The effect of angiotensin receptor neprilysin inhibitor, sacubitril/valsartan, on central nervous system amyloid-ß concentrations and clearance in the cynomolgus monkey.

Sacubitril/valsartan (LCZ696) is the first angiotensin receptor neprilysin inhibitor approved to reduce cardiovascular mortality and hospitalization in patients with heart failure with reduced ejection fraction. As neprilysin (NEP) is one of several enzymes known to degrade amyloid-ß (Aß), there is a theoretical risk of Aß accumulation following long-term NEP inhibition. The primary objective of this study was to evaluate the potential effects of sacubitril/valsartan on central nervous system clearance of Aß isoforms in cynomolgus monkeys using the sensitive Stable Isotope Labeling Kinetics (SILK™)-Aß methodology. The in vitro selectivity of valsartan, sacubitril, and its active metabolite sacubitrilat was established; sacubitrilat did not inhibit other human Aß-degrading metalloproteases. In a 2-week study, sacubitril/valsartan (50mg/kg/day) or vehicle was orally administered to female cynomolgus monkeys in conjunction with SILK™-Aß. Despite low cerebrospinal fluid (CSF) and brain penetration, CSF exposure to sacubitril was sufficient to inhibit NEP and resulted in an increase in the elimination half-life of Aß1-42 (65.3%; p=0.026), Aß1-40 (35.2%; p=0.04) and Aßtotal (29.8%; p=0.04) acutely; this returned to normal as expected with repeated dosing for 15days. CSF concentrations of newly generated Aß (AUC(0-24h)) indicated elevations in the more aggregable form Aß1-42 on day 1 (20.4%; p=0.039) and day 15 (34.7%; p=0.0003) and in shorter forms Aß1-40 (23.4%; p=0.009), Aß1-38 (64.1%; p=0.0001) and Aßtotal (50.45%; p=0.00002) on day 15. However, there were no elevations in any Aß isoforms in the brains of these monkeys on day 16. In a second study cynomolgus monkeys were administered sacubitril/valsartan (300mg/kg) or vehicle control for 39weeks; no microscopic brain changes or Aß deposition, as assessed by immunohistochemical staining, were present. Further clinical studies are planned to address the relevance of these findings.

Characterization and Prediction of Cardiovascular Effects of Fingolimod and Siponimod Using a Systems Pharmacology Modeling Approach.

Sphingosine 1-phosphate (S1P) receptor agonists are associated with cardiovascular effects in humans. This study aims to develop a systems pharmacology model to identify the site of action (i.e., primary hemodynamic response variable) of S1P receptor agonists, and to predict, in a quantitative manner, the cardiovascular effects of novel S1P receptor agonists in vivo. The cardiovascular effects of once-daily fingolimod (0, 0.1, 0.3, 1, 3, and 10 mg/kg) and siponimod (3 and 15 mg/kg) were continuously recorded in spontaneously hypertensive rats and Wistar-Kyoto rats. The results were analyzed using a recently developed systems cardiovascular pharmacology model, i.e. the CVS model; total peripheral resistance and heart rate were identified as the site of action for fingolimod. Next, the CVS model was interfaced with an S1P agonist pharmacokinetic-pharmacodynamic (PKPD) model. This combined model adequately predicted, in a quantitative manner, the cardiovascular effects of siponimod using in vitro binding assays. In conclusion, the combined CVS and S1P agonist PKPD model adequately describes the hemodynamic effects of S1P receptor agonists in rats and constitutes a basis for the prediction, in a strictly quantitative manner, of the cardiovascular effects of novel S1P receptor agonists.

trans-3,4-Disubstituted pyrrolidines as inhibitors of the human aspartyl protease renin. Part II: prime site exploration using an oxygen linker.

