Randy Lee Webb

Effects of Aliskiren on Blood Pressure, Albuminuria, and (Pro)Renin Receptor Expression in Diabetic TG(mRen-2)27 Rats

The aim of this study was to explore the effects of the renin inhibitor aliskiren in streptozotocin-diabetic TG(mRen-2)27 rats. Furthermore, we investigated in vitro the effect of aliskiren on the interactions between renin and the (pro)renin receptor and between aliskiren and prorenin. Aliskiren distributed extensively to the kidneys of normotensive (non)diabetic rats, localizing in the glomeruli and vessel walls after 2 hours exposure. In diabetic TG(mRen-2)27 rats, aliskiren (10 or 30 mg/kg per day, 10 weeks) lowered blood pressure, prevented albuminuria, and suppressed renal transforming growth factor-&bgr; and collagen I expression versus vehicle. Aliskiren reduced (pro)renin receptor expression in glomeruli, tubules, and cortical vessels compared to vehicle (in situ hybridization). In human mesangial cells, aliskiren (0.1 &mgr;mol/L to 10 &mgr;mol/L) did not inhibit binding of 125I-renin to the (pro)renin receptor, nor did it alter the activation of extracellular signal-regulated kinase 1/2 by renin (20 nmol/L) preincubated with aliskiren (100 nmol/L) or affect gene expression of the (pro)renin receptor. Evidence was obtained that aliskiren binds to the active site of prorenin. The above results demonstrate the antihypertensive and renoprotective effects of aliskiren in experimental diabetic nephropathy. The evidence that aliskiren can reduce in vivo gene expression for the (pro)renin receptor and that it may block prorenin-induced angiotensin generation supports the need for additional work to reveal the mechanism of the observed renoprotection by this renin inhibitor.

Aliskiren, a novel, orally effective renin inhibitor, lowers blood pressure in marmosets and spontaneously hypertensive rats

Objectives Aliskiren is a new renin inhibitor of a novel structural class that has recently been shown to be efficacious in hypertensive patients after once-daily oral dosing. We report the results of animal experiments performed in marmosets and rats in order to characterize aliskiren before its recent investigation in humans. Methods The effects of aliskiren were investigated in sodium-depleted marmosets (oral dosing) and in spontaneously hypertensive rats (dosing via subcutaneous osmotic minipumps). Blood pressure (BP) and heart rate were measured by radiotelemetry. Results In sodium-depleted marmosets, single oral doses of aliskiren (1-30 mg/kg) dose-dependently lowered BP. At a dose of 3 mg/kg, peak effects were observed 1 h after dosing (−30 ± 4 mmHg, n = 6) and the response persisted for more than 12 h. A single oral dose of 3 mg/kg aliskiren was more effective than the same dose of either remikiren or zankiren, two orally active renin inhibitors previously tested in humans. Aliskiren (10 mg/kg) was at least as effective as equal doses of the AT1-receptor blocker valsartan or the angiotensin-converting enzyme inhibitor benazepril. In spontaneously hypertensive rats, aliskiren dose-dependently (10-100 mg/kg per day) decreased BP. Aliskiren also potentiated the antihypertensive effects of low doses of valsartan or benazeprilat (1 or 3 mg/kg per day). Conclusions Aliskiren is an orally effective, long-lasting renin inhibitor that shows antihypertensive efficacy in animals superior to previous renin inhibitors and at least equivalent to angiotensin-converting enzyme inhibitors and AT1-receptor blockers. Aliskiren may therefore represent an effective, novel approach to the treatment of hypertension and related disorders, alone or in combination with other antihypertensive agents.

Synergistic effects of combined converting enzyme inhibition and angiotensin II antagonism on blood pressure in conscious telemetered spontaneously hypertensive rats

