monkeys. Data represent individual % change from vehicle in plasma concentration (measured at 1 hour post-adrenocorticotropic hormone challenge) for aldosterone and precursors (A) and cortisol and precursors (B) by dose group. Means by dose group are connected by a line (n=2/dose group). 11-DOC indicates 11-deoxycorticosterone. Bogman et al An Oral Selective Inhibitor of Aldosterone Synthase 193 Table 2. Comparison of Human and Monkey In Vivo and In Vitro Potency and Selectivity Aldosterone Synthase 11β-Hydroxylase RO6836191-Free C Max Change in RO6836191-Free C Max Change in avg avg Ki In Vitro, at Minimum Effective Aldosterone Ki In Vitro, at Dose With Max 11-Deoxycortisol Species ng/mL Dose, ng/mL (% Chg From Ctrl) ng/mL Observed Effect, ng/mL (% Chg From Ctrl) a b c c Monkey 1.45 0.74; 0.94 −84.6; −94.1 1144 1891; 1972 + 266; +519 d e f f Human 4.7 5.2 (0.57) −84.9 (−86.6 to −82.8) 476 793 (79.3) +193 (157 to 234) Ki (see Table 1) units were converted to ng/mL for comparison with RO6836191 plasma concentrations. RO6836191 free C was calculated from total C , taking avg avg into account the free fraction of 0.26 for human and of 0.33 for monkey; C is expressed as individual data per monkey or mean and SD for human. Change in plasma avg exposure for aldosterone and 11-deoxycortisol is expressed as % change from vehicle for monkey (individual values per monkey, n=2) and as % change from day 1 baseline for human (represented as mean and 90% confidence interval). The minimum effective dose was defined as the dose at which ≥75% of the maximum observed effect was reached. a, b, c, d, e, and f are parameters for dose groups 0.035 mg/kg, 3 mg/kg, 30 mg/kg, 3 mg, 10 mg, and 360 mg, respectively. aldosterone to a similar level as induced by the ACTH chal- CI, 157 to 234]). 18-Hydroxycorticosterone decreased at 1 mg lenge. After RO6836191 administration, plasma aldosterone with a maximum effect at 10 mg onward (P<0.01; maximum was decreased dose-dependently with the maximum effect mean change in AUC from baseline −58.6% [90% CI, −64.3 reached by 10 mg onward (mean change in AUC from baseline to −52.0]). These results were consistent with the in vitro and −84.9% [90% CI, −86.6 to −82.8%]; different from placebo monkey findings, with the absence of change in cortisol levels P<0.0001). Corticosterone and cortisol were unchanged at all post-ACTH challenge across the 360-fold dose range predicted doses compared with placebo, and there were no reductions in by the drug’s selectivity for CYP11B2. The increase in plasma plasma cortisol on ACTH challenge. At doses >90 mg, there concentration of precursors 11-DOC (substrate for CYP11B1 were dose-dependent (and different from placebo P<0.01) and CYP11B2) and 11-deoxycortisol (substrate for CYP11B1) increases in 11-DOC (maximum mean change in AUC from at doses >90 mg were proportionately less than the reduction baseline +194% [90% CI, 160 to 232]) and 11-deoxycortisol in aldosterone and as in monkey was absent or insubstantial (maximum mean change in AUC from baseline +193% [90% except at concentrations of drug inhibiting 11β-hydroxylase. Figure 3. Dose-dependent effect of RO6836191 on plasma aldosterone, cortisol, and precursors in healthy subjects (study part 1). Aldosterone (A) and cortisol (B) plasma concentration-time profiles after single ascending doses of RO6836191 (0–360 mg) administered orally to healthy subjects 1 hour before the adrenocorticotropic hormone (ACTH) challenge. Downward arrow indicates time of ACTH challenge (1 hour postdose) and upward arrow time of postural change (6 hours postdose). Data represent mean plasma concentrations (+SD) by dose group (n=6/dose group and n=12 for placebo). Below limit of quantification (BLQ) values were set to lower limit of quantification (LLOQ; 5 pg/mL), for doses >10 mg, aldosterone concentrations were BLQ from 2.5 hours postdose onward. Effect of RO6836191 on plasma aldosterone (C), cortisol (D), and precursors after a single oral dose (0–360 mg) in healthy subjects administered 1 hour before the ACTH challenge. Data represent mean (and 90% confidence interval) % change from baseline in plasma exposure (AUC ) 0-24 for aldosterone and precursors (left graph) and cortisol and precursors (right graph) by dose group (n=6/dose group and n=12 for placebo). Filled symbols indicate P<0.01 compared with placebo. 11-DOC indicates 11-deoxycorticosterone. 194 Hypertension January 2017 Consistent with the decrease in plasma aldosterone, uri- nary excretion of both aldosterone and its metabolite, tetrahy- droaldosterone, was decreased dose-dependently (Figure SV). No dose-dependent effect on urinary cortisol was apparent. The pattern of RO6836191-induced changes in aldoste- rone, cortisol, and precursors was qualitatively and quanti- tatively comparable between monkey and human, relative to the respective Ki values. A comparison of monkey and human RO6836191 plasma exposures leading to a similar effect on aldosterone and 11-deoxycortisol is provided in Table 2 and demonstrate that the effective free plasma concentrations are consistent with the in vitro potency and selectivity of RO6836191 in the respective species. PKPD Characterization in Healthy Subjects and Comparison With In Vitro Cell-Based Potency Figure 4. Urinary sodium-to-potassium ratio after single-dose Assessment of the effect of RO6836191 on aldosterone and pre- administration of 1, 3, 10 mg RO6836191 or placebo to healthy cursors in subjects who received study drug under normal-salt or subjects under low-salt diet (study part 2). Box whiskers show interquartile range (box) and min–max (whiskers) for Na/K ratio low-salt conditions without an ACTH challenge (part 2) resulted from 24-hour urine collections, by dose group (n=6/dose group) in a similar dose response as observed with ACTH challenge at baseline (BL), on day 1 and day 2. (part 1). The exploratory PKPD analysis indicated that aldo- sterone decreases, and both 11-DOC and 11-deoxycortisol lev- However, a high suppression is achieved by single doses of 3 els increase RO6836191 concentration dependently as a direct mg and virtually complete aldosterone suppression at 10 mg. effect. The PKPD model parameters provide a good estimate of By contrast, the precursors only start to increase at a dose the concentration range where RO6836191 is both efficacious of 90 mg and cortisol remained unchanged up to the highest and selectively inhibits aldosterone but not cortisol production. dose tested (360 mg). The half-maximal inhibitory concentration (IC ) values were We think that of critical importance in the evaluation similar to the Ki values from human in vitro assays (Table SIII). of selectivity, and its impact on the potential clinical util- ity of an AS inhibitor, is the demonstration of a wide dose Effect on Electrolytes, Renin, and ACTH in Healthy Subjects range at which aldosterone itself is suppressed without any increase in its precursor, 11-DOC. Aldosterone and 11-DOC Low-salt diet conditions caused the expected increases in have approximately equal and high affinities for the MR and plasma aldosterone and PRA levels (Figures SIVB and circulate at roughly similar total concentrations under nor- SVIID). Urinary sodium excretion was dose-dependently mal conditions; however, the free fraction of aldosterone (fu (0–10 mg) increased by RO6836191 (up to 100% from base- 0.30–0.50) is greater than that of 11-DOC (fu <0.05). In the line) under low-salt conditions (Figure SVIB). An increase case of imbalance between free aldosterone and free 11-DOC, in urinary sodium (0–360 mg) was also observed under the mineralocorticoid effect of 11-DOC may become relevant, uncontrolled, normal-salt conditions, but without any differ- with high levels of circulating 11-DOC negating the value of ence from placebo (see Figure SVIA). Only