Inhibition of the aspartyl protease renin is considered as an efficient approach for treating hypertension. Lately, we described the discovery of a novel class of direct renin inhibitors which comprised a pyrrolidine scaffold (e.g., 2). Based on the X-ray structure of the lead compound 2 bound to renin we predicted that optimization of binding interactions to the prime site could offer an opportunity to further expand the scope of this chemotype. Pyrrolidine-based inhibitors were synthesized in which the prime site moieties are linked to the pyrrolidine core through an oxygen atom, resulting in an ether or a carbamate linker subseries. Especially the carbamate derivatives showed a pronounced increase in in vitro potency compared to 2. Here we report the structure-activity relationship of both subclasses and demonstrate blood pressure lowering effects for an advanced prototype in a hypertensive double-transgenic rat model after oral dosing.

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.

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.

Aldosterone synthase inhibition improves cardiovascular function and structure in rats with heart failure: a comparison with spironolactone.

AIMS: Inhibition of aldosterone synthase, the key enzyme in aldosterone formation, could be an alternative strategy for mineralocorticoid-receptor antagonists in congestive heart failure (CHF), but its effect in CHF is unknown. METHODS AND RESULTS: We compared, in rats with CHF, the effects of a 7 day and a 12 week treatment with the aldosterone synthase inhibitor FAD286 (4 mg kg(-1) day(-1)) with those induced by spironolactone (80 mg kg(-1) day(-1)). FAD286/spironolactone increased cardiac output without modifying arterial pressure. Long-term FAD286 and spironolactone reduced left ventricular (LV) end-diastolic pressure, LV relaxation constant, and LV dilatation, and these effects were more marked with FAD286, whereas both drugs reduced LV hypertrophy and collagen accumulation to the same extent. Long-term FAD286/spironolactone prevented CHF-related enhancement in LV ACE and reduction in LV ACE-2, but only FAD286 prevented the reduction in LV AT(2) receptors. FAD286, but not long-term spironolactone, reduced the CHF-related enhancements in LV reactive oxygen species, reduced-oxidized glutathione ratio, and aortic nicotinamide adenine dinucleotide phosphate oxidase activity. FAD286 normalized the CHF-induced impairment of endothelium-dependent vasodilatation. CONCLUSION: In experimental CHF, FAD286 and spironolactone improve LV haemodynamics, remodelling, and function, but only FAD286 persistently normalizes LV 'redox status'. These results suggest that aldosterone synthase inhibition is a potential therapeutic strategy for the treatment of CHF.

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-beta 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 micromol/L to 10 micromol/L) did not inhibit binding of (125)I-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.

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.

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-alpha (TNF-alpha) 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-alpha in sham animals did not change significantly. In animals with ischemia, CIF Ang II and TNF-alpha increased progressively. Amlodipine alone, benazepril alone, or combined benazepril-amlodipine significantly reduced TNF-alpha (P < 0.01 for monotherapies and P < 0.001 for combination therapy). Hydrochlorothiazide did not cause significant changes in NOX, cGMP, or TNF-alpha. Combination benazepril-amlodipine may be beneficial for managing cardiac ischemia.

Aliskiren, a human renin inhibitor, ameliorates cardiac and renal damage in double-transgenic rats.

We tested the hypothesis that the renin inhibitor aliskiren ameliorates organ damage in rats transgenic for human renin and angiotensinogen genes (double transgenic rat [dTGR]). Six-week-old dTGR were matched by albuminuria (2 mg per day) and divided into 5 groups. Untreated dTGR were compared with aliskiren (3 and 0.3 mg/kg per day)-treated and valsartan (Val; 10 and 1 mg/kg per day)-treated rats. Treatment was from week 6 through week 9. At week 6, all groups had elevated systolic blood pressure (BP). Untreated dTGR showed increased BP (202+/-4 mm Hg), serum creatinine, and albuminuria (34+/-5.7 mg per day) at week 7. At week 9, both doses of aliskiren lowered BP (115+/-6 and 139+/-5 mm Hg) and albuminuria (0.4+/-0.1 and 1.6+/-0.6 mg per day) and normalized serum creatinine. Although high-dose Val lowered BP (148+/-4 mm Hg) and albuminuria (2.1+/-0.7 mg per day), low-dose Val reduced BP (182+/-3 mm Hg) and albuminuria (24+/-3.8 mg per day) to a lesser extent. Mortality was 100% in untreated dTGR and 26% in Val (1 mg/kg per day) treated rats, whereas in all other groups, survival was 100%. dTGR treated with low-dose Val had cardiac hypertrophy (4.4+/-0.1 mg/g), increased left ventricular (LV) wall thickness, and diastolic dysfunction. LV atrial natriuretic peptide and beta-myosin heavy chain mRNA, albuminuria, fibrosis, and cell infiltration were also increased. In contrast, both aliskiren doses and the high-dose Val lowered BP to a similar extent and more effectively than low-dose Val. We conclude that in dTGR, equieffective antihypertensive doses of Val or aliskiren attenuated end-organ damage. Thus, renin inhibition compares favorably to angiotensin receptor blockade in reversing organ damage in dTGR.