Objective To investigate the chronic effects of combined administration of an angiotensin II receptor antagonist (valsartan) and an angiotensin converting enzyme inhibitor (benazeprilat) on blood pressure and heart rate in conscious telemetered spontaneously hypertensive rats. Methods Blood pressure and heart rate were monitored (by radiotelemetry) during 2-week infusions of 0.5–10 mg/kg valsartan per day and 0.5–10 mg/kg benazeprilat per day, alone or in combination, into conscious spontaneously hypertensive rats. Also, responses of blood pressure in conscious spontaneously hypertensive rats to exogenous angiotensin I and II were determined. Results Synergistic antihypertensive effects were observed when valsartan and benazeprilat were coadministered at submaximal monotherapy doses in the range 0.5–1.5 mg/kg per day. For all combination groups, the area over the curve (mmHg × days) for lowering of blood pressure was significantly greater (synergy) than that predicted from the sum of the monotherapy responses. Combination therapy abrogated pressor responses to angiotensin I more effectively than did comparable doses of the monotherapies. Conclusions These results demonstrate that combination therapy aimed at interrupting operation of the renin–angiotensin system simultaneously at multiple sites can prevent the partial escape which occurs during chronic angiotensin converting enzyme inhibitor monotherapy. Furthermore, multiple-site intervention results in a more efficacious antihypertensive response than that achieved with high doses of the individual monotherapies.

Beneficial effects of combined benazepril-amlodipine on cardiac nitric oxide, cGMP, and TNF-alpha production after cardiac ischemia.

The aim of this study was to determine if myocardial inflammation is increased after myocardial ischemia and whether angiotensin-converting enzyme inhibitors, calcium channel blockers, or diuretics decrease mediators of inflammation in rats with induced myocardial ischemia. Changes in cardiac interstitial fluid (CIF) levels of nitric oxide metabolites (NOX), cyclic guanosine 3′,5′-monophosphate (cGMP), angiotensin II (Ang II), and tumor necrosis factor-α (TNF-α) were monitored with/without oral administration of benazepril, amlodipine, combined benazepril-amlodipine, or hydrochlorothiazide. Using a microdialysis technique, levels of several mediators of inflammation were measured after sham operation or 30-minute occlusion of the left anterior descending coronary artery. Compared with sham animals, levels of CIF NOX and cGMP were decreased in animals with ischemia (P<0.001). Benazepril or amlodipine significantly increased NOX levels (P<0.05 vs. untreated ischemia), but only benazepril significantly increased cGMP (P<0.05). Combined benazepril-amlodipine further increased CIF NOX and cGMP (P<0.001), compared with either drug alone. CIF Ang II and TNF-α in sham animals did not change significantly. In animals with ischemia, CIF Ang II and TNF-α increased progressively. Amlodipine alone, benazepril alone, or combined benazepril-amlodipine significantly reduced TNF-α (P<0.01 for monotherapies and P<0.001 for combination therapy). Hydrochlorothiazide did not cause significant changes in NOX, cGMP, or TNF-α. Combination benazepril-amlodipine may be beneficial for managing cardiac ischemia.

Effects of combined AT1 receptor antagonist/NEP inhibitor on vascular remodeling and cardiac fibrosis in SHRSP.

Background The association of an angiotensin-converting enzyme inhibitor (ACEI) with a neutral endopeptidase inhibitor (NEPI) has potent blood pressure (BP) lowering action, but is associated with side-effects. We evaluated the effects of combining an angiotensin II type 1 (AT1) receptor blocker (ARB, valsartan) and a NEPI (CGS 25354) in comparison with a dual ACEI/NEPI (CGS 30440) in stroke-prone spontaneously hypertensive rats (SHRSP). Methods and results Ten-week-old SHRSP were treated with valsartan (10 mg/kg per day), valsartan + CGS 25354 (100 mg/kg per day), CGS 25354, CGS 30440 (10 mg/kg per day) or hydralazine (25 mg/kg per day) for 10 weeks. Mesenteric resistance arteries were studied on a pressurized myograph, whereas cardiac effects were assessed by histology and immunohistochemistry. BP of SHRSP was lowered by combined valsartan/NEPI and ACEI/NEPI slightly more than valsartan, whereas NEPI was ineffective. Valsartan, valsartan/NEPI and ACEI/NEPI normalized resistance artery relaxation responses to acetylcholine, and significantly decreased media/lumen ratio and collagen deposition. All treatments decreased vascular NAD(P)H oxidase-mediated superoxide production. Valsartan/NEPI and ACEI/NEPI decreased media/lumen ratio of intramyocardial coronary arteries, while valsartan alone had no effect. Valsartan/NEPI and ACEI/NEPI increased vascular matrix metalloproteinase-2 activity, and decreased tissue inhibitors of metalloproteinase-2 activity and macrophage infiltration. Conclusion Combined valsartan/NEPI was almost as effective as a dual ACEI/NEPI in lowering BP and improving vascular remodeling in SHRSP. These findings suggest the potential therapeutic value of combining ARB and NEPI in the treatment of hypertension.

