Aldosterone synthase (CYP11B2) inhibitors (ASIs) represent an attractive therapeutic approach for mitigating the untoward effects of aldosterone. We characterized the pharmacokinetic/pharmacodynamic relationships of a prototypical ASI, (+)-(5R)-4-(5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-5-yl]benzonitrile hydrochloride (CGS020286A, FAD286, FAD) and compared these profiles to those of the 11β-hydroxylase inhibitor metyrapone (MET) in two rodent models of secondary hyperaldosteronism and corticosteronism. In chronically cannulated Sprague-Dawley rats, angiotensin II (ANG II) (300 ng/kg bolus + 100 ng/kg/min infusion) or adrenocorticotropin (100 ng/kg + 30 ng/kg/min) acutely elevated plasma aldosterone concentration (PAC) from ∼0.26 nM to a sustained level of ∼2.5 nM for 9 h. Adrenocorticotropin but not ANG II elicited a sustained increase in plasma corticosterone concentration (PCC) from ∼300 to ∼1340 nM. After 1 h of Ang II or adrenocorticotropin infusion, FAD (0.01–100 mg/kg p.o.) or MET (0.1–300 mg/kg p.o.) dose- and drug plasma concentration-dependently reduced the elevated PACs over the ensuing 8 h. FAD was ∼12 times more dose-potent than MET in reducing PAC but of similar or slightly greater potency on a plasma drug concentration basis. Both agents also decreased PCC in the adrenocorticotropin model at relatively higher doses and with similar dose potencies, whereas FAD was 6-fold weaker based on drug exposures. FAD was ∼50-fold selective for reducing PAC versus PCC, whereas MET was only ∼3-fold selective. We conclude that FAD is a potent, orally active, and relatively selective ASI in two rat models of hyperaldosteronism. MET is an order of magnitude less selective than FAD but is, nevertheless, more potent as an ASI than as an 11β-hydroxylase inhibitor.
Aldosterone is not only a major regulator of extracellular fluid volume and electrolytes but is also linked to the pathogenesis of hypertension and congestive heart failure (Weber, 2001; Rocha and Funder, 2002). Aldosterone (mineralocorticoid) receptor antagonists (MRAs), such as spironolactone (Aldactone), and eplerenone (Inspra), exhibit antihypertensive, cardiac antihypertrophic, and anti-inflammatory properties, organ protection, and reduction of mortality in patients with congestive heart failure receiving standard therapies (Pitt et al., 1999; Fiebeler et al., 2005; Cohn and Colucci, 2006; Funder, 2006; Fiebeler et al., 2007).
An alternative therapeutic approach to prevent the deleterious effects of aldosterone is to suppress its production with inhibitors (ASI) of aldosterone synthase (CYP11B2), which catalyzes the final steps of aldosterone biosynthesis (Funder, 2006; Ménard and Pascoe, 2006). However, the preferred mechanism to block the effects of aldosterone is unknown. ASIs reduce aldosterone and thereby mitigate both its MR- and non-MR-mediated actions (Connell and Davies, 2005; Fiebeler et al., 2005; Ménard et al., 2006; Marney and Brown, 2007). MRAs can elevate circulating and tissue levels of aldosterone, which may amplify the untoward effects of this steroid mediated by MR-independent pathways (Connell and Davies, 2005; Marney and Brown, 2007). In contrast, treatment with an ASI could allow activation of the unprotected MR by glucocorticoids (Funder, 2009). Resolution of this debate has been hindered by the limited availability of potent, effective, selective, and orally active ASIs.
(+)-(5R)-4-(5,6,7,8-Tetrahydroimidazo [1,5-a]pyridin-5-yl] benzonitrile hydrochloride (CGS020286A, FAD286, FAD) is a prototypical ASI that has undergone limited in vivo preclinical testing (Fiebeler et al., 2005; Ménard et al., 2006; Minnaard-Huiban et al., 2008; Mulder et al., 2008; Lea et al., 2009) and has been recommended for clinical evaluation (Funder, 2006; Ménard and Pascoe, 2006; Siragy and Xue, 2008). FAD is the R-(+)-enantiomer of the racemic CYP19 (aromatase) inhibitor CGS016949A (fadrozole), which is marketed in Japan as a treatment for estrogen-dependent breast cancer. Although its antipode is an aromatase inhibitor, FAD potently inhibits human AS in vitro (Fiebeler et al., 2005; Müller-Vieira et al., 2005; Mulder et al., 2008; LaSala et al., 2009). Its potency against AS from nonhuman species is unknown. Likewise, clinical evaluation of FAD has not been reported. However, at or somewhat above marketed doses, the racemate fadrozole lowers basal or stimulated plasma aldosterone in humans (Dowsett et al., 1990; Stein et al., 1990; Trunet et al., 1992). FAD also lowers plasma aldosterone concentration or urinary aldosterone excretion in transgenic or genetic rat models of hypertension or heart failure (Fiebeler et al., 2005; Ménard et al., 2006; Minnaard-Huiban et al., 2008).
