Gary Michael Ksander

Pharmacokinetics and Pharmacodynamics of LCZ696, a Novel Dual-Acting Angiotensin Receptor−Neprilysin Inhibitor (ARNi)

Angiotensin receptor blockade and neprilysin (NEP) inhibition together offer potential benefits for the treatment of hypertension and heart failure. LCZ696 is a novel single molecule comprising molecular moieties of valsartan and NEP inhibitor prodrug AHU377 (1:1 ratio). Oral administration of LCZ696 caused dose‐dependent increases in atrial natriuretic peptide immunoreactivity (due to NEP inhibition) in Sprague‐Dawley rats and provided sustained, dose‐dependent blood pressure reductions in hypertensive double‐transgenic rats. In healthy participants, a randomized, double‐blind, placebo‐controlled study (n = 80) of single‐dose (200–1200 mg) and multiple‐dose (50–900 mg once daily for 14 days) oral administration of LCZ696 showed that peak plasma concentrations were reached rapidly for valsartan (1.6–4.9 hours), AHU377 (0.5–1.1 hours), and its active moiety, LBQ657 (1.8–3.5 hours). LCZ696 treatment was associated with increases in plasma cGMP, renin concentration and activity, and angiotensin II, providing evidence for NEP inhibition and angiotensin receptor blockade. In a randomized, open‐label crossover study in healthy participants (n = 56), oral LCZ696 400 mg and valsartan 320 mg were shown to provide similar exposure to valsartan (geometric mean ratio [90% confidence interval]: AUC0‐∞ 0.90 [0.82–0.99]). LCZ696 was safe and well tolerated. These data support further clinical development of LCZ696, a novel, orally bioavailable, dual‐acting angiotensin receptor—NEP inhibitor (ARNi) for hypertension and heart failure.

Benzofused Macrocyclic Lactams as Triple Inhibitors of Endothelin-converting Enzyme, Neutral Endopeptidase 24.11, and Angiotensin-converting Enzyme

The purpose of this study was to identify endothelin-converting enzyme (ECE) inhibitors that also possess inhibitory activity for neutral endopeptidase 24.11 (NEP) and angiotensin-converting enzyme (ACE). The ortho-substituted benzofused macrocyclic lactams, such as CGS 26670, are generally potent NEP inhibitors but poor ACE inhibitors. CGS 26670 inhibited ECE activity with an IC50 of 600 nM, whereas it inhibited NEP and ACE activities with IC50 values of 0.9 and > 10,000 nM, respectively. This compound also prevented the conversion of big endothelin-1 (big ET-1) to ET-1 by denuded porcine coronary arterial smooth muscle with an IC50 of 200 nM. The ACE inhibitory activity is greatly is greatly improved in metasubstituted benzofused macrocyclic lactams. For example, CGS 26582 inhibited ECE, NEP, and ACE activities with IC50 values of 620, 4, and 175 nM, respectively. When injected at 30 mg/kg i.v. in conscious rats, followed by a challenge with big ET-1 at 1 nmol/kg i.v., this compound suppressed by 44% the increase in mean arterial blood pressure owing to the generation of ET-1 by ECE. Because ECE, NEP, and ACE play regulatory roles in cardiovascular and renal function, triple inhibitors of these enzymes may represent a novel class of agents for treatment of cardiovascular and renal diseases.

Thromboxane synthase inhibition enhances action of converting enzyme inhibitors.

Mean arterial blood pressure was measured over a 24-hour period from the femoral artery of conscious, unrestrained spontaneously hypertensive rats. Oral administration of the angiotensin converting enzyme inhibitor CGS 16617 significantly lowered mean arterial pressure. In contrast, both the thromboxane synthase inhibitor CGS 12970 and the thromboxane receptor antagonist BM 13505 lacked an antihypertensive action in the spontaneously hypertensive rat. When administered concurrently, the thromboxane synthase inhibitor CGS 12970 potentiated the antihypertensive action of the angiotensin converting enzyme inhibitor CGS 16617. This effect was not observed with the thromboxane receptor antagonist BM 13505. In addition to CGS 16617, CGS 12970 also potentiated the hypotensive effect of two structurally dissimilar angiotensin converting enzyme inhibitors, benazapril HCL and captopril. Indomethacin blocked the thromboxane synthase inhibition-induced potentiation of the antihypertensive action of angiotensin converting enzyme inhibitors. The thromboxane synthase inhibitor CGS 12970 had no effect on the hypotension induced by hydralazine, indicating that the hypotension is not a nonspecific action related to the fall in blood pressure. These results may suggest that converting enzyme inhibition augments the levels and actions of a hormone that stimulates prostaglandin formation. It is well established that thromboxane synthase inhibitors eliminate the formation of the vasoconstrictor thromboxane A2 and allow reorientation of eicosanoid production toward the formation of vasodilating prostaglandins, which could enhance the antihypertensive action of angiotensin converting enzyme inhibitors.

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.

Discovery of N-[5-(6-Chloro-3-cyano-1-methyl-1H-indol-2-yl)-pyridin-3-ylmethyl]-ethanesulfonamide, a Cortisol-Sparing CYP11B2 Inhibitor that Lowers Aldosterone in Human Subjects.