A Novel Class of Oral Direct Renin Inhibitors: Highly Potent 3,5-Disubstituted Piperidines Bearing a Tricyclic P3–P1 Pharmacophore

A small library of fragments comprising putative recognition motifs for the catalytic dyad of aspartic proteases was generated by in silico similarity searches within the corporate compound deck based on rh-renin active site docking and scoring filters. Subsequent screening by NMR identified the low-affinity hits 3 and 4 as competitive active site binders, which could be shown by X-ray crystallography to bind to the hydrophobic S3-S1 pocket of rh-renin. As part of a parallel multiple hit-finding approach, the 3,5-disubstituted piperidine (rac)-5 was discovered by HTS using a enzymatic assay. X-ray crystallography demonstrated the eutomer (3S,5R)-5 to be a peptidomimetic inhibitor binding to a nonsubstrate topography of the rh-renin prime site. The design of the potent and selective (3S,5R)-12 bearing a P3(sp)-tethered tricyclic P3-P1 pharmacophore derived from 3 is described. (3S,5R)-12 showed oral bioavailability in rats and demonstrated blood pressure lowering activity in the double-transgenic rat model.

Combination of Low-Dose Valsartan and Enalapril Improves Endothelial Dysfunction and Coronary Reserve in Nω-Nitro-l-Arginine Methyl Ester–Treated Spontaneously Hypertensive Rats

Combination of nonhypotensive doses of valsartan and enalapril markedly improved survival (+87%) compared with untreated animals (37%) in spontaneously hypertensive rats (SHRs) with endothelial dysfunction. However, the combination had no effect on kidney function, and proteinuria persisted over the 12 weeks of the study. It was hypothesized that the greater survival was due to improvement in endothelial function or coronary vasculature despite blockade of nitric oxide synthase and high blood pressure. Therefore, endothelial function was evaluated in isolated aortic ring and maximal coronary blood flow was studied in isolated perfused SHR hearts (20–24 weeks) treated with Nω-nitro-l-arginine methyl ester (l-NAME) (50 mg/l) for 4 weeks. The animals received vehicle, valsartan 5 mg/kg/d, enalapril 1 mg/kg/d, valsartan 50 mg/kg/d, or the combination valsartan 5 mg/kg/d with enalapril 1 mg/kg/d in drinking water. Normotensive Wistar-Kyoto (WKY) rats were used as control. Blood pressure was measured by telemetry. Histopathology was performed on heart, kidney (hematoxylin-eosin), and aorta (Masson trichrome). l-NAME elevated blood pressure by 50 mm Hg after vehicle (199 ± 5 mm Hg). Valsartan 50 mg/kg/d completely abolished this increase (150 ± 4 mm Hg) whereas the valsartan-enalapril combination synergistically decreased blood pressure (−37 mm Hg at 162 ± 7 mm Hg) compared with monotherapy (valsartan 5 mg/kg/d −10 mm Hg; enalapril 1 mg/kg/d −15 mm Hg). All treatments improved the histopathology, most markedly in those receiving the valsartan-enalapril combination. The severity mean grades for lesions were 2.1, 1.9, 1.7, 1.1, and 0.9 in vehicle-treated SHRs, enalapril 1 mg/kg/d, valsartan 5 mg/kg/d, valsartan 5 or 50 mg/kg/d, and the valsartan-enalapril combination, respectively, compared with 0.02 in WKY rats. Acetylcholine-induced relaxation was significantly greater in treated SHRs than after vehicle (−40% at 0.1 mmol acetylcholine) but the combination induced the maximal relaxation (−85%). The ratio of maximal tension induced by serotonin in rings with and without endothelium was 1.4 and 1.3 in vehicle and valsartan 5 mg/kg/d–treated rats whereas it did not differ from 1 in WKY rats and all other treated groups. The cardiac hypertrophy (+27%) was prevented by valsartan 50 mg/kg/d and the valsartan-enalapril combination. Coronary reserve was significantly increased by valsartan 50 mg/kg/d (+85% at 7.8 ± 0.7 ml/min/g) and the valsartan-enalapril combination (+42% at 6.0 ± 0.4 ml/min/g) compared with 4.2 ± 0.5 (vehicle). This was not different of 8.8 ± 0.5 (WKYs). Despite the maintenance of a high blood pressure, low-dose valsartan-enalapril significantly improved endothelial function and histopathology and increased coronary reserve in SHRs chronically receiving l-NAME.