The optimal dose of FAD for inhibiting aldosterone production in rats is unknown. In a transgenic rat model of angiotensin II (ANG II)-dependent hypertension, 3.4 mg/kg/day of FAD free base exhibited cardiac and renal protection and reduced the postweaning rise in blood pressure compared with that in untreated controls (Fiebeler et al., 2005). Adrenalectomy reduced serum and cardiac aldosterone levels considerably more than did FAD and further ameliorated the cardiac and renal injury, suggesting that the dose of FAD used (Lea et al., 2009) was submaximal. This notion is supported by a study in spontaneously hypertensive rats in which 86 mg/kg/day (100 mg/kg HCl salt) FAD was required to maximally lower 24-h urinary aldosterone excretion (Ménard et al., 2006). None of these studies could determine the time course of aldosterone reduction or the pharmacokinetics of FAD because only a single terminal blood sample was withdrawn or the time-integrated urinary aldosterone excretion was measured. Likewise, the FAD exposure-response relationships and the extent of CYP11B1 (11β-hydroxylase) inhibition and, thus, the lowering of corticosterone at optimal therapeutic doses of FAD are unclear (Fiebeler et al., 2005; Minnaard-Huiban et al., 2008; Lea et al., 2009).
The purpose of this study was severalfold: 1) to develop facile and robust ASI profiling models in rats, 2) to use these models to characterize the dose-dependent magnitude and duration of action of FAD in vivo, 3) to assess the PK/PD relationships and traditional PK parameters of FAD in vivo, 4) to evaluate the CYP11B2/CYP11B1 selectivity of FAD in vivo, and 5) to compare these in vivo results with in vitro potency/selectivity profiles in a rat adrenal cortical CYP11B2/CYP11B1 preparation. Furthermore, these profiles of the CYP11B2 inhibitor FAD were compared with those of metyrapone (MET), which has been used for many years as a CYP11B1 inhibitor for clinical diagnosis and treatment of hypothalamic-pituitary-adrenal axis disorders.
Materials and Methods
In Vivo Experiments
All animal procedures were conducted in accordance with an approved Institutional Animal Care and Use Committee protocol and the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996).
Experiments were conducted on 121 male, adult, Sprague-Dawley rats (mean body weights 517 g; range 404–715 g) purchased from Taconic Farms (Germantown, NY). After arrival at the Novartis vivarium, rats were acclimated for at least several weeks before being used in the experiments. They were housed on a 12-h light/dark cycle (light 6:00 AM–6:00 PM) at temperature and relative humidity set points of 72°F and 55%, respectively. Rats were provided normal chow (Harlan Teklad 8604; Harlan, Indianapolis, IN) and water ad libitum except for a partial fast before and during each experiment, in which case all but two chow pellets were removed the preceding evening (∼5:00 PM). On the morning of the experiment, any remaining food was removed. Food was again provided ad libitum at the end of the experiment.
Rats were surgically instrumented to allow direct measurement of arterial blood pressure, repeated blood sampling, and intravenous or intra-arterial administration of substances. Under isoflurane anesthesia, a femoral artery and vein were isolated and catheterized. Catheters consisted of 55 cm of polyvinylchloride (Tygon) microbore tubing (0.020 inch i.d. × 0.060 inch o.d.) bonded with cyclohexanone to 4.5 cm of polyvinylchloride (0.011 inch i.d. × 0.024 inch o.d.; Biocorp Australia Pty. Ltd., Huntingdale, VIC, Australia) or Micro-Renathane (type MRE-025 polyurethane, 0.012 inch i.d. × 0.025 inch o.d.; Braintree Scientific Inc., Braintree, MA) tubing. The catheters were tunneled subcutaneously and exteriorized in the mid-dorsal thoracic/abdominal region. Catheters exited through a subcutaneously anchored tether/swivel system that allowed the animal to move unrestrained in a specialized plastic wire-bottom cage. Ketoprofen (1 mg/kg i.m.) was administered for preemptive analgesia before the surgical procedure was begun and again on the first postoperative day. In addition, penicillin G (50,000 U/kg i.m.) was administered preoperatively to prevent infection. The rats were allowed to recover for a minimum of 1 week before being studied while conscious and unrestrained. Catheters were flushed with sterile 0.9% saline and locked with 200 U/ml heparin in sterile 0.9% saline after the surgery was completed and at least twice per week thereafter.
Rats were allowed to remain undisturbed in their home cages before, during, and after each experiment. On the morning of the study, the catheters were flushed and prepared for use. In some experiments, arterial pressure was monitored via the arterial catheter, a calibrated blood pressure transducer (Statham P23; Becton Dickinson, Franklin Lakes, NJ), and a digital data acquisition system (Modular Instruments Inc., West Chester, PA).
Blood samples were withdrawn on heparin (15 U/ml final concentration) from the arterial cannula. Samples were centrifuged at 20,000g for ∼20 min to generate plasma, which was aliquoted and frozen (−70°C) for later analysis of plasma aldosterone (PAC), corticosterone (PCC), or drug concentrations. ANG II or adrenocorticotropin was infused into the venous catheter. FAD, MET, or vehicle was administered as a bolus intra-arterially or by oral gavage.
Infusion doses of ANG II and adrenocorticotropin were determined from pilot dose-response experiments (3–100 ng/kg/min ANG II; 0.3–300 ng/kg/min adrenocorticotropin). The final doses (see below) were selected to achieve sustained increases in PAC to the target level of 1 to 5 nM (360–1800 pg/ml). These PACs represent the middle to upper range reported in patients with primary aldosteronism (Phillips et al., 2000; Perschel et al., 2004; Unger et al., 2004). Rats with baseline PAC values outside this range were excluded from analyses (see Results). Most doses of ANG II also elevated mean arterial pressure by ∼40 mm Hg, whereas adrenocorticotropin had no evident effect on blood pressure.