minor changes aldosterone inhibition. Because 11-DOC is a substrate for in potassium urinary excretion (≤20%) were observed (see both CYP11B1 and CYP11B2 (Figure SI), intact activity of Figure SVIC and SVID). However, an increase in the urinary the former now seems essential in preventing accumulation sodium/potassium ratio was apparent, under controlled low- of the precursor after inhibition of the latter. This observation salt diet (Figure 4). No change in urine creatinine or in plasma may explain the relatively low clinical effect on blood pres- electrolytes (potassium, sodium) was apparent. The single- sure, when compared with MR blockade, of an AS inhibitor dose administration of RO6836191 resulted in minor (<2- with less selectivity than RO6836191. Thus, after treatment fold increase) changes in PRA (Figure SVIIC and SVIID), and there were no dose- or diet-dependent changes in ACTH with LCI699, the ACTH-stimulated adrenal gland produced a 10-fold increase in 11-DOC. RO6836191 did not affect (Figure SVIIA and SVIIB). the ability of CYP11B1 to transform 11-DOC into corticos- Discussion terone; however, to date, we do not have data to confirm that the 11β-hydroxylase activity of CYP11B2 is inhibited by Our studies demonstrate that, despite the close homology of the 2 enzymes catalyzing the final steps in aldosterone RO683619. The large selectivity ratio of RO6836191 in vitro led us and cortisol synthesis, it is possible for 1 molecule to com- pletely inhibit the former without effect on the latter and that to study a wide range of doses in vivo, allowing the quanti- preclinical experiments conducted on cell lines and then in fication of the dose range over which it can be expected to monkeys predict to a remarkable degree the doses in humans, suppress aldosterone but not cortisol. Although the single- which selectively inhibit aldosterone production. The thera- dose first-in-man study cannot establish the clinical range, the use of conventional PKPD analyses enabled us to cor- peutic range of RO6836191 for optimal plasma aldosterone suppression has to be defined in subsequent clinical studies. relate doses at which biochemical responses were observed Bogman et al An Oral Selective Inhibitor of Aldosterone Synthase 195 with the previously measured Ki of RO6836191 for the 2 natriuresis would lower blood pressure, and several doses enzymes, CYP11B1 and CYP11B2. Some other selective AS may be required for a compensatory activation of PRA to be inhibitors have recently been reported, but the only one to detectable. We have yet to show any effect on blood pressure, date tested in humans did not reduce plasma aldosterone by which was not measured in the cynomolgus studies addressing >70%, even at high doses. Previous AS inhibitors to enter biochemical and pathological effects of RO6836191 and did clinical development were LCI699 and fadrozole. Of these, not change after the single dose in healthy volunteers. Clearly, LCI699 has been repurposed for the treatment of Cushing therefore, predictions regarding clinical potential are untested disease, where excess of cortisol is the cardinal clinical fea- until multiple-dose studies have been undertaken, and the ture, and fadrozole is an aromatase inhibitor used for the effect of selective AS inhibition on blood pressure is demon- 24,25 treatment of breast cancer. strated in patients with primary aldosteronism. However, an We undertook extensive preclinical studies in the cyno- important first step was to show whether the hurdle of 93% molgus monkey to understand changes observed in adre- homology between AS and 11β-hydroxylase could be over- nal ZG. Interestingly, RO6836191 reproduced the adrenal come, and if so, what the consequences are likely to be for −/− changes of the AS mouse, namely, expansion of the ZG; circulating levels of substrate and product of the 2 enzymes. increased expression of AS (or of enhanced green fluo- Our findings open the door to suppression of aldosterone pro- rescent protein, which was used to disrupt the CYP11B2 duction being investigated as a treatment option and provide −/− gene in the AS some confidence that further selective inhibitors of AS can be mouse); hypertrophy, proliferation, and apoptosis of ZG cells. The partial amelioration of apop- developed using the monkey as the relevant preclinical species totic and other adaptive changes in the ZG layer, by elec- for mechanistic exploration of aldosterone inhibition. trolyte supplementation and ACE inhibition, is consistent In conclusion, selective AS inhibition can be achieved in with a counteraction of the RO6836191-induced salt loss at humans, with selectivity preventing not only the inhibition doses ≥1 mg/kg. However, not all the effects on ZG cells of of cortisol production but also the compensatory increase in RO6836191 were reversed by ACE inhibition and electrolyte 11-DOC. Preclinical data underline the probable importance supplementation. We cannot exclude the possibility that the of aldosterone in regulating ZG cell turnover, with the pos- dose of the ACE inhibitor, or the electrolyte replacement, sibility that accelerated turnover may contribute to chronic was insufficient to fully prevent sodium depletion. However, efficacy of AS inhibitors in comparison with the receptor the residual increase in CYP11B2 expression and apoptosis blockers, which elevate aldosterone secretion. in the ZG layer are consistent with adrenal changes reported −/− Perspectives in the AS mouse, which suggested that ZG cells migrate to the medullary junction and undergo apoptosis when unable The homology between the 2 enzymes catalyzing the final to synthesize aldosterone. Increased renin production is steps in aldosterone and cortisol synthesis has been a block −/− observed in the AS mouse, in response to sodium deple- in the development of drugs, which inhibit aldosterone tion, as in our monkeys treated with RO6836191 but not secretion rather than block its response. We show that in receiving saline supplementation. But the increased PRA, vitro selectivity can translate into clinical practice, provid- reported in Figure SIII, is unlikely to be the sole or prin- ing that, as in our cynomolgus studies, the single-dose find- cipal cause of changes in the adrenal, especially when the ings are predictive of those on multiple dosing. An important renin elevation is partly because of the inhibition by lisino- discovery is that the plasma concentration of deoxycortico- pril of angiotensin production. Only minor increases in PRA sterone, the biologically active substrate for AS, does not were observed after a single dose in the clinical study. We increase at selective doses of inhibitor. This predicts greater would anticipate an increase in PRA in future, multiple dos- natriuretic and antihypertensive efficacy than achieved by ing, studies, given the expected natriuresis and the trend we less selective inhibitors. observed both in the preclinical data and the low-salt diet group in the clinical study. Although there was no significant Acknowledgments We thank Nathalie Lambert for operational support during the study evidence of natriuresis after a single dose of RO6836191, we conduct, personnel from PRA Groningen, the Netherlands, for the did observe a similar increase in the Na/K ratio as reported clinical study conduct, and Kevin Smart for critical review of the ex- after a single dose of MR antagonist. ploratory PKPD analysis. A degree of apoptosis may be beneficial in patients with hyperaldosteronism, particularly if this occurred in the so- Sources of Funding called aldosterone-producing cell clusters that are often the All studies were funded by F. Hoffmann-La Roche Ltd. only residual sites of aldosterone production or in actual aldo- sterone-producing adenomas. By contrast, drugs that block Disclosures the MR (ie, spironolactone, eplerenone, and newer aldoste- M.J.B. received external consultant honoraria from F. Hoffmann-La Roche Ltd. K.A., K.B., S.M., M.C.D.V.M., G.P., M.