Aldosterone synthase inhibitor ameliorates angiotensin II-induced organ damage.

BACKGROUND: Aldosterone and angiotensin (Ang) II both may cause organ damage. Circulating aldosterone is produced in the adrenals; however, local cardiac synthesis has been reported. Aldosterone concentrations depend on the activity of aldosterone synthase (CYP11B2). We tested the hypothesis that reducing aldosterone by inhibiting CYP11B2 or by adrenalectomy (ADX) may ameliorate organ damage. Furthermore, we investigated how much local cardiac aldosterone originates from the adrenal gland. METHODS AND RESULTS: We investigated the effect of the CYP11B2 inhibitor FAD286, losartan, and the consequences of ADX in transgenic rats overexpressing both the human renin and angiotensinogen genes (dTGR). dTGR-ADX received dexamethasone and 1% salt. Dexamethasone-treated dTGR-salt served as a control group in the ADX protocol. Untreated dTGR developed hypertension and cardiac and renal damage and had a 40% mortality rate (5/13) at 7 weeks. FAD286 reduced mortality to 10% (1/10) and ameliorated cardiac hypertrophy, albuminuria, cell infiltration, and matrix deposition in the heart and kidney. FAD286 had no effect on blood pressure at weeks 5 and 6 but slightly reduced blood pressure at week 7 (177+/-6 mm Hg in dTGR+FAD286 and 200+/-5 mm Hg in dTGR). Losartan normalized blood pressure during the entire study. Circulating and cardiac aldosterone levels were reduced in FAD286 or losartan-treated dTGR. ADX combined with dexamethasone and salt treatment decreased circulating and cardiac aldosterone to barely detectable levels. At week 7, ADX-dTGR-dexamethasone-salt had a 22% mortality rate compared with 73% in dTGR-dexamethasone-salt. Both groups were similarly hypertensive (190+/-9 and 187+/-4 mm Hg). In contrast, cardiac hypertrophy index, albuminuria, cell infiltration, and matrix deposition were significantly reduced after ADX (P<0.05). CONCLUSIONS: Aldosterone plays a key role in the pathogenesis of Ang II-induced organ damage. Both FAD286 and ADX reduced circulating and cardiac aldosterone levels. The present results show that aldosterone produced in the adrenals is the main source of cardiac aldosterone.

Increased renal production of angiotensin II and thromboxane B2 in conscious diabetic rats.