The final dosing regimens selected for the experiments were bolus injections (300 ng/kg ANG II; 100 ng/kg adrenocorticotropin) followed by a 9-h intravenous infusion (100 or 30 ng/kg/min for ANG II or adrenocorticotropin, respectively). In some studies, pre-ANG II or adrenocorticotropin blood samples were withdrawn. After 1 h of ANG II or adrenocorticotropin infusion, a blood sample was collected for determining the post-ANG II or adrenocorticotropin “baseline” (i.e., secretagogue-elevated) PAC and PCC. FAD (0.01–100 mg/kg p.o.; 1 mg/kg i.a.), MET (0.1–300 mg/kg; 10 mg/kg i.a.), vehicle, or no treatment was administered, and the ANG II or adrenocorticotropin infusion was continued for an additional 8 h. Blood samples were withdrawn at 5 (intra-arterial dosing only), 15, and 30 min, and 1, 2, 3, 4, 5, 6, 7, 8, and 24 h after dosing for assessment of PAC, PCC, and plasma drug concentrations.
Some of the ANG II-treated rats were used for more than one experiment after at least 1 week of recovery from the previous experiment was allowed. Model validation experiments confirmed that results with this experimental paradigm were highly reproducible. In contrast, in rats administered adrenocorticotropin, corticosteroid responses to a second adrenocorticotropin infusion were not reproducible even after several weeks of recovery from the initial experiment. Therefore, rats receiving adrenocorticotropin were used for only one experiment and then euthanized.
In Vitro Experiments
Rat Adrenal Tissue CYP11B1 and CYP11B2 Assays.
Male Sprague-Dawley rats were anesthetized with isoflurane, and both adrenal glands were surgically removed. The glands were decapsulated, and the capsular tissue was frozen in liquid nitrogen and stored at −80°C. Thawed adrenal capsules were pooled and homogenized in 1 ml of ice-cold homogenization buffer [8.5 mM MgCl2, 3.13 mM KCl, 7.59 mM NaCl, 2.7 mM CaCl2, 50 mM Tris/HCl, pH 7.4, and 1 Complete EDTA-Free Protease Inhibitor Cocktail Tablet (Roche Applied Science, Indianapolis, IN) per 50 ml of buffer] per 37.5 mg of tissue. The homogenized material was then centrifuged at 450g for 5 min, and the supernatant was collected. The supernatant was brought to a final glycerol concentration of 5%, flash-frozen in liquid nitrogen, and stored at −80°C.
Material from frozen adrenal homogenate preparations was thawed on ice on the day of experiment and then diluted in ice-cold assay buffer (8.5 mM MgCl2, 3.13 mM KCl, 7.59 mM NaCl, 2.7 mM CaCl2, and 50 mM Tris/HCl, pH 7.4) to a protein concentration of approximately 3 mg/ml. The CYP11B1 and CYP11B2 assays were performed in white 96-well clear, flat-bottom, nontreated, assay plates. For the CYP11B1 assay, 1 to 2 μg of protein in 20 μl was incubated with 10 μl of assay buffer or a compound at the desired concentration and 20 μl of substrate mix (2.5× NADPH Regeneration Solution A, 2.5× NADPH Regeneration Solution B, and 1.25 μM 11-deoxycorticosterone) for 4 h at 25°C in a shaking incubator. Final substrate concentration was 0.5 μM. The reaction was stopped by freezing the plates over dry ice. Production of corticosterone was measured by a scintillation proximity assay (SPA).
The CYP11B2 assay was carried out similarly, except that 15 to 20 μg of protein and 1 μM 11-deoxycorticosterone (final concentration) were used. Production of aldosterone was also determined by a SPA.
Measurement of aldosterone and corticosterone was performed using a 96-well plate format. Test samples were incubated with 0.02 μCi of either d-[1,2,6,7-3H(N)]corticosterone (CYP11B1) or d-[1,2,6,7-3H(N)]aldosterone (CYP11B2) and 0.2 μg of either anti-corticosterone or anti-aldosterone antibody in PBS containing 0.1% Triton X-100, 0.1% bovine serum albumin, and 12% glycerol in a total volume of 200 μl at room temperature for 1 h. Anti-sheep (corticosterone) or anti-mouse (aldosterone) PVT SPA beads (50 μl) were then added to each well and incubated overnight at room temperature before counting in a MicroBeta Plate Counter. The amount of corticosterone or aldosterone in each sample was calculated by comparing with standard curves generated using known quantities of the respective steroid. Full concentration-response curves of an inhibitor were generated from at least three independent experiments.
Plasma Protein Binding Assay.
In vitro plasma protein binding of FAD, MET, racemic metyrapol, and the two metyrapol enantiomers was assessed in triplicate using an equilibrium dialysis method (the Rapid Equilibrium Dialysis (RED) Device System; Pierce Biotechnology, Rockford, IL). Compound was added to rat or human plasma at a final concentration of 1 or 10 μM (in 1% dimethyl sulfoxide). The plasma was incubated at 37°C for 4 h in the RED Device System. Parent compound concentrations in the plasma and PBS compartments were measured at time 0 and 4 h by LC-MS/MS. Percentages of parent compound bound in plasma and percent recovery were calculated as
Ex Vivo Analyses
PACs and PCCs were assayed with commercially available radioimmunoassay kits (Coat-A-Count Aldosterone and Coat-A-Count Rat Corticosterone; Diagnostic Products, Los Angeles, CA) with unextracted plasma according to the manufacturer's instructions. The intra- and interassay coefficients of variation were ≤3% for aldosterone and ≤12% for corticosterone.