Structure-based design of substituted piperidines as a new class of highly efficacious oral direct Renin inhibitors.

A cis-configured 3,5-disubstituted piperidine direct renin inhibitor, (syn,rac)-1, was discovered as a high-throughput screening hit from a target-family tailored library. Optimization of both the prime and the nonprime site residues flanking the central piperidine transition-state surrogate resulted in analogues with improved potency and pharmacokinetic (PK) properties, culminating in the identification of the 4-hydroxy-3,5-substituted piperidine 31. This compound showed high in vitro potency toward human renin with excellent off-target selectivity, 60% oral bioavailability in rat, and dose-dependent blood pressure lowering effects in the double-transgenic rat model.

The Discovery of Novel Potent trans-3,4-Disubstituted Pyrrolidine Inhibitors of the Human Aspartic Protease Renin from in Silico Three-Dimensional (3D) Pharmacophore Searches

The small-molecule trans-3,4-disubstituted pyrrolidine 6 was identified from in silico three-dimensional (3D) pharmacophore searches based on known X-ray structures of renin-inhibitor complexes and demonstrated to be a weakly active inhibitor of the human enzyme. The unexpected binding mode of the more potent enantiomer (3S,4S)-6a in an extended conformation spanning the nonprime and S1 pockets of the recombinant human (rh)-renin active site was elucidated by X-ray crystallography. Initial structure-activity relationship work focused on modifications of the hydrophobic diphenylamine portion positioned in S1 and extending toward the S2 pocket. Replacement with an optimized P3-P1 pharmacophore interacting to the nonsubstrate S3(sp) cavity eventually resulted in significantly improved in vitro potency and selectivity. The prototype analogue (3S,4S)-12a of this new class of direct renin inhibitors exerted blood pressure lowering effects in a hypertensive double-transgenic rat model after oral administration.

Effects of the novel dual inhibitor of neutral endopeptidase and angiotensin-converting enzyme, CGS 30440, on blood pressure and cardiac hypertrophy in spontaneously hypertensive rats.

This study examined the long-term effects of CGS 30440 on blood pressure, heart rate, cardiac hypertrophy, and urinary parameters in conscious spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats. Initial studies with CGS 30440 produced dose-related reductions in mean arterial pressure, with a dose of 30 mg/kg/day of CGS 30440 producing a maximal sustained response of 40 mm Hg. CGS 30440 significantly inhibited plasma angiotensin-converting enzyme (ACE) activity by 82% in WKY rats. In SHRs, lung ACE and renal neutral endopeptidase (NEP) were inhibited by >60 and >90%, respectively. Urinary cyclic guanosine monophosphate (cGMP) excretion was significantly increased by CGS 30440 in SHRs but was unaltered in WKY rats. One hour after the final dose of an 8-week regimen, blood pressure was 122 +/- 4 and 189 +/- 5 mm Hg in CGS 30440-treated (30 mg/kg/day) and vehicle-treated SHRs, respectively. Heart-rate responses were not different between treatment groups. Left ventricular hypertrophy (LV weight/body weight ratio) was reduced significantly in SHRs to 2.45 +/- 0.08 mg/g at 10 mg/kg/day and 2.26 +/- 0.07 mg/g at 30 mg/kg/day versus 2.91 +/- 0.09 mg/g in rats receiving only vehicle. These results demonstrate that CGS 30440 is a potent, orally active antihypertensive agent with a long duration of action. The cardiac hypertrophy of established hypertension in the SHRs was attenuated by CGS 30440. Thus CGS 30440, an orally active prodrug, has been shown to be a novel antihypertensive agent with dual ACE/NEP inhibitory activity in SHRs.