Michael E. Beil

Pharmacodynamic and pharmacokinetic characterization of the aldosterone synthase inhibitor FAD286 in two rodent models of hyperaldosteronism: comparison with the 11β-hydroxylase inhibitor metyrapone

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.

Antiarrhythmic evaluation of naloxone against acute coronary occlusion-induced arrhythmias in pigs.

Naloxone pretreatment (1.0, 3.0 and 10.0 mg/kg i.v.) failed to protect anesthetized pigs from cardiac arrhythmias including ventricular fibrillation (VF) and death following acute occlusion (20 min) or reperfusion of the left anterior descending coronary artery. These findings suggest that opiate-like substances possibly released by the ischemic myocardium do not contribute significantly to the etiology of cardiac arrhythmias, or sudden death associated with the early stages of myocardial infarction in pigs. The effectiveness of naloxone in preventing acute ischemia-induced arrhythmias in rats may be due to mechanisms other than opiate-receptor blockade.

PKPD modelling of the interrelationship between mean arterial BP, cardiac output and total peripheral resistance in conscious rats

The homeostatic control of arterial BP is well understood with changes in BP resulting from changes in cardiac output (CO) and/or total peripheral resistance (TPR). A mechanism‐based and quantitative analysis of drug effects on this interrelationship could provide a basis for the prediction of drug effects on BP. Hence, we aimed to develop a mechanism‐based pharmacokinetic‐pharmacodynamic (PKPD) model in rats that could be used to characterize the effects of cardiovascular drugs with different mechanisms of action (MoA) on the interrelationship between BP, CO and TPR.

CGS 34226, a thiol-based dual inhibitor of endothelin converting enzyme-1 and neutral endopeptidase 24.11

Endothelins (ETs) are potent vasoconstrictors and have been implicated in the pathogenesis of various cardiovascular and renal diseases. In contrast, atrial natriuretic peptide (ANP) is a potent vasorelaxant and diuretic agent, which is mainly degraded by neutral endopeptidase 24.11 (NEP) in vivo. Thus, compounds that can suppress the biosynthesis of ETs by inhibiting endothelin converting enzymes (ECEs), which catalyse the final step of post-translational processing of the vasoconstrictors, while simultaneously potentiating the levels of ANP by inhibiting NEP may have novel therapeutic utility. Through targeted screening of our compound library and subsequent optimization, CGS 34226 was identified as a potent, dual inhibitor of ECE-1 and NEP, inhibiting the enzymes with respective IC(50) values of 11 and 4.6 nM. In vivo, CGS 34226 suppressed the big endothelin-1 (big ET-1)-induced pressor response dose-dependently. At 15 and 90 min after an intravenous dose of 30 mg/kg in anaesthetized rats, this compound inhibited the big ET-1-induced effect by 79% and 65% respectively. In addition, CGS 34226 increased plasma ANP immunoreactivity by 120% up to 4 h after an intravenous dose of 10 mg/kg in conscious rats infused with ANP at a rate of 450 ng/kg per min, intravenously. These results show that CGS 34226 is a potent dual inhibitor of ECE-1 and NEP in vitro and in vivo and that the compound may represent a novel agent for the treatment of cardiovascular and renal dysfunction.

Cgs 35601 and its Orally Active Prodrug Cgs 37808 as Triple Inhibitors of Endothelin-converting Enzyme-1, Neutral Endopeptidase 24.11, and Angiotensin-converting Enzyme

CGS 35601 is a potent triple inhibitor of endothelinconverting enzyme-1, neutral endopeptidase 24.11, and angiotensinconverting enzyme. It inhibited the activities of these three enzymes with IC50 values of 55, 2 and 22 nM, respectively. In conscious rats, CGS 35601 suppressed the big endothelin-1-induced pressor response by 82% and 72% at 30 and 120 minutes, respectively, following injection at a dose of 10 mg/kg, intravenously. At the same dose, CGS 35601 increased plasma atrial natriuretic peptide (ANP) immunoreactivity by 170% for up to 4 hours in conscious rats infused with ANP, and it inhibited the angiotensin I-induced pressor response by 74-94% within the first 2 hours after dosing. Similar in vivo activities were also observed with its orally active prodrug, CGS 37808. This compound blocked the big endothelin-1- induced pressor response by 71% and 67% at 30 and 120 minutes, respectively, after an oral dose of 10 mgEq/kg in conscious rats. It also increased plasma ANP immunoreactivity by 103% for up to 4 hours and inhibited the angiotensin I-induced pressor response by an average of 49% within the first 4 hours after the same dosing regimen. By suppressing the biosyntheses of endothelin-1 and angiotensin II, two potent vasoconstrictors, while simultaneously potentiating the circulating levels of ANP, a vasorelaxant and diuretic, CGS 35601 and CGS 37808 may represent novel agents for the treatment of cardiovascular and renal diseases.