Plasma concentrations of FAD and MET were measured by an LC-MS/MS method [lower limit of quantification (LLOQ) = 0.5 ng/ml = 2.24 nM and 1 ng/ml = 4.4 nM, for FAD and MET, respectively]. Likewise, the concentrations of the two primary reduced metabolites of MET [(+)- and (−)-MOH] were measured by LC-MS/MS with a Chiral PAK 4.6 × 150-mm chiral column (LLOQ = 1 ng/ml = 4.4 nM). Quantification of the analytes was based on a calibration curve with at least five points and a dynamic range set from the respective LLOQs to 10 μg/ml. The bias of all calibration standards and quality control samples was within 30%. In each case, samples with drug concentrations below the LLOQ were treated as zero for computations of means.
Drugs and Reagents
Stock solutions of ANG II (Sigma-Aldrich, St. Louis, MO) and adrenocorticotropin(1–24) (herein referred to as “adrenocorticotropin”; American Peptide Company, Inc., Sunnyvale, CA) were prepared in sterile saline or water based on the respective purities and peptide contents stated by the manufacturers. Aliquots were frozen at −70°C. On the day of the experiment, an infusion solution was prepared in sterile saline at a calculated concentration (5 and 1.5 ng/μl for ANG II and adrenocorticotropin, respectively) to allow an infusion volume of 20 μl/kg/min.
FAD (hydrochloride or hydrogentartrate salt) was synthesized within Novartis [see Fiebeler et al. (2005) for FAD structure]. MET (2-methyl-1,2-di-3-pyridyl-1-propanone) was purchased from Sigma-Aldrich and its primary racemic metabolite (MOH) was generated within Novartis by sodium borohydride reduction of MET [see Nagamine et al. (1997) for MET and MOH structures]. The (+)- and (−)-enantiomers of MOH were resolved from the racemate by chiral chromatography (CHIRALPAK IA; Daicel Chemical Industries, Ltd., Fort Lee, NJ; mobile phase: 15% ethanol-heptane). Formulated FAD and MET were prepared freshly from the powders before each experiment. Vehicles (2 ml/kg volume) were water for oral administration or sterile 0.9% saline for intra-arterial injection. Doses of both salt forms of FAD are reported as milligram per kilogram of the free base of the drug.
NADPH Regeneration Solution A and Solution B were purchased from BD Biosciences Clontech (Mountain View, CA). Anti-sheep and anti-mouse Amersham PVT SPA beads were acquired from GE Healthcare Life Sciences (Piscataway, NJ). [1,2,6,7-3H(N)]Corticosterone and d-[1,2,6,7-3H(N)]aldosterone were products of PerkinElmer Life and Analytical Sciences (Waltham, MA).
Data and Statistical Analyses.
For each individual rat, the PAC and PCC time course data were expressed as a percentage of the respective baseline value. For dose- and concentration-response analyses, the “pharmacodynamic response” was defined as the peak lowering of PAC or PCC (“peak”) or the time-weighted average of this baseline-normalized PAC and PCC over the 8-h experiment (TWA = area under the curve ÷ 8 h). Likewise, the TWAs of the plasma FAD and MET concentrations were estimated from the 8-h area under the concentration-time curve (AUC).
ED50, EC50, and IC50 values (in vivo dose or plasma concentration at 50% of “baseline” response or half-maximally effective in vitro concentration) were estimated by fitting the individual dose- or concentration-response data to a four-parameter sigmoidal dose-response model (model 205) in XLfit (version 4; ID Business Solutions, Inc., Guildford, UK): where A and B represent the responses at supramaximally effective and subthreshold concentrations/doses, respectively, C is the concentration/dose that produces a half-maximal response, D is a slope constant, and X is the test concentration or dose.
PK parameters were calculated with the computer program WinNonlin (Enterprise version 5.2; Pharsight Corporation, Palo Alto, CA) using a noncompartmental model for the parent drug. PK data were calculated based on individual concentrations. The AUC was calculated using the linear trapezoidal rule. Likewise, the time-weighted average drug concentration, PAC, and PCC were computed as the AUC divided by the integration time (8 h).
Results are presented as means ± S.E.M. An unpaired t test (Microsoft Excel) was used to discern statistically significant differences (P < 0.05) in values between two experimental groups. One-way analysis of variance was applied to determine whether the control PAC and PCC responses were sustained at 100% over the 8-h experiment. Multiple comparisons versus the time 0 values were conducted with Dunnett's test.
Inhibition of Aldosterone and Corticosterone Production by FAD, MET, and MOH In Vitro.
Aldosterone and corticosterone products were time-dependently generated from the 11-deoxycorticosterone substrate by rat adrenal capsular preparations with respective rates of 43 ± 4 pmol/mg of protein/h (mean ± S.E.M., n = 4) and 4.9 ± 0.5 nmol/mg of protein/h up to 4 h (results not shown). These results show that corticosterone is produced at a rate approximately 2 orders of magnitude greater than that of aldosterone.
All four compounds dose-dependently inhibited the biosynthesis of aldosterone (CYP11B2) and corticosterone (CYP11B1) in vitro (Fig. 1). FAD, MET, and (+)-MOH exhibited similar potency against CYP11B2, whereas (−)-MOH was ∼6 to 7 times less potent (Table 1). MET was the most potent compound against CYP11B1 but was, nevertheless, 2 times more selective for CYP11B2 than CYP11B1. (−)-MOH was equipotent against both enzymes, whereas FAD and (+)-MOH were 5- and 4-fold more selective, respectively, for CYP11B2 (Table 1).