BACKGROUND: The mechanisms involved in development of cardiovascular complications associated with diabetes mellitus are not well elucidated. Among the vasoactive factors that may play a role in development of these complications are angiotensin II and thromboxane B2 (TXB2). We hypothesized that diabetes increases renal production of TXB2 through stimulation of angiotensin type-1 receptor. METHODS: We used a microdialysis technique to monitor changes in renal interstitial fluid (RIF) TXB2 in conscious streptozotocin-induced diabetes rat model. The RIF levels of angiotensin II and TXB2 were monitored before and during 6 weeks after development of diabetes and during treatment with the angiotensin type-1 receptor blocker valsartan at 10 mg/kg. Measurement of the urinary albumin excretion (UAE) was used to monitor the development and progression of diabetic nephropathy. RESULTS: The UAE was 81.62 +/- 1.31 ng/min, 184.75 +/- 9.41 ng/min (P < .01), and 229.84 +/- 4.49 ng/min (P < .0001) at baseline, week 3, and week 6, respectively, after induction of diabetes. Basal levels of RIF angiotensin II were 4.28 +/- 0.02 pg/mL and significantly increased to 6.24 +/- 0.31 pg/mL (P < .001) and 7.66 +/- 0.05 pg/mL (P < .001) at 3 and 6 weeks after development of diabetes. Similarly, basal RIF TXB2 was 197 +/- 27 pg/mL and increased to 488 +/- 80 pg/mL (P < .01) and 703 +/- 130 pg/mL (P < .01) at 3 and 6 weeks after development of diabetes. Valsartan caused further increase in RIF angiotensin II levels. In contrast, valsartan decreased RIF TXB2 levels at baseline to 85 +/- 11 pg/mL (P < .01), at 3 weeks to 141 +/- 17 pg/mL (P < .01), and at 6 weeks to 255 +/- 45 pg/mL (P < .01) after development of diabetes. CONCLUSIONS: These results demonstrate that diabetes mellitus is accompanied by increased renal production of angiotensin II and TXB2. The increase in TXB2 is mediated through stimulation of angiotensin type-1 receptor.

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.

Renal nitric oxide production is decreased in diabetic rats and improved by AT1 receptor blockade.

OBJECTIVE: Diabetes mellitus is associated with increased incidence of cardiovascular complications. Lack of nitric oxide production may exacerbate these complications. We hypothesized that diabetes decreases renal nitric oxide (NO) production, an effect that is reversed via inhibition of angiotensin subtype-1 receptor. METHODS: We monitored changes in renal interstitial fluid nitric oxide by a microdialysis technique in the renal cortex of conscious Sprague-Dawley rats. Rats (n = 8 each group) were given streptozotocin 30 mg/kg intravenously to induce diabetes. Changes in renal interstitial fluid angiotensin II and NO were evaluated at baseline before and over 12 weeks during the development of diabetes and at 4 and 8 h after oral administration of the angiotensin subtype-1 (AT1) receptor blockers, losartan (30 mg/kg) or valsartan (10 mg/kg). RESULTS: Renal interstitial fluid angiotensin II significantly increased after development of diabetes. In contrast, basal renal interstitial fluid nitric oxide decreased significantly over 12 weeks after development of diabetes. Both losartan and valsartan caused a further increase in renal angiotensin II levels. Some 4 h after administration, there was significantly greater increase in renal nitric oxide after administration of valsartan than of losartan. At 8 h post- treatment, only valsartan caused a significant increase in renal nitric oxide levels. CONCLUSION: These results demonstrate that diabetes mellitus is associated with an increase in renal production of angiotensin II, while renal production of nitric oxide is reduced. The decrease in renal NO is reversed by AT1 receptor blockade.

Effects of valsartan and valeryl 4-hydroxy valsartan on human platelets: a possible additional mechanism for clinical benefits.