Human clinical studies conducted with LCI699 established aldosterone synthase (CYP11B2) inhibition as a promising novel mechanism to lower arterial blood pressure. However, LCI699s low CYP11B1/CYP11B2 selectivity resulted in blunting of adrenocorticotropic hormone-stimulated cortisol secretion. This property of LCI699 prompted its development in Cushings disease, but limited more extensive clinical studies in hypertensive populations, and provided an impetus for the search for cortisol-sparing CYP11B2 inhibitors. This paper summarizes the discovery, pharmacokinetics, and pharmacodynamic data in preclinical species and human subjects of the selective CYP11B2 inhibitor 8.

The discovery of potent inhibitors of aldosterone synthase that exhibit selectivity over 11-β-hydroxylase

Aldosterone, the final component of the renin-angiotensin-aldosterone system, plays an important role in the pathophysiology of hypertension and congestive heart failure. Aldosterone synthase (CYP11B2) catalyzes the last three steps of aldosterone biosynthesis, and as such appears to be a target for the treatment of these disorders. A sulfonamide-imidazole scaffold has proven to be a potent inhibitor of CYP11B2. Furthermore, this scaffold can achieve high levels of selectivity for CYP11B2 over CYP11B1, a key enzyme in the biosynthesis of cortisol.

Structure of neprilysin in complex with the active metabolite of sacubitril

Sacubitril is an ethyl ester prodrug of LBQ657, the active neprilysin (NEP) inhibitor, and a component of LCZ696 (sacubitril/valsartan). We report herein the three-dimensional structure of LBQ657 in complex with human NEP at 2 Å resolution. The crystal structure unravels the binding mode of the compound occupying the S1, S1’ and S2’ sub-pockets of the active site, consistent with a competitive inhibition mode. An induced fit conformational change upon binding of the P1’-biphenyl moiety of the inhibitor suggests an explanation for its selectivity against structurally homologous zinc metallopeptidases.

Delineation of endothelin receptor subtypes in rat and rabbit aortas

Summary: The receptor subtypes mediating tension responses to endothelin-1 (ET-1) in isolated rat and rabbit aortic preparations were examined with various ET receptor antagonists. In the presence of 3 u,M IRL 2500 (an ETB-selective antagonist), SB 209670 (an ETA/ETB antagonist) induced monophasic inhibitions of the ET-1 dose-response curves of both rat (pKB 9.1) and rabbit aortas (pKB 8.9). In the presence of IRL 2500, PD 156707 (an ETA antagonist) also caused a similar monophasic inhibition of the agonist-induced dose-response curve of the rat aorta (pKB 7.7) but produced a biphasic inhibition of the curve obtained with the rabbit aorta. PD 156707, however, produced monophasic inhibitions of the ET-1-induced dose-response curve of rabbit aortas pretreated either with BQ 788 (an ETB-selective antagonist) or desensitized with sarafotoxin S6c (an ETB-selective agonist). The receptor subtypes mediating the contractile responses to ET-1 were further characterized by examining the effects of the antagonists on the binding of [125I]ET-1 to these two tissues. In the presence of 1 nM IRL 2500, SB 209670 displaced [125I]ET-1 binding in both rat and rabbit aortic membrane preparations in a monophasic manner, with similar potencies (IC50 1.2–1.6 nM). Similar results were also obtained with PD 156707 in rat aortic membrane (IC50 0.27 nM). In contrast, the data obtained with PD 156707 using rabbit aortic membrane were best-fitted with a two-site model (site 1, IC50 0.21 nM, 67% of Bmax; site 2, 100 nM and 33%, respectively). Therefore, the constrictor response to ET-1 in the rat aorta is mediated exclusively by the ETA receptor, whereas that in the rabbit aorta is additionally mediated by an ETB receptor subtype that is sensitive to SB 209670 and BQ 788 but insensitive to IRL 2500.

Effects of the ETB-selective antagonist IRL 2500 in conscious spontaneously hypertensive and Wistar-Kyoto rats.

Summary: The pharmacologic profile of a novel and selective ETB antagonist, IRL 2500 (JV-(3,5-dimeth-ylbenzoyl)-N-methyl-(D)-(4-phenylphenyl)-alanyl-L-tryptophan) was examined in conscious spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats. The initial vasodepressor response to endothelin-1 (ET-1) and IRL 1620 (0.5 nmol/kg, i.v.) was significantly reduced in conscious WKY rats pretreated with IRL 2500 (10 mg/ kg, i.v.). The secondary and sustained pressor response to these agonists, however, was not altered by IRL 2500. The linear peptide antagonist BQ 788, although also inhibiting the initial depressor responses, attenuated the secondary pressor response to IRL 1620 and potentiated the pressor response to ET-1. IRL 2500, administered alone to naive conscious SHRs produced a - 37 ± 8 mm Hg reduction in blood pressure, followed by a secondary pressor response (+ 38 ± 7 mm Hg) with a duration exceeding 90 min. Pretreatment with either the ETA-selective antagonist BQ 123 or with the nonselective ETA/ ETB antagonist SB 209670 resulted in marked potentiation of the depressor response and significant attenuation of the secondary rise in pressure. These results indicate that in the conscious rat, IRL 2500 acts as an ETB1-selective antagonist. In addition, ETA receptor activation contributes to the sustained pressor response to IRL 2500 in the conscious SHR. Furthermore, IRL 2500 may also exert a non-ET receptor-mediated vasodilatation in the SHR.