Inhibition of Aldosterone and Corticosterone Production by FAD and MET In Vivo.
One-hundred forty-eight experiments were conducted on 121 rats. Of these, 6 were excluded from the analyses because the baseline PAC deviated from the target range of 1 to 5 nM and 1 was excluded because of technical problems.
Basal and Baseline PACs and PCCs.
Before administration of ANG II or adrenocorticotropin, the basal PAC and PCC values were ∼0.28 and ∼300 nM, respectively, reflecting the expected ∼3 orders of magnitude higher levels of glucocorticoid than mineralocorticoid. ANG II and adrenocorticotropin bolus/infusions rapidly increased PACs and PCCs, which reached a steady state within 1 h (“baseline” PAC or PCC; i.e., time 0). These baseline values were similar for all treatment groups (PAC, ∼2.5 nM; PCC, ∼1300 nM). Thus, the secretagogues elevated PAC and PCC by ∼10- and ∼4- to 5-fold, respectively, over basal levels (results not shown).
Time Courses of PAC and PCC in the Control Experiments with ANG II and Adrenocorticotropin.
Each rat's PAC and PCC time course data were normalized to its own baseline PAC or PCC by expressing the results as a percentage of the baseline value. Responses in untreated and vehicle-treated control rats were similar and therefore combined for each secretagogue. Infusion of ANG II in control rats resulted in a sustained increase in PAC (∼100%) for at least 9 h (Fig. 2). In contrast, PCC remained elevated for only the first 2 h and then significantly declined (P = 0.001) at 3 h to achieve a steady state of 50% by 5 h. Because of this transient increase in PCC, the ANG II model was used to evaluate the effects of the pharmacologic agents on only PAC. Adrenocorticotropin maintained the stimulated level of both PAC and PCC at ∼100% and was, therefore, used to evaluate the PAC/PCC selectivity of the inhibitors.
PAC and PK Time-Course Responses to FAD and MET in the ANG II Model.
Figure 3 displays the PAC time courses as a percentage of baseline PAC (mean ± S.E.M.) for each of the oral doses of FAD (Fig. 3, top left) and MET (Fig. 3, top right) as well as the plasma compound concentrations (Fig. 3, bottom) in the ANG II-infused rats.
FAD dose- and time-dependently lowered PAC. At the two lowest doses of FAD, PAC was transiently elevated above baseline followed by a ∼20% decrease and then a recovery. All of the remaining doses elicited a sustained, dose-dependent decrease in PAC for the duration of the experiment. The maximally effective dose seemed to be 3 to 10 mg/kg. At doses of 1 mg/kg and higher, PAC declined with a characteristic temporal pattern over the first 2 to 3 h after dosing.
MET also dose- and time-dependently lowered PAC in this model. At the two lowest doses, the reduction of PAC was transient and followed by a rebound above the baseline. Likewise, at doses of 1 to 10 mg/kg, the PAC began to recover after ∼5 h. In contrast, the two highest doses of MET decreased PAC for the duration of the 8-h experiment. The pattern of PAC decline during the first 2 to 3 h was almost identical to that of FAD. This common profile suggests that it is attributable to the intrinsic metabolism/elimination characteristics of aldosterone and not to the tissue distribution and enzyme kinetic properties of the two compounds.
Oral treatment with FAD dose- and time-dependently elevated plasma FAD concentrations (Fig. 3). Plasma FAD concentrations were nearly constant throughout the 8-h experiment, whereas MET levels declined by ∼1 order of magnitude. Twenty-four hours after dosing, plasma concentrations decreased even more for MET than for FAD.
PAC, PCC, and PK Time-Course Responses to FAD and MET in the Adrenocorticotropin Model.
Figure 4 displays the time courses of PAC and PCC as a percentage of the respective baseline values (mean ± S.E.M.) for each of the oral doses of FAD (Fig. 4, two top left panels) and MET (Fig. 4, two top right panels) and the plasma compound concentrations (Fig. 4, bottom panels) in the adrenocorticotropin-infused rats.
As in the ANG II model, both FAD and MET dose- and time-dependently lowered PAC. However, one striking difference was the shorter duration of action and overshoot above baseline in PAC at the low-to-moderate doses of FAD and MET. This effect was not evident with PCC. The reason for this difference is not clear. However, pilot experiments with renin-angiotensin system blockade suggest that this pattern was not due to ASI-induced counterregulatory activation of the renin-angiotensin system, which was intact in the adrenocorticotropin model but “clamped” in the ANG II infusion model. Accordingly, it appears as though higher doses of FAD or MET were required to elicit a PAC response comparable to that in the ANG II model or to yield a sustained lowering of PAC. Again, the characteristic pattern of the decline in PAC during the first 2 to 3 h after FAD or MET was similar to that in the ANG II model. Oral treatment with FAD and MET dose- and time-dependently elevated plasma compound concentrations similar to those in the ANG II model.
Dose-Response Relationships of FAD and MET in the ANG II and Adrenocorticotropin Models.
The PD responses for FAD and MET were derived as the peak (Fig. 5, top panels) or TWA (Fig. 5, bottom panels) of the baseline-normalized PAC or PCC over the 8-h experiment. In the control experiments (gray symbols in Fig. 5), there was no net effect on PAC or PCC in the two models.