-L.D., and D.S. rone receptor antagonists) cause an increase in aldosterone 28,29 are employees of F. Hoffmann-La Roche Ltd. P.F. is currently an em- production. The MR knockout mouse has shown increased ployee of Novartis Pharma. circulating aldosterone levels, and no reports were found of involution of aldosterone-producing cells. References A limitation of our study for predicting clinical util- 1. Calhoun DA. Hyperaldosteronism as a common cause of resis- ity of AS inhibition is the single-dose nature of the clinical tant hypertension. Annu Rev Med. 2013;64:233–247. doi: 10.1146/ study. It was not expected that in healthy subjects a transient annurev-med-042711-135929. 196 Hypertension January 2017 2. Connell JM, Fraser R, MacKenzie S, Davies E. Is altered adrenal steroid requirements for a new therapeutic strategy. J Hypertens. 2013;31:2085– biosynthesis a key intermediate phenotype in hypertension? 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Specific nonge- Endocrinol. 2014;10:127. doi: 10.1038/nrendo.2013.266. nomic actions of aldosterone. Kidney Int. 2000;57:1390–1394. doi: 20. Martin RE, Aebi JD, Hornsperger B, et al. Discovery of 4-Aryl-5,6,7,8- 10.1046/j.1523-1755.2000.00980.x. tetrahydroisoquinolines as potent, selective, and orally active aldosterone 6. Brown NJ. Contribution of aldosterone to cardiovascular and renal inflam- synthase (CYP11B2) inhibitors: in vivo evaluation in rodents and cyno- mation and fibrosis. Nat Rev Nephrol. 2013;9:459–469. doi: 10.1038/ molgus monkeys. J Med Chem. 2015;58:8054–8065. doi: 10.1021/acs. nrneph.2013.110. jmedchem.5b00851. 7. Azizi M, Amar L, Menard J. Aldosterone synthase inhibition in humans. 21. Cerny MA. Progress towards clinically useful aldosterone synthase inhib- Nephrol Dial Transplant. 2013;28:36–43. doi: 10.1093/ndt/gfs388. itors. Curr Top Med Chem. 2013;13:1385–1401. 8. Bassett MH, White PC, Rainey WE. The regulation of aldosterone syn- 22. 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[Clinical evaluation of CGS16949A in advanced 162. doi: 10.1016/j.mce.2011.07.034. or recurrent breast cancer—a multi-institutional late phase II clinical 11. Gardner DG, Shoback D. Greenspan’s Basic and Clinical Endocrinology. trial]. Gan To Kagaku Ryoho. 1994;21:477–484. 8th. New York, NY: McGraw Hill Medical; 2007. 25. Wada T, Nomura Y, Oohashi Y, Abe O, Koyama H, Takashima S. [Late 12. Menard J, Watson C, Rebello S, Zhang YM, Dole WP. Hormonal and elec- phase II study of CGS16949A, a new aromatase inhibitor—a multicen- trolyte responses to the aldosterone synthase inhibitor LCI699 in sodium tral cooperative study (Western Japan Group)]. Gan To Kagaku Ryoho. depleted healthy subjects. JACC. 2010;55:A61.E583. 1994;21:485–493. 13. Amar L, Azizi M, Menard J, Peyrard S, Watson C, Plouin PF. Aldosterone 26. Lee G, Makhanova N, Caron K, Lopez ML, Gomez RA, Smithies O, synthase inhibition with LCI699: a proof-of-concept study in patients with Kim HS. 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Eplerenone: a review of its use in essential hyper- Lefkowitz M, Trapani A. The effects of aldosterone synthase inhi- tension. Am J Cardiovasc Drugs. 2005;5:51–69. bition on aldosterone and cortisol in patients with hypertension: 29. Pitt B, Kober L, Ponikowski P, Gheorghiade M, Filippatos G, Krum H, a phase II, randomized, double-blind, placebo-controlled, multi- Nowack C, Kolkhof P, Kim SY, Zannad F. Safety and tolerability of the center study. J Clin Hypertens (Greenwich). 2012;14:580–587. doi: novel non-steroidal mineralocorticoid receptor antagonist BAY 94-8862 10.1111/j.1751-7176.2012.00667.x. in patients with chronic heart failure and mild or moderate chronic kidney 16. Karns AD, Bral JM, Hartman D, Peppard T, Schumacher C. Study of disease: a randomized, double-blind trial. Eur Heart J. 2013;34:2453– aldosterone synthase inhibition as an add-on therapy in resistant hyper- 2463. doi: 10.1093/eurheartj/eht187. tension. J Clin Hypertens (Greenwich). 2013;15:186–192. doi: 10.1111/ 30. Berger S, Bleich M, Schmid W, Cole TJ, Peters J, Watanabe H, Kriz jch.12051. W, Warth R, Greger R, Schütz G. Mineralocorticoid receptor knockout 17. Schumacher CD, Steele RE, Brunner HR. Aldosterone synthase inhi- mice: pathophysiology of Na+ metabolism. Proc Natl Acad Sci U S A. bition for the treatment of hypertension and the derived mechanistic 1998;95:9424–9429. Novelty and Significance What Is New? Summary We report that it is possible to completely inhibit the enzyme-producing An inhibitor of aldosterone synthase was tested over multiple doses aldosterone without causing any inhibition of the 93% homologous en- in cynomolgus monkeys and after a single dose in humans. Its 100- zyme-producing cortisol. fold selectivity in vitro (for aldosterone versus cortisol inhibition) This inhibition reduced plasma aldosterone to below detectable levels, translated into 100-fold difference in doses required to inhibit aldo- without accumulation of deoxycorticosterone, the substrate of aldoste- sterone and cortisol in vivo. rone synthase. What Is Relevant? No drug is currently available for the treatment of hyperaldosteronism, which works through inhibition of aldosterone production rather than blocking aldosterone response. This study now permits the consequenc- es of selective aldosterone inhibition to be investigated in patients. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Hypertension (Dallas, Tex. : 1979) Pubmed Central

Preclinical and Early Clinical Profile of a Highly Selective and Potent Oral Inhibitor of Aldosterone Synthase (CYP11B2)

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10.1161/HYPERTENSIONAHA.116.07716
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Abstract

Aldosterone Synthase Inhibitor Preclinical and Early Clinical Profile of a Highly Selective and Potent Oral Inhibitor of Aldosterone Synthase (CYP11B2) Katrijn Bogman, Dietmar Schwab, Marie-Laure Delporte, Giuseppe Palermo, Kurt Amrein, Susanne Mohr, Maria Cristina De Vera Mudry, Morris J. Brown,* Philippe Ferber* See Editorial Commentary, pp 11–12 Abstract—Primary hyperaldosteronism is a common cause of resistant hypertension. Aldosterone is produced in the adrenal by aldosterone synthase (AS, encoded by the gene CYP11B2). AS shares 93% homology to 11β-hydroxylase (encoded by the gene CYP11B1), responsible for cortisol production. This homology has hitherto impeded the development of a drug, which selectively suppresses aldosterone but not cortisol production, as a new treatment for primary hyperaldosteronism. We now report the development of RO6836191 as a potent (Ki 13 nmol/L) competitive inhibitor of AS, with in vitro selectivity >100- fold over 11β-hydroxylase. In cynomolgus monkeys challenged with synthetic adrenocorticotropic hormone, single doses of RO6836191 inhibited aldosterone synthesis without affecting the adrenocorticotropic hormone–induced rise in cortisol. In −/− repeat-dose toxicity studies in monkeys, RO6836191 reproduced the adrenal changes of the AS mouse: expansion of the −/− zona glomerulosa; increased expression of AS (or disrupted green fluorescent protein gene in the AS mouse); hypertrophy, proliferation, and apoptosis of zona glomerulosa cells. These changes in the monkey were partially reversible and partially preventable by electrolyte supplementation and treatment with an angiotensin-converting enzyme inhibitor. In healthy subjects, single doses of RO6836191, across a 360-fold dose range, reduced plasma and urine aldosterone levels with maximum suppression at a dose of 10 mg, but unchanged cortisol, on adrenocorticotropic hormone challenge, up to 360 mg, and increase in the precursors 11-deoxycorticosterone and 11-deoxycortisol only at or >90 mg. In conclusion, RO6836191 demonstrates that it is possible to suppress aldosterone production completely in humans without affecting cortisol production. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifier: NCT01995383. (Hypertension. 2017;69:189-196. DOI: 10.1161/HYPERTENSIONAHA.116.07716.) Online Data Supplement Key Words: adrenal cortex ■ aldosterone ■ CYP11B2 ■ cytochrome P-450 ■ hyperaldosteronism ■ hydrocortisone ■ zona glomerulosa rimary hyperaldosteronism is recognized as a common cortex, catalyzes the final 3 steps in aldosterone synthesis from Pcause of resistant hypertension and contributes to heart and 11-deoxycorticosterone (11-DOC; sequentially to corticoste- 1–3 kidney diseases. Mineralocorticoid receptor (MR) antago- rone and 18-OH-corticosterone) to aldosterone. Aldosterone nists are currently included among the treatments of choice. synthesis shares several of the same enzymes as that of cortisol However, they are not always well tolerated and induce a synthesis, whose final step (from the precursor 11-deoxycorti- counterregulatory increase in aldosterone production, which sol) is mediated by 11β-hydroxylase, encoded by the CYP11B1 3,4 may limit the efficacy of the MR blockade. Furthermore, gene in the zona fasciculata and zona reticularis (see Figure experimental data suggest that some of the deleterious effects SI in the online-only Data Supplement for a schematic repre- 5,6 8 of aldosterone may occur through nongenomic pathways. sentation of the synthetic pathways). Selective AS inhibition These limitations have led to the exploration of alternative has foundered on the 93% homology between the transcripts approaches to antagonize aldosterone effects by inhibition of of CYP11B2 and CYP11B1 genes. Angiotensin II, potassium, aldosterone synthase (AS, encoded by the gene CYP11B2). and adrenocorticotropic hormone (ACTH) are the main physi- AS, expressed within the zona glomerulosa (ZG) of the adrenal ological agonists that regulate aldosterone secretion. ACTH Received April 28, 2016; first decision May 23, 2016; revision accepted September 13, 2016. From the Clinical Pharmacology (K.B., D.S., M.-L.D.), Biostatistics (G.P.), Discovery (K.A.), Pharmaceutical Sciences (S.M., M.C.D.V.M.), and Translational Medicine, Cardiovascular Diseases (P.F.), Pharma Research and Early Development, Roche Innovation Center Basel, Switzerland; and Clinical Pharmacology, William Harvey Research Institute, Queen Mary University of London, United Kingdom (M.J.B). Current address for P.F.: Novartis Pharma, Basel, Switzerland. *These authors contributed equally to this work. The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA. 116.07716/-/DC1. Correspondence to Katrijn Bogman, F. Hoffmann-La Roche Ltd, Grenzacherstrasse 124, 4070 Basel, Switzerland. E-mail katrijn.bogman@roche.com © 2016 The Authors. Hypertension is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDervis License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made. Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.116.07716 189 190 Hypertension January 2017 inhibitor. In both studies, recovery animals were dosed for 4 weeks stimulates the adrenal glands to release cortisol, and negative and then maintained undosed for a further 4 weeks to assess recovery. feedback of cortisol on ACTH secretion occurs at the hypotha- The studies were performed at contract research organizations lamic and pituitary levels. accredited by the Association for Assessment and Accreditation of The only AS inhibitor to enter phase 2 clinical development Laboratory Animal Care International (AAALAC) study procedures was LCI699. This compound reduced plasma aldosterone in such as necropsy and are in compliance with AAALAC accreditation. Further details are provided in the online-only Data Supplement. healthy subjects and in patients with primary hyperaldosteron- 7,12,13 ism. It decreased blood pressure in patients with primary Histology and Immunohistochemistry hyperaldosteronism, essential hypertension, and resistant Serial sections (nominal thickness of ≈5 µm) were prepared from 13–17 hypertension. However, lack of sufficient selectivity led to formalin-fixed, paraffin-embedded adrenal gland (left) of all animals. cortisol suppression within the clinical efficacious dose range The adrenal gland was stained routinely with hematoxylin-eosin, and consequently apparent termination of the development as well as for Ki-67 (marker for proliferative activity), CYP11B2 (marker for AS), and TUNEL (terminal deoxynucleotidyl transfer- program in hypertension. Indeed, LCI699 is currently under 18,19 ase-mediated deoxyuridine triphosphate nick end labeling; marker development for the treatment of Cushing syndrome. for apoptosis). Further details are provided in the online-only Data RO6836191 was designed as a highly selective and potent Supplement. AS inhibitor, belonging to the series with a novel tetrahy- droisoquinoline structure. We have investigated the selectiv- First-In-Man Study ity, efficacy, and safety of RO6836191 in various preclinical This was an adaptive, single-center, randomized, blinded, single ascend- ing dose, placebo-controlled study to investigate safety, tolerability, test systems, including in vitro cells expressing recombinant pharmacokinetic effects, and pharmacodynamic effects of RO6836191 CYP11B1 and CYP11B2 enzymes. Because there is only 68% in healthy male subjects (unique identifier: NCT01995383) conducted homology between rat and human CYP11B2, preclinical phar- in the Netherlands. RO6836191 was administered as oral solution to macological characterization and chronic toxicity studies were fasted subjects on day 1. Subjects were advised to consume a minimum of 1.5 L fluid daily while in clinic. performed in cynomolgus monkeys. We then conducted a The study was conducted in 2 parts. In part 1, a total of 64 subjects first-in-man single ascending dose study during which we mea- received single ascending oral doses of 1, 3, 10, 30, 90, 180, or 360 mg sured the extent of translation from preclinical into clinical. RO6836191 or matching placebo (the active treatment/placebo ratio was 3/1) under normal-salt diet conditions with ACTH challenge (n=56) or Methods without ACTH challenge (n=8). In part 2, a total of 24 subjects received single oral doses of 1, 3, or 10 mg RO6836191 or matching placebo (the In Vitro Assay of CYP11B2 and CYP11B1 active treatment/placebo ratio was 3/1). Each subject was studied under Inhibition low-salt and normal-salt diet conditions, in a 2-way crossover design The in vitro effect of RO6836191 on CYP11B2 and CYP11B1 proteins with a 2- to 3-week wash out, with no change in treatment assignment. was assessed in human renal leiomyoblastoma cells (ATCC CRL1440) Dosing under low-salt diet conditions required a 3-day in-clinic run-in period in which subjects adhered to a low-salt diet of 50 mmol sodium/d expressing recombinant human or cynomolgus CYP11B1 and and 70 to 100 mmol potassium/d, which was confirmed by urinary so- CYP11B2 enzymes as described for an earlier compound in the series. dium and creatinine assessments. The low-salt diet was continued until Further details are provided in the online-only Data Supplement. 48 hours postdose. Adverse events, vital signs, orthostatic vital signs, ECG, standard laboratory safety data (hematology, coagulation, blood In Vivo Monkey Pharmacology Study chemistry, and urinalysis), and body weight were monitored at baseline The effect of RO6836191 in cynomolgus monkeys was assessed as and postdose. The AS inhibition was assessed with different procedures described for an earlier compound in the series. Animals received to stimulate aldosterone release: ACTH challenge (intravenous admin- an oral gavage of vehicle and RO6836191 (0.035, 3, or 30 mg/kg, istration of 0.25 mg Cortrosyn, 1 hour postdose in part 1 only), postural 2 monkeys per dose). At 1 hour postdose, 0.0145 mg/kg Synacthen change (assessed at 6 hours postdose in parts 1 and 2, subjects were re- (ACTH challenge) was given intramuscularly. Serial blood samples quired to be standing or moving for at least 30 minutes before sampling, were collected pre- and postdose for analysis of RO6836191, cortisol, whereas at other timepoints pharmacodynamic samples were taken in aldosterone, and precursors. supine position), and low-salt diet. Serial blood samples (predose and up to maximum 96 hours post- In Vivo Monkey Preclinical Safety Studies dose) were taken for the assessment of plasma aldosterone, cortisol, and their precursors, plasma renin activity (PRA), plasma ACTH, and A 4-week high-dose study and 4-week mechanistic study were per- plasma electrolytes. Serial blood samples were taken before and up formed. The high-dose study explored doses from 0 to 40 mg/kg and to 4 hours post-ACTH challenge for the measurement of cortisol and included a treatment-free period to assess the reversibility, persis- precursors. Urine (collected from day −1 up to 48 hours postdose) tence, or delayed occurrence of any changes noted at 40 mg/kg. At 40 was analyzed for electrolytes, aldosterone, tetrahydroaldosterone, mg/kg, clinical signs of dehydration (skin tenting, urine drinking, and and cortisol. All pharmacodynamic assessments and procedures increase water consumption) after 16 days of dosing necessitated a were performed at baseline (day −1, time-matched with day 1 assess- suspension of dosing for 3 days. Animals received free access to elec- ments) and at multiple time points postdose. Serial blood samples for trolyte solution bottles from day 17 onward, which resulted in rapid RO6836191 pharmacokinetic analysis were collected up to 96 hours reversal of dehydration status and allowed redosing of the animals postdose, and a sample was taken on day 9 and at the follow-up visit. until end of the 4-week treatment period. The study was approved by the local independent ethics committees A 4-week mechanistic study explored doses from 0 to 1 mg/ and health authorities and conducted according to the provisions of the kg, testing whether adrenal changes in the high-dose study were Declaration of Helsinki. Written informed consent was obtained from entirely because of salt depletion and consequent activation of the each study participant before conducting any protocol-related proce- renin–angiotensin system, and investigating the reversibility of any dures. Further details are provided in the online-only Data Supplement. remaining changes in the adrenal ZG. Animals received either ve- hicle alone or RO6836191 0.15 or 1 mg/kg, with electrolyte supple- Pharmacokinetic and Statistical Analysis mentation and with or without angiotensin-converting enzyme (ACE) inhibitor (lisinopril 0.5 mg/kg). An additional group received 1 mg/ Results are reported as mean and standard deviation. Estimates of kg RO6836191 alone without electrolyte supplementation or ACE ANOVA models are reported together with 90% confidence interval (CI). Bogman et al An Oral Selective Inhibitor of Aldosterone Synthase 191 Percentage change from baseline was calculated as follows: (PD Table 1. In Vitro Potency of RO6836191 as Inhibitor of treated − PD baseline) × 100/PD baseline, where PD refers to a phar- CYP11B1 and CYP11B2 in Human Renal Leiomyoblastoma macodynamic marker concentration at a specific time point or over a Cells Expressing Recombinant Human and Cynomolgus specific time interval. Monkey Enzymes Pharmacokinetic parameters for RO6836191 and area under the plasma concentration versus time curve (AUC) for the pharmacody- Monkey Ki, Human Ki, namic markers were estimated by noncompartmental methods, and Enzyme Substrate nmol/L nmol/L exploratory pharmacokinetic and pharmacodynamic (PKPD) analy- Aldosterone 11-DOC 4±2.2 13±2.2 sis was performed using WinNonlin software (Phoenix version 6.2; synthase Pharsight Corporation, Mountain View, CA). (CYP11B2) Plasma concentrations for aldosterone and precursors that were be- low limit of quantification were set to the lower limit of quantification 11β-Hydroxylase 11-deoxycortisol 3150±1410 1310±533 value to allow further calculations. (CYP11B1) GraphPad Prism 6, version 6.04 (GraphPad Software, Inc, La Jolla, CA) was used for creation of figures and for calculation of in vitro Ki values. To calculate the inhibition constant (Ki) value, the amount Selectivity ratio 800 100 produced was plotted versus the substrate concentration for every in- Cells were incubated in the presence of 11-deoxycorticosterone (11-DOC) hibitor concentration tested; the resulting data set was then analyzed or 11-deoxycortisol as substrates for CYP11B2 and CYP11B1, respectively. Ki using the mixed model equation that is part of the statistic program values are means of replicates ±SD (n=5 and 8–13 for CYP11B2 and CYP11B1, GraphPad Prism. respectively). Selectivity ratio is defined as the ratio of Ki for CYP11B1 versus For the monkey study, individual percentage change from vehicle Ki for CYP11B2. in analytes in plasma at 2 hours postdose (or 1 hour after Synacthen application) was calculated. To test the effect of dose on plasma aldosterone, cortisol, and their present in vehicle-treated monkeys was absent or indistinct precursors in the clinical study, an ANOVA model was applied on re- in RO6836191-treated animals. Furthermore, both apoptosis sults from part 1 on the change from baseline for the log-transformed and proliferation of ZG cells were increased in dosed animals AUC. Baseline AUC (as a continuous variable) and dose (as a cat- compared with vehicle controls. After a 4-week treatment- egorical variable) were included as fixed effects in the model. The 90% CIs were derived for the percentage change from baseline on free period, there was a trend toward reversibility of increased AUC for each dose and for the placebo-adjusted percentage change CYP11B2 expression, while apoptosis and proliferation of from baseline on AUC for each active dose of RO6836191. ZG cells persisted (see Figure 1A). An exploratory pharmacokinetic and pharmacodynamic analysis Changes in clinical chemistry and urinalysis were consistent using data from the clinical study was applied on percentage change with the predicted natriuretic action of RO6836191, notably PRA from baseline in plasma aldosterone, 11-DOC, and 11-deoxycortisol concentration versus the corresponding RO6836191 plasma concen- was increased in both sexes at 1 and 7 mg/kg. At these dose lev- tration (details of the analysis are in Table SIII in the online-only Data els, aldosterone levels were clearly decreased without changing Supplement). cortisol. At the highest dose of 40 mg/kg, there was no change in renin, most probably because high-dose animals received rescue Analytical Methods electrolyte supplementation. At this dose, a decrease in cortisol Details are provided in the online-only Data Supplement. was observed in addition to the expected decrease in aldoste- rone, indicating a loss of selectivity against 11β-hydroxylase Results (CYP11B1). All values returned to control group values by the Estimation of In Vitro Selectivity of RO6836191 end of the 4-week treatment-free period (see Figure SIIIA). Cortisol or aldosterone production by human renal leiomyo- In the mechanistic study, supplementation with electro- blastoma cells expressing recombinant human or monkey lytes ameliorated the histopathologic findings in the adrenal CYP11B1 or CYP11B2 was measured in the presence of the gland (see Figure 1B), which had occurred in the previous respective substrate for these enzymes. The in vitro Ki values study at doses of 1 mg/kg and