Structure–Activity Relationships, Pharmacokinetics, and in Vivo Activity of CYP11B2 and CYP11B1 Inhibitors

CYP11B2, the aldosterone synthase, and CYP11B1, the cortisol synthase, are two highly homologous enzymes implicated in a range of cardiovascular and metabolic diseases. We have previously reported the discovery of LCI699, a dual CYP11B2 and CYP11B1 inhibitor that has provided clinical validation for the lowering of plasma aldosterone as a viable approach to modulate blood pressure in humans, as well normalization of urinary cortisol in Cushings disease patients. We now report novel series of aldosterone synthase inhibitors with single-digit nanomolar cellular potency and excellent physicochemical properties. Structure-activity relationships and optimization of their oral bioavailability are presented. An illustration of the impact of the age of preclinical models on pharmacokinetic properties is also highlighted. Similar biochemical potency was generally observed against CYP11B2 and CYP11B1, although emerging structure-selectivity relationships were noted leading to more CYP11B1-selective analogs.

Discovery and in Vivo Evaluation of Potent Dual CYP11B2 (Aldosterone Synthase) and CYP11B1 Inhibitors.

Aldosterone is a key signaling component of the renin-angiotensin-aldosterone system and as such has been shown to contribute to cardiovascular pathology such as hypertension and heart failure. Aldosterone synthase (CYP11B2) is responsible for the final three steps of aldosterone synthesis and thus is a viable therapeutic target. A series of imidazole derived inhibitors, including clinical candidate 7n, have been identified through design and structure-activity relationship studies both in vitro and in vivo. Compound 7n was also found to be a potent inhibitor of 11β-hydroxylase (CYP11B1), which is responsible for cortisol production. Inhibition of CYP11B1 is being evaluated in the clinic for potential treatment of hypercortisol diseases such as Cushings syndrome.

Design and synthesis of a potent and selective endothelin-converting enzyme inhibitor, CGS 35066.

CGS 26303 has previously been shown to inhibit human endothelin converting enzyme-1 (ECE-1) with an IC50 of 410 nM and to be efficacious in several animal disease models. However, it is a more potent inhibitor of neutral endopeptidase 24.11 (NEP) with an IC50 of 1 nM. The aim of this study was to optimize CGS 26303 for greater potency and selectivity towards ECE-1 inhibition. The in vivo activity of the compounds was assessed by inhibition of the big endothelin-1 (ET-1)-induced pressor response in anesthetized rats at 90 min after treatment with a dose of 10 mg/kg, i.v. Under these conditions, CGS 26303 inhibited the pressor response to big ET-1 by 50%. Replacement of the biphenyl and tetrazol groups in CGS 26303 with a dibenzofuran and carboxylic acid, respectively, yielded CGS 35066, a potent ECE-1 inhibitor having an IC50 of 22 nM. In contrast, these substitutions markedly weakened the NEP inhibitory activity of the compound to an IC50 of 2.3 microM. CGS 35066 also exhibited a potent and sustained ECE-1 inhibitory activity in vivo, blocking the pressor response to big ET-1 by 84%. Its orally active prodrug, CGS 35339, was obtained by introducing two phenyl groups at the phosphonic acid substituent in CGS 35066. Therefore, CGS 35066 and CGS 35339 represent novel compounds for assessing the pathogenic role of ET-1 overproduction in various disease states.

Drug effects on the CVS in conscious rats: separating cardiac output into heart rate and stroke volume using PKPD modelling

Previously, a systems pharmacology model was developed characterizing drug effects on the interrelationship between mean arterial pressure (MAP), cardiac output (CO) and total peripheral resistance (TPR). The present investigation aims to (i) extend the previously developed model by parsing CO into heart rate (HR) and stroke volume (SV) and (ii) evaluate if the mechanism of action (MoA) of new compounds can be elucidated using only HR and MAP measurements.

Pharmacological properties of CGS 35066, a potent and selective endothelin-converting enzyme inhibitor, in conscious rats.

The purpose of this study was to examine the pharmacologic properties of CGS 35066, a novel aminophosphonate inhibitor of endothelin-converting enzyme-1 (ECE-1). CGS 35066 inhibited the activity of human ECE-1 and rat kidney neutral endopeptidase 24.11 (NEP) in vitro with IC50 values of 22 +/- 0.9 nM and 2.3 +/- 0.03 microM, respectively. The in vivo effects of CGS 35066 were characterized in conscious, catheterized rats. At 30 and 120 min after treatment with vehicle, big endothelin-1 (big ET-1, 0.3 nmol/kg i.v.) produced increases in mean arterial pressure (MAP) of 982 +/- 31 and 992 +/- 43 mmHg x min (area under the curve), respectively. Doses of 0.3, 1.0, 3.0 and 10.0 mg/kg i.v., of CGS 35066 blocked these pressor responses by 61 +/- 7, 78 +/- 4, 93 +/- 4 and 98 +/- 2% at 30 min (p < 0.05 compared with vehicle controls, all doses), and by 29 +/- 7, 63 +/- 5, 63 +/- 5 and 84 +/- 10% at 120 min (p < 0.05, all doses). In contrast, the pressor effect (58 +/- 6 mmHg) of angiotensin-I (300 ng/kg i.v.) was unaffected by the ECE-1 inhibitor (10 mg/kg i.v.) indicating the absence of activity against angiotensin-converting enzyme. In rats infused with atrial natriuretic peptide (ANP), CGS 35066, at 1 mg/kg, had no effect on plasma irANP; however, irANP levels were doubled at a dose of 30 mg/kg. These results demonstrate that CGS 35066 is the most potent and selective ECE inhibitor identified to date.