FAD and MET dose-dependently lowered peak and TWA PAC and PCC (Fig. 5). Both compounds tended to be less potent in lowering PAC in the adrenocorticotropin model (i.e., rightward shift in dose-response relationships) than in the ANG II model. This finding is consistent with the observation that the magnitude and duration of PAC lowering was attenuated in the adrenocorticotropin model compared with that in the ANG II model (see 3.4 and 4). Both FAD and MET were more potent in lowering PAC than PCC in the adrenocorticotropin model (Fig. 5). Thus, FAD is a relatively selective ASI, whereas MET is clearly not a selective 11β-hydroxylase inhibitor in this model. Note that these in vivo rat PAC/PCC results reflect the relative CYP11B2/CYP11B1 profiles observed in the in vitro rat adrenocortical assays (Fig. 1). Likewise, both compounds tended to be more efficacious in lowering PAC than PCC (Fig. 5).
Dose-Exposure Relationships of FAD and MET in the ANG II and Adrenocorticotropin Models.
Oral administration of FAD and MET dose-dependently elevated the plasma concentrations of the respective parent compounds (Fig. 6). Exposures were similar between the two models at the highest doses but tended to be slightly higher in the adrenocorticotropin model at the lowest doses tested. Peak concentrations of both compounds increased dose-proportionally. Eight-hour TWA concentrations of FAD tended to increase dose-overproportionally, whereas those for MET increased dose-proportionally.
After intra-arterial administration of FAD (1 mg/kg), the 8-h TWA plasma concentrations of FAD were similar to those after the same oral dose (Fig. 6, bottom), suggesting a relatively high oral bioavailability (BAV) for this compound. In contrast, there was a greater separation between the intra-arterial (10 mg/kg) and oral exposures of MET, indicating a lower oral BAV (see PK results below).
Exposure-Response Relationships of FAD and MET in the ANG II and Adrenocorticotropin Models.
The exposure-response relationships (Fig. 7) were derived by combining the dose-response (Fig. 5) and dose-exposure (Fig. 6) relationships. FAD and MET concentration-dependently lowered PAC and PCC. As described for the dose-response relationships, both compounds tended to be less potent in lowering PAC in the adrenocorticotropin model than in the ANG II model. This pattern is particularly striking for the 8-h TWA for MET because of the shorter duration of action in the adrenocorticotropin model. As observed for the dose-response relationships (Fig. 5), both FAD and MET were also more potent on a plasma exposure basis in lowering PAC than PCC in the adrenocorticotropin model (Fig. 7).
ED50, EC50 (Total and Unbound), and PAC/PCC Selectivity Values In Vivo.
ED50 and EC50 values for lowering PAC and PCC in the two models are displayed in Table 2. FAD was ∼12 times (i.e., ED50 values = 0.5 versus 5.5 mg/kg in the ANG II model and 0.8 versus 10.6 mg/kg in the adrenocorticotropin model) more potent than MET on a dose basis in reducing PAC and of similar potency on a plasma drug concentration basis. In the adrenocorticotropin model, both agents also decreased PCC at relatively higher doses and with similar dose potencies (ED50 values = 45 versus 40 mg/kg), whereas FAD was 6-fold weaker than MET based on drug exposures (EC50 values = 23,000 versus 3600 nM). Accordingly, based on both ED50 and EC50 values, FAD was ∼50-fold selective for reducing PAC than PCC but MET was only ∼3-fold selective.
In vitro protein binding in rat and human plasma was low and similar for FAD and MET (rat: FAD, 57%; MET, 33%; human: FAD, 55%; MET, 27%). Likewise, plasma protein binding for the MOH enantiomers was nearly identical to that for MET. Thus, the “unbound” EC50 values for FAD and MET were ∼43 and 67%, respectively, of the “total” EC50 values (Table 2) and FAD was only slightly (1.6-fold = 67%/43%) more potent than MET based on unbound concentrations relative to the total concentration EC50 values.
(+)- and (−)-MOH Concentrations in the ANG II and Adrenocorticotropin Models.
At each MET dose, individual-time, peak, and 8-h TWA concentrations of (−)-MOH were ∼3 to 10 times higher than the corresponding (+)-MOH levels (Supplemental Fig. S1, top right versus top left), indicating an enantioselective generation and/or metabolism of the two MOH metabolites. Concentrations of (+)- or (−)-MOH equaled or exceeded those of MET at the three to five highest doses of MET (Supplemental Figs. S1 and S2).
Based on the relative in vitro potencies of MET and its two metabolites (Table 1) and the relative in vivo concentrations of each (Fig. 6; Supplemental Fig. S1), we estimated the contributions of (+)- and (−)-MOH to the overall pharmacodynamic response of orally administered MET (30–300 mg/kg). These calculations suggest that the combined (+)- and (−)-MOH effect on PAC and PCC was 4 to 6 and 4 to 7 times higher, respectively, than that of MET alone, indicating the important pharmacologic contribution of these active metabolites.
Oral BAV of FAD increased dose-dependently and ranged from 16 to 100% depending on the model and dose (Supplemental Fig. S3, left). Oral BAV for MET was low (∼10%), independent of dose, and similar between the two models (Supplemental Fig. S3, right).