Valsartan selectively blocks angiotensin II binding to the AT1 receptor. ince platelet activation plays a key role in the pathogenesis of vascular disease, and because AT1 receptors are present on the platelet surface, we assessed the in vitro effects of valsartan and its metabolite, valeryl 4-hydroxy valsartan (V4HV), on platelets in 30 subjects with multiple risk factors for cardiovascular disease. Platelet characteristics in blood samples pretreated and incubated with 10 nmol to 100 micromol concentrations of valsartan and V4HV were assessed by aggregometry, rapid platelet analyzers, and by flow cytometry. Pretreatment of blood with valsartan and V4HV resulted in inhibition of conventional plasma (ADP, P = 0.0001, valsartan; epinephrine, P = 0.0001, V4HV) and whole blood collagen-induced (P = 0.01, valsartan; P =.0001, V4HV) platelet aggregation. Closure time was delayed (P = 0.02, valsartan; P = 0.03, 4VHV), indicating platelet inhibition in whole blood under high shear conditions. Expression of many surface platelet receptors, namely GP IIb/IIIa antigen, and activity, vitronectin, p-selectin, and LAMP-1 was significantly reduced compared with autologous baseline activity. Intensity of platelet-leukocyte formation and other platelet activation markers remained unchanged. Platelet inhibition was not dose dependent and was more potent for 4VHV than valsartan in the therapeutic range.Valsartan and 4VHV exhibited significant in vitro inhibition of human platelets. Their antiplatelet properties, especially more potent activity of the metabolite appear to be independent of those of other antiplatelet agents. Whether valsartan reduces vascular ischemic events via additional pathways of platelet inhibition in patients with myocardial infarction and ischemic stroke requires further clinical research.

Synergistic stimulation of aldosterone production in human adrenocortical carcinoma NCI-H295R cells by endothelin-1 and angiotensin II.

Aldosterone has recently been implicated in the pathogenesis of heart failure. The purpose of the present study was to determine the effect of endothelin-1 (ET-1) and angiotensin II (Ang II), two potent vasoconstrictors that are also involved in heart failure, on aldosterone secretion by human adrenocortical carcinoma NCIH295R cells grown in 96-well plates. Ang II stimulated the production of aldosterone dose-dependently in serum-free medium, and the presence of serum drastically decreased aldosterone secretion. In contrast, ET-1-stimulated aldosterone production absolutely required serum. Under optimal conditions, ET-1 was more effective than Ang II as an aldosterone secretagogue. In a suboptimal condition of 2.5% serum, ET-1 and Ang II at 1 microM produced 63 and 76 pmol aldosterone/mg protein, respectively, while 230 pmol aldosterone/mg protein was generated upon coincubation with ET-1 and Ang II. The effect of ET-1 was inhibited dose-dependently by the selective ETA receptor antagonist BQ-123 with an IC50 of 23 nM, but the selective ETB receptor antagonist RES-701 had no effect up to 10 microM. These results suggest that ET-1 and Ang II stimulated aldosterone secretion synergistically in NCIH295R cells and that the effect of ET-1 was mediated via the ETA receptor subtype.

Combination of low-dose valsartan and enalapril improves endothelial dysfunction and coronary reserve in Nomega-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 -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.

Dipeptide sulfonamides as endothelin ETA/ETB receptor antagonists.

Endothelin-1 (ET-1) is a potent mitogen and modulator of vascular tone. It is synthesized and released from endothelial cells and acts upon two receptor subtypes designated as ETA and ETB. In this study, a series of potent dipeptide sulfonamide dual-endothelin ETA/ETB receptor antagonists were prepared to investigate their potential benefit in vascular diseases. CGS 31398 inhibited [125I]ET-1 binding to human ETA and ETB receptors expressed in Chinese hamster ovary (CHO) cells (ETA/CHO, ETB/CHO) with respective IC50 values of 0.26 and 0.12 nM. However, in anesthetized rats, this compound markedly potentiated ET-1-induced renal vascular resistance, a response normally observed with selective ETB receptor antagonists. To determine whether species differences account for these results, a direct comparison was made between binding to rat and rabbit aortic membranes versus functional antagonism in isolated rat aortic rings. It was found that CGS 31398 had potent affinity for the ETA receptor in rat and rabbit aorta with IC50 values of 0.87 and 0.79 nM, respectively. Inhibition of ET-1-induced contractions of rat aorta by the compound was considerably weaker than expected (pKB = 6.4), while that of sarafotoxin S6c induced contraction of dog saphenous vein (100% inhibition at 100 nM) was consistent with corresponding binding data. These results suggest that although CGS 31398 is a potent dual inhibitor of ETA/ETB receptor binding, it surprisingly displays potent ETB and weak ETA receptor antagonism in functional assays.