higher, namely, resulting in of RO6836191 in these cell lines showed that RO6836191 is only minimally reduced ZG expansion, CYP11B2 expression, a potent, selective, and competitive inhibitor of AS (Table 1 and cell proliferation. In addition, coadministration of an ACE and Figure SII). Its inhibition of AS was 800- and 100-fold inhibitor in monkeys treated with 1 mg/kg RO6836191 with more potent than that of 11β-hydroxylase (CYP11B1) in the electrolytes conferred further protection, notably with the monkey and human cell lines, respectively. retention of the transitional zone. At 0.15 mg/kg, no expan- sion of the ZG was observed. PRA was increased only for Histopathologic Effect of RO6836191 on Adrenal doses >0.15 mg/kg. PRA was still high when electrolyte sup- ZG in Monkey plementation was present, but almost comparable with control The ZG of the adrenal gland was the main affected tissue, values in the group receiving electrolyte supplementation and and changes occurred at the lowest dose tested, that is, 1 mg/ ACE inhibitor treatment. kg, which was then used as the high dose in the mechanis- Measurements of aldosterone and cortisol showed the tic study. Results of the semiquantitative analysis from both expected decrease and no change, respectively, at all doses. studies are in Table SI. There was drug-related expansion of The expected decrease of aldosterone was also observed in the ZG, hypertrophy, and increased AS (CYP11B2) expres- the group treated with ACE inhibitor alone (see Figure SIIIB). sion of ZG cells at 1 or 7 mg/kg. At 40 mg/kg RO6836191, Table SII shows that the efficacy and selectivity of the 0.15 the width of the ZG and CYP11B2 expression was reduced and 1 mg/kg doses are consistent with the RO6836191 expo- compared with the lower doses. The typically unstained tran- sures being equal or above the Ki for CYP11B2 and below Ki sitional zone at the junction of the ZG and zona fasciculata for CYP11B1. 192 Hypertension January 2017 but absence of change in cortisol levels post-ACTH chal- lenge across the 1000-fold dose range of RO6836191, was consistent with the selectivity ratio predicted from in vitro experiments. The observed aldosterone reduction at the low- est dose and the related free drug exposure are in line with the in vitro Ki value for AS. By contrast, the rise in 11-DOC and 11-deoxycortisol was consistent with the free drug exposure at the highest dose being comparable with the in vitro Ki value for 11β-hydroxylase (see Table 2). RO6836191 plasma expo- sure data are provided in Table SII. Proof of Mechanism in Healthy Subjects Safety, Tolerability, and Pharmacokinetics RO6836191 was well tolerated at all doses tested. The most Figure 1. Histopathologic changes in the cynomolgus monkey frequently reported adverse events were headache, nasophar- zona glomerulosa in the high-dose (A) and mechanistic (B) safety studies. A, Histopathology of representative monkeys in the high- yngitis, asthenia, and diarrhea, which were also reported in dose study. Animals were administered RO6836191 at 0, 1, 7, or the placebo group and which were all of mild intensity. There 40 mg/kg for 4 weeks and recovery after 40 mg/kg. Staining with was no dose-related increase in incidence or in the severity of hematoxylin-eosin (upper); immunostaining with human anti- reported adverse events. There were no other apparent treat- CYP11B2 antibody to visualize the zona glomerulosa (lower). Scale bar=100 µm. Dotted lines delineate the demarcation ment-related trends or any clinically significant findings in between the zona glomerulosa from the underlying zonal any other safety assessments. Mean time-matched blood pres- fasciculata. B, Histopathology of representative monkeys in the sure and pulse rate changes from baseline up to day 3 did not mechanistic study. Animals were administered RO6836191 at 1 mg/kg for 4 weeks, with or without electrolytes (E) or angiotensin- show a trend with time, dose, or diet, and no difference from converting enzyme inhibitor lisinopril 0.5 mg/kg (A), recovery after placebo was observed. treatment with 1 mg/kg with electrolytes (E*), control monkeys RO6836191 was rapidly absorbed reaching maximum were administered electrolytes alone. Staining with hematoxylin- plasma concentrations between 0.5 and 2 hours postdose. eosin (scale bar=100 µm; upper), double-sided arrows delineate width of the zona glomerulosa (ZG), single-sided arrows indicate Exposure of RO6836191 increased dose-proportionally. Mean the transitional zone of the ZG. Immunostaining with human half-life was 29 hours, and ≈10% of the dose was recovered anti-CYP11B2 antibody to visualize the zona glomerulosa (scale unchanged in the urine (RO6836191 plasma exposure data are bar=50 µm; lower). provided in Table SII). Pharmacological Characterization in Cynomolgus Pharmacological Characterization in Healthy Subjects Monkeys With ACTH Challenge and Comparison With Monkey Data The effect of a single oral dose RO6836191 (0–30 mg/kg) The time course of plasma aldosterone and cortisol in healthy on aldosterone, cortisol, and precursors after an ACTH chal- subjects after single ascending doses of RO6836191 or pla- lenge is shown in Figure 2. RO6836191 blunted aldosterone cebo with ACTH challenge is shown in Figure 3A and 3B. production at all doses tested compared with vehicle, with a The dose-dependent change from baseline in plasma expo- 70% to 90% decrease in aldosterone from the 0.035 mg/kg sure (AUC ) to aldosterone, cortisol, and their precursors is 0-24 to the 30 mg/kg dose, respectively. The precursors 11-DOC shown in Figure 3C and 3D. In the absence of drug, the ACTH and 11-deoxycortisol were increased only by high doses (up challenge induced a plasma aldosterone and cortisol surge to 600% at 30 mg/kg), but cortisol and corticosterone levels peaking ≈0.5 and 2 hours after the challenge, respectively. were not different from vehicle. The reduction in aldosterone, The postural change induced an immediate surge in plasma Figure 2. Dose-dependent effect of RO6836191 on plasma aldosterone, cortisol, and precursors in cynomolgus monkeys. Data represent individual % change from vehicle in plasma concentration (measured at 1 hour post-adrenocorticotropic hormone challenge) for aldosterone and precursors (A) and cortisol and precursors (B) by dose group. Means by dose group are connected by a line (n=2/dose group). 11-DOC indicates 11-deoxycorticosterone. Bogman et al An Oral Selective Inhibitor of Aldosterone Synthase 193 Table 2. Comparison of Human and Monkey In Vivo and In Vitro Potency and Selectivity Aldosterone Synthase 11β-Hydroxylase RO6836191-Free C Max Change in RO6836191-Free C Max Change in avg avg Ki In Vitro, at Minimum Effective Aldosterone Ki In Vitro, at Dose With Max 11-Deoxycortisol Species ng/mL Dose, ng/mL (% Chg From Ctrl) ng/mL Observed Effect, ng/mL (% Chg From Ctrl) a b c c Monkey 1.45 0.74; 0.94 −84.6; −94.1 1144 1891; 1972 + 266; +519 d e f f Human 4.7 5.2 (0.57) −84.9 (−86.6 to −82.8) 476 793 (79.3) +193 (157 to 234) Ki (see Table 1) units were converted to ng/mL for comparison with RO6836191 plasma concentrations. RO6836191 free C was calculated from total C , taking avg avg into account the free fraction of 0.26 for human and of 0.33 for monkey; C is expressed as individual data per monkey or mean and SD for human. Change in plasma avg exposure for aldosterone and 11-deoxycortisol is expressed as % change from vehicle for monkey (individual values per monkey, n=2) and as % change from day 1 baseline for human (represented as mean and 90% confidence interval). The minimum effective dose was defined as the dose at which ≥75% of the maximum observed effect was reached. a, b, c, d, e, and f are parameters for dose groups 0.035 mg/kg, 3 mg/kg, 30 mg/kg, 3 mg, 10 mg, and 360 mg, respectively. aldosterone to a similar level as induced by the ACTH chal- CI, 157 to 234]). 18-Hydroxycorticosterone decreased at 1 mg lenge. After RO6836191 administration, plasma aldosterone with a maximum effect at 10 mg onward (P<0.01; maximum was decreased dose-dependently with the maximum effect mean change in AUC from baseline −58.6% [90% CI, −64.3 reached by 10 mg onward (mean change in AUC from baseline to −52.0]). These results were consistent with the in vitro and −84.9% [90% CI, −86.6 to −82.8%]; different from placebo monkey findings, with the absence of change in cortisol levels P<0.0001). Corticosterone and cortisol were unchanged at all post-ACTH challenge across the 360-fold dose range predicted doses compared with placebo, and there were no reductions in by the drug’s selectivity for CYP11B2. The increase in plasma plasma cortisol on ACTH challenge. At doses >90 mg, there concentration of precursors 11-DOC (substrate for CYP11B1 were dose-dependent (and different from placebo P<0.01) and CYP11B2) and 11-deoxycortisol (substrate for CYP11B1) increases in 11-DOC (maximum mean change in AUC from at doses >90 mg were proportionately less than the reduction baseline +194% [90% CI, 160 to 232]) and 11-deoxycortisol in aldosterone and as in monkey was absent or insubstantial (maximum mean change in AUC from baseline +193% [90% except at concentrations of drug inhibiting 11β-hydroxylase. Figure 3. Dose-dependent effect of RO6836191 on plasma aldosterone, cortisol, and precursors in healthy subjects (study part 1). Aldosterone (A) and cortisol (B) plasma concentration-time profiles after single ascending doses of RO6836191 (0–360 mg) administered orally to healthy subjects 1 hour before the adrenocorticotropic hormone (ACTH) challenge. Downward arrow indicates time of ACTH challenge (1 hour postdose) and upward arrow time of postural change (6 hours postdose). Data represent mean plasma concentrations (+SD) by dose group (n=6/dose group and n=12 for placebo). Below limit of quantification (BLQ) values were set to lower limit of quantification (LLOQ; 5 pg/mL), for doses >10 mg, aldosterone concentrations were BLQ from 2.5 hours postdose onward. Effect of RO6836191 on plasma aldosterone (C), cortisol (D), and precursors after a single oral dose (0–360 mg) in healthy subjects administered 1 hour before the ACTH challenge. Data represent mean (and 90% confidence interval) % change from baseline in plasma exposure (AUC ) 0-24 for aldosterone and precursors (left graph) and cortisol and precursors (right graph) by dose group (n=6/dose group and n=12 for placebo). Filled symbols indicate P<0.01 compared with placebo. 11-DOC indicates 11-deoxycorticosterone. 194 Hypertension January 2017 Consistent with the decrease in plasma aldosterone, uri- nary excretion of both aldosterone and its metabolite, tetrahy- droaldosterone, was decreased dose-dependently (Figure SV). No dose-dependent effect on urinary cortisol was apparent. The pattern of RO6836191-induced changes in aldoste- rone, cortisol, and precursors was qualitatively and quanti- tatively comparable between monkey and human, relative to the respective Ki values. A comparison of monkey and human RO6836191 plasma exposures leading to a similar effect on aldosterone and 11-deoxycortisol is provided in Table 2 and demonstrate that the effective free plasma concentrations are consistent with the in vitro potency and selectivity of RO6836191 in the respective species. PKPD Characterization in Healthy Subjects and Comparison With In Vitro Cell-Based Potency Figure 4. Urinary sodium-to-potassium ratio after single-dose Assessment of the effect of RO6836191 on aldosterone and pre- administration of 1, 3, 10 mg RO6836191 or placebo to healthy cursors in subjects who received study drug under normal-salt or subjects under low-salt diet (study part 2). Box whiskers show interquartile range (box) and min–max (whiskers) for Na/K ratio low-salt conditions without an ACTH challenge (part 2) resulted from 24-hour urine collections, by dose group (n=6/dose group) in a similar dose response as observed with ACTH challenge at baseline (BL), on day 1 and day 2. (part 1). The exploratory PKPD analysis indicated that aldo- sterone decreases, and both 11-DOC and 11-deoxycortisol lev- However, a high suppression is achieved by single doses of 3 els increase RO6836191 concentration dependently as a direct mg and virtually complete aldosterone suppression at 10 mg. effect. The PKPD model parameters provide a good estimate of By contrast, the precursors only start to increase at a dose the concentration range where RO6836191 is both efficacious of 90 mg and cortisol remained unchanged up to the highest and selectively inhibits aldosterone but not cortisol production. dose tested (360 mg). The half-maximal inhibitory concentration (IC ) values were We think that of critical importance in the evaluation similar to the Ki values from human in vitro assays (Table SIII). of selectivity, and its impact on the potential clinical util- ity of an AS inhibitor, is the demonstration of a wide dose Effect on Electrolytes, Renin, and ACTH in Healthy Subjects range at which aldosterone itself is suppressed without any increase in its precursor, 11-DOC. Aldosterone and 11-DOC Low-salt diet conditions caused the expected increases in have approximately equal and high affinities for the MR and plasma aldosterone and PRA levels (Figures SIVB and circulate at roughly similar total concentrations under nor- SVIID). Urinary sodium excretion was dose-dependently mal conditions; however, the free fraction of aldosterone (fu (0–10 mg) increased by RO6836191 (up to 100% from base- 0.30–0.50) is greater than that of 11-DOC (fu <0.05). In the line) under low-salt conditions (Figure SVIB). An increase case of imbalance between free aldosterone and free 11-DOC, in urinary sodium (0–360 mg) was also observed under the mineralocorticoid effect of 11-DOC may become relevant, uncontrolled, normal-salt conditions, but without any differ- with high levels of circulating 11-DOC negating the value of ence from placebo (see Figure SVIA). Only minor changes aldosterone inhibition. Because 11-DOC is a substrate for in potassium urinary excretion (≤20%) were observed (see both CYP11B1 and CYP11B2 (Figure SI), intact activity of Figure SVIC and SVID). However, an increase in the urinary the former now seems essential in preventing accumulation sodium/potassium ratio was apparent, under controlled low- of the precursor after inhibition of the latter. This observation salt diet (Figure 4). No change in urine creatinine or in plasma may explain the relatively low clinical effect on blood pres- electrolytes (potassium, sodium) was apparent. The single- sure, when compared with MR blockade, of an AS inhibitor dose administration of RO6836191 resulted in minor (<2- with less selectivity than RO6836191. Thus, after treatment fold increase) changes in PRA (Figure SVIIC and SVIID), and there were no dose- or diet-dependent changes in ACTH with LCI699, the ACTH-stimulated adrenal gland produced a 10-fold increase in 11-DOC. RO6836191 did not affect (Figure SVIIA and SVIIB). the ability of CYP11B1 to transform 11-DOC into corticos- Discussion terone; however, to date, we do not have data to confirm that the 11β-hydroxylase activity of CYP11B2 is inhibited by Our studies demonstrate that, despite the close homology of the 2 enzymes catalyzing the final steps in aldosterone RO683619. The large selectivity ratio of RO6836191 in vitro led us and cortisol synthesis, it is possible for 1 molecule to com- pletely inhibit the former without effect on the latter and that to study a wide range of doses in vivo, allowing the quanti- preclinical experiments conducted on cell lines and then in fication of the dose range over which it can be expected to monkeys predict to a