PK parameters (Table 3) were similar between the two models for both compounds. The only exception was clearance, which was significantly higher (∼2-fold) for FAD in the adrenocorticotropin model than in the ANG II model. In addition, the elimination half-life of MET was significantly longer than that of FAD in the adrenocorticotropin model and the steady-state volume of distribution was significantly higher for MET than for FAD in both models.
Pharmacologic suppression of aldosterone synthesis with ASIs is an attractive therapeutic alternative to MRAs. However, appropriate in vivo models for pharmacologically characterizing such agents are uncommon (Fiebeler et al., 2005; Ménard et al., 2006). MRAs have traditionally been evaluated by assessing urinary electrolytes or organ injury in rodent models with exogenously administered aldosterone (Rudolph et al., 2004; Brandish et al., 2008; McManus et al., 2008). In contrast, profiling ASIs requires stimulation of endogenous aldosterone production either by pharmacologic or dietary activation of biosynthetic pathways (Rowland and Morian, 1999; Ye et al., 2003; Ménard et al., 2006), exogenous administration of natural or synthetic secretagogues for corticosteroids (Komor and Müller, 1979; Mazzocchi et al., 1989; Roesch et al., 2000; Ye et al., 2003; Lea et al., 2009), or transgenic manipulations (Sander et al., 1992; Garnier et al., 2004; Fiebeler et al., 2005; Makhanova et al., 2008).
Our first goal was to develop a model of hyperaldosteronism in conscious, chronically cannulated rats to simultaneously assess the PD and PK profiles and corticosteroid selectivity of ASIs. We sought to increase PAC to the middle to upper range (1–5 nM = 360-1800 pg/ml) reported in patients with primary hyperaldosteronism (Phillips et al., 2000; Perschel et al., 2004; Unger et al., 2004). We initially explored a low-sodium diet, dietary potassium supplementation, diuretic/natriuretic treatment, or combinations thereof to amplify endogenous aldosterone. Although each of these strategies was effective (Rowland and Morian, 1999; Ye et al., 2003; Ménard et al., 2006), the resultant PAC increases were gradual, highly variable, and not temporally stable over a single- or multiple-day study. Moreover, these interventions exaggerated diurnal variations in corticosteroid levels and stress responses to oral gavage and generated no or small elevations in corticosterone (Rowland and Morian, 1999; Ye et al., 2003). Together, these factors greatly limited the utility of these approaches. In contrast, continuous intravenous infusion of either of the primary aldosterone secretagogues (ANG II or adrenocorticotropin) overcame these limitations; PAC was consistently elevated to a steady level for at least 8 h. Adrenocorticotropin infusion also stably increased corticosterone, thereby allowing simultaneous evaluation of the CYP11B2/CYP11B1 selectivity of the compounds.
In both models, FAD dose- and concentration-dependently attenuated the secretagogue-elevated PAC. This inhibitory response was sustained for at least 8 h at doses ≥1 mg/kg. The maximally effective dose (∼3–30 mg/kg) within this time window depended on the “response” (peak or 8-h TWA) and the model (ANG II or adrenocorticotropin). Doses required to block AS for 24 h might be even higher. Indeed, in spontaneously hypertensive rats, 86 mg/kg/day (100 mg/kg HCl salt) of FAD was needed to maximally lower 24-h urinary aldosterone excretion (Ménard et al., 2006). Likewise, in a transgenic rat model of ANG II-dependent hyperaldosteronism, 3.4 mg/kg/day (4 mg/kg HCl salt) of FAD only partly lowered PAC relative to bilateral adrenalectomy (Fiebeler et al., 2005). Thus, our comprehensive characterization of FAD is critical for defining the effective dose range of the drug and suggests that its therapeutic benefits were underestimated by studies using suboptimal doses (0.24–3.4 mg/kg/day) (Minnaard-Huiban et al., 2008; Mulder et al., 2008; Siragy and Xue, 2008; Lea et al., 2009).
The effective in vitro FAD concentrations appear to reflect the effective plasma exposure-response relationships; the total (120 or 480 nM) and unbound (54 or 210 nM) EC50 values for reducing PAC in our two models are comparable with the IC50 (670 nM) for inhibiting CYP11B2 in the rat adrenal homogenate assay. Concentrations at AS within adrenal mitochondria are probably higher than the plasma levels because FAD is preferentially distributed into the adrenal cortical tissue (D. F. Rigel, unpublished observations). Nevertheless, the consistent in vivo exposure-response relationships suggest that the plasma FAD concentrations do predict the extent of PAC suppression in rats and, presumably, in humans or other species.
The effective dose of FAD for lowering PAC in rats appears to be considerably higher than that in humans based on clinical studies with the racemic fadrozole (CGS016949A). Daily doses of ∼4 mg (∼0.06 mg/kg) of fadrozole suppressed basal and adrenocorticotropin-stimulated PAC in postmenopausal women with breast cancer or in healthy males (Dowsett et al., 1990; Stein et al., 1990; Trunet et al., 1992). Thus, FAD is several hundred-fold more potent on a body weight-normalized dose basis in humans than in rats. This in vivo species disparity is consistent with the ∼20- to 400-fold greater potency of FAD in human adrenocortical carcinoma (NCI-H295R) cells (IC50 = 37 nM) and recombinant human AS (IC50 = 1.6 nM) (Fiebeler et al., 2005; LaSala et al., 2009) versus our IC50 (670 nM) in rat adrenal cortical homogenates.