remarkable degree the doses in humans, suppress aldosterone but not cortisol. Although the single- which selectively inhibit aldosterone production. The thera- dose first-in-man study cannot establish the clinical range, the use of conventional PKPD analyses enabled us to cor- peutic range of RO6836191 for optimal plasma aldosterone suppression has to be defined in subsequent clinical studies. relate doses at which biochemical responses were observed Bogman et al An Oral Selective Inhibitor of Aldosterone Synthase 195 with the previously measured Ki of RO6836191 for the 2 natriuresis would lower blood pressure, and several doses enzymes, CYP11B1 and CYP11B2. Some other selective AS may be required for a compensatory activation of PRA to be inhibitors have recently been reported, but the only one to detectable. We have yet to show any effect on blood pressure, date tested in humans did not reduce plasma aldosterone by which was not measured in the cynomolgus studies addressing >70%, even at high doses. Previous AS inhibitors to enter biochemical and pathological effects of RO6836191 and did clinical development were LCI699 and fadrozole. Of these, not change after the single dose in healthy volunteers. Clearly, LCI699 has been repurposed for the treatment of Cushing therefore, predictions regarding clinical potential are untested disease, where excess of cortisol is the cardinal clinical fea- until multiple-dose studies have been undertaken, and the ture, and fadrozole is an aromatase inhibitor used for the effect of selective AS inhibition on blood pressure is demon- 24,25 treatment of breast cancer. strated in patients with primary aldosteronism. However, an We undertook extensive preclinical studies in the cyno- important first step was to show whether the hurdle of 93% molgus monkey to understand changes observed in adre- homology between AS and 11β-hydroxylase could be over- nal ZG. Interestingly, RO6836191 reproduced the adrenal come, and if so, what the consequences are likely to be for −/− changes of the AS mouse, namely, expansion of the ZG; circulating levels of substrate and product of the 2 enzymes. increased expression of AS (or of enhanced green fluo- Our findings open the door to suppression of aldosterone pro- rescent protein, which was used to disrupt the CYP11B2 duction being investigated as a treatment option and provide −/− gene in the AS some confidence that further selective inhibitors of AS can be mouse); hypertrophy, proliferation, and apoptosis of ZG cells. The partial amelioration of apop- developed using the monkey as the relevant preclinical species totic and other adaptive changes in the ZG layer, by elec- for mechanistic exploration of aldosterone inhibition. trolyte supplementation and ACE inhibition, is consistent In conclusion, selective AS inhibition can be achieved in with a counteraction of the RO6836191-induced salt loss at humans, with selectivity preventing not only the inhibition doses ≥1 mg/kg. However, not all the effects on ZG cells of of cortisol production but also the compensatory increase in RO6836191 were reversed by ACE inhibition and electrolyte 11-DOC. Preclinical data underline the probable importance supplementation. We cannot exclude the possibility that the of aldosterone in regulating ZG cell turnover, with the pos- dose of the ACE inhibitor, or the electrolyte replacement, sibility that accelerated turnover may contribute to chronic was insufficient to fully prevent sodium depletion. However, efficacy of AS inhibitors in comparison with the receptor the residual increase in CYP11B2 expression and apoptosis blockers, which elevate aldosterone secretion. in the ZG layer are consistent with adrenal changes reported −/− Perspectives in the AS mouse, which suggested that ZG cells migrate to the medullary junction and undergo apoptosis when unable The homology between the 2 enzymes catalyzing the final to synthesize aldosterone. Increased renin production is steps in aldosterone and cortisol synthesis has been a block −/− observed in the AS mouse, in response to sodium deple- in the development of drugs, which inhibit aldosterone tion, as in our monkeys treated with RO6836191 but not secretion rather than block its response. We show that in receiving saline supplementation. But the increased PRA, vitro selectivity can translate into clinical practice, provid- reported in Figure SIII, is unlikely to be the sole or prin- ing that, as in our cynomolgus studies, the single-dose find- cipal cause of changes in the adrenal, especially when the ings are predictive of those on multiple dosing. An important renin elevation is partly because of the inhibition by lisino- discovery is that the plasma concentration of deoxycortico- pril of angiotensin production. Only minor increases in PRA sterone, the biologically active substrate for AS, does not were observed after a single dose in the clinical study. We increase at selective doses of inhibitor. This predicts greater would anticipate an increase in PRA in future, multiple dos- natriuretic and antihypertensive efficacy than achieved by ing, studies, given the expected natriuresis and the trend we less selective inhibitors. observed both in the preclinical data and the low-salt diet group in the clinical study. Although there was no significant Acknowledgments We thank Nathalie Lambert for operational support during the study evidence of natriuresis after a single dose of RO6836191, we conduct, personnel from PRA Groningen, the Netherlands, for the did observe a similar increase in the Na/K ratio as reported clinical study conduct, and Kevin Smart for critical review of the ex- after a single dose of MR antagonist. ploratory PKPD analysis. A degree of apoptosis may be beneficial in patients with hyperaldosteronism, particularly if this occurred in the so- Sources of Funding called aldosterone-producing cell clusters that are often the All studies were funded by F. Hoffmann-La Roche Ltd. only residual sites of aldosterone production or in actual aldo- sterone-producing adenomas. By contrast, drugs that block Disclosures the MR (ie, spironolactone, eplerenone, and newer aldoste- M.J.B. received external consultant honoraria from F. Hoffmann-La Roche Ltd. 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J Clin Hypertens (Greenwich). 2013;15:186–192. doi: 10.1111/ 30. Berger S, Bleich M, Schmid W, Cole TJ, Peters J, Watanabe H, Kriz jch.12051. W, Warth R, Greger R, Schütz G. Mineralocorticoid receptor knockout 17. Schumacher CD, Steele RE, Brunner HR. Aldosterone synthase inhi- mice: pathophysiology of Na+ metabolism. Proc Natl Acad Sci U S A. bition for the treatment of hypertension and the derived mechanistic 1998;95:9424–9429. Novelty and Significance What Is New? Summary We report that it is possible to completely inhibit the enzyme-producing An inhibitor of aldosterone synthase was tested over multiple doses aldosterone without causing any inhibition of the 93% homologous en- in cynomolgus monkeys and after a single dose in humans. Its 100- zyme-producing cortisol. fold selectivity in vitro (for aldosterone versus cortisol inhibition) This inhibition reduced plasma aldosterone to below detectable levels, translated into 100-fold difference in doses required to inhibit aldo- without accumulation of deoxycorticosterone, the substrate of aldoste- sterone and cortisol in vivo. rone synthase. What Is Relevant? No drug is currently available for the treatment of hyperaldosteronism, which works through inhibition of aldosterone production rather than blocking aldosterone response. This study now permits the consequenc- es of selective aldosterone inhibition to be investigated in patients.

Journal

Hypertension (Dallas, Tex. : 1979)Pubmed Central

Published: Dec 7, 2016

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