FAD was 5-fold more selective for inhibiting biosynthesis of aldosterone (via CYP11B2) versus corticosterone (via CYP11B1) in our rat adrenocortical assay, similar to its selectivity (6-fold) reported in the recombinant human enzyme preparations (LaSala et al., 2009). In our rat adrenocorticotropin model, FAD exhibited 50-fold selectivity for lowering PAC versus PCC on both a dose (ED50) and plasma concentration (EC50) basis. This observation suggests that the translation from in vitro to in vivo conferred to this ASI an additional CYP11B2/CYP11B1 selectivity (∼10×). However, we observed no such in vitro to in vivo amplification for MET, possibly because MET also inhibits the conversion of cholesterol to pregnenolone (Carballeira et al., 1974), an upstream step that is common to both mineralocorticoid and glucocorticoid biosynthesis. Because our in vitro rat adrenal assays used the immediate precursor substrate (11-deoxycorticosterone) for the CYP11B2 and CYP11B1 enzymes, this off-target activity of MET would be evident only in vivo. Overall, these results indicate the feasibility of developing ASIs that are relatively selective in vitro and in vivo, despite the high sequence homology between the CYP11B2/CYP11B1 genes and proteins within a particular species (Roumen et al., 2007).
A seemingly surprising finding is that MET, a so-called corticosterone inhibitor, was actually severalfold more selective both in vitro and in vivo for inhibiting CYP11B2 than CYP11B1. Although this lack of CYP11B1 selectivity has been recognized previously (e.g., Gomez-Sanchez et al., 1997), it does not appear to be widely appreciated. The major reduced metabolites of MET [(+)- and (−)-MOH] (Nagamine et al., 1997) were unlikely to significantly alter the selectivity profile of MET in vivo because both enantiomers exhibited in vitro selectivities similar to that of MET itself. However, we estimate that ∼80 to 85% of the PAC- and PCC-lowering effects of therapeutic doses of MET were attributable to these abundant and potent metabolites. In summary, although ∼100 to 300 mg/kg MET effectively reduced corticosteroid production in vivo, MET clearly is not a reliable tool compound for selectively attenuating glucocorticoid production.
Interesting physiologic observations could be gleaned from AS inhibition in our models. For example, the temporal decline of PAC after the highest doses of FAD or MET followed a biphasic pattern similar to the clearance profile of exogenously (intravenously) administered radiolabeled aldosterone in rats (fast and slow elimination half-lives of 6 and 36 min, respectively) (Morris et al., 1975). Based on a biexponential curve fit to our data, the estimated elimination half-lives of endogenously generated aldosterone were 8 and 66 min, respectively, and independent of the model or compound. The similar fast-phase half-lives (i.e., 8 versus 6 min) indicate rapid drug absorption, distribution to, and inhibition of the target enzyme. The somewhat longer slow-phase half-life may reflect a delayed compensatory up-regulation of aldosterone biosynthesis in response to pharmacologic inhibition of AS, a factor that would not affect the elimination of labeled exogenous aldosterone.
Several limitations of our models can be noted. First, as performed, they do not allow the assessment of the duration of action of a compound beyond 8 h. However, the ANG II or adrenocorticotropin infusion time could readily be extended to achieve this goal. Second, although the primary site of action of these compounds is presumably AS, any off-target inhibition of the ANG II or adrenocorticotropin signaling pathways or upstream steroidogenic cascade would also affect the PAC/PCC response. Nevertheless, similar responses in both models would rule out possible off-target effects that are specific to distinct signaling pathways of either of the two secretagogues. Likewise, observing an in vivo selectivity that is narrower than that predicted by the in vitro CYP11B2/CYP11B1 inhibitory profile (e.g., MET in our study) would suggest that the compound also blocks an upstream step common to the mineralocorticoid/glucocorticoid steroidogenic pathways (e.g., cholesterol to pregnenolone). Third, the secretagogues or their biosynthetic products may alter the PK properties of the test compound. For example, ANG II (but not adrenocorticotropin) is a potent vasoconstrictor that can alter renal or liver blood flow and, therefore, the elimination/metabolism profile of the ASI.
Despite these modest limitations, our two models demonstrate utility for robustly and simultaneously assessing the PK/PD profiles of test compounds. When combined with in vitro rat and human data, the models can be used to identify selective and effective clinical ASI candidates such as FAD. These agents are expected to be novel treatments for diseases such as hyperaldosteronism, resistant hypertension, and heart failure.
We thank Drs. Gary Ksander and Jim Zhou for preparing, separating, and conducting the optical rotation analysis of the MOH enantiomers, Ye Lu, Jakal Amin, Shaoyong Li, and Wieslawa Maniara for their skillful bioanalytic/PK assistance, and Drs. Keith DiPetrillo, Gary Ksander, and Joel Ménard for their valuable feedback on the article.
This study was supported by Novartis Pharmaceuticals Corporation.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- mineralocorticoid receptor
- mineralocorticoid receptor antagonist
- aldosterone synthase
- aldosterone synthase inhibitor
- FAD286, (+)-(5R)-4-(5,6,7,8-tetrahydroimidazo [1,5-a]pyridin-5-yl]benzonitrile hydrochloride
- CGS016949A, fadrozole
- ANG II
- angiotensin II
- plasma aldosterone concentration
- plasma corticosterone concentration
- scintillation proximity assay
- phosphate-buffered saline
- liquid chromatography
- tandem mass spectroscopy
- area under the concentration-time curve
- time-weighted average
- lower limit of quantification
- Received February 9, 2010.
- Accepted March 29, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics