Leoluca Criscione

Pharmacological profile of valsartan: a potent, orally active, nonpeptide antagonist of the angiotensin II AT1-receptor subtype

1 The pharmacological profile of valsartan, (S)‐N‐valeryl‐N‐{[2′‐(1H‐tetrazol‐5‐yl)biphenyl‐4‐yl]‐methyl}‐valine, a potent, highly selective, and orally active antagonist at the angiotensin II (AII) AT1‐receptor, was studied in vitro and in vivo. 2 Valsartan competed with [125I]‐AII at its specific binding sites in rat aortic smooth muscle cell membranes (AT1‐receptor subtype) with a Ki of 2.38 nm, but was about 30,000 times less active in human myometrial membranes (AT2‐receptor subtype). 3 In rabbit aortic rings incubated for 5 min with valsartan, at concentrations of 2, 20 and 200 nm, the concentration‐response curve of AII was displaced to the right and the maximum response was reduced by 33%, 36% and 40%, respectively. Prolongation of the incubation time with valsartan to 1 h or 3 h, further reduced the maximum response by 48% or 59% (after 20 nm) and by 59% or 60% (after 200 nm) respectively. After 3 h incubation an apparent pKB value of 9.26 was calculated. Contractions induced by noradrenaline, 5‐hydroxytryptamine, or potassium chloride were not affected by valsartan. No agonistic effects were observed in the rabbit aorta at concentrations of valsartan up to 2 μm. 4 In bovine adrenal glomerulosa, valsartan inhibited All‐stimulated aldosterone release without affecting the maximum response (pA2 8.4). 5 In the pithed rat, oral administration of valsartan (10 mg kg−1) shifted the All‐induced pressor response curves to the right, without affecting responses induced by the electrical stimulation of the sympathetic outflow or by noradrenaline. Animals treated with valsartan 24 h before pithing also showed significant inhibition of the response to AII. 6 In conscious, two‐kidney, one‐clip renal hypertensive rats (2K1C), valsartan decreased blood pressure in a dose‐dependent manner after single i.v. or oral administration. The respective ED30 values were 0.06 mg kg−1 (i.v.) and 1.4 mg kg−1 (p.o.). The antihypertensive effect lasted for at least 24 h after either route of administration. After repeated oral administration for 4 days (3 and 10 mg kg−1 daily), in 2K1C renal hypertensive rats, systolic blood pressure was consistently decreased, but heart rate was not significantly affected. 7 In conscious, normotensive, sodium‐depleted marmosets, valsartan decreased mean arterial pressure, measured by telemetry, after oral doses of 1–30 mg kg−1. The hypotensive effect persisted up to 12 h after 3 and 10 mg kg−1 and up to 24 h after 30 mg kg−1. 8 In sodium‐depleted marmosets, the hypotensive effect of valsartan lasted longer than that of losartan (DuP 753). In renal hypertensive rats, both agents had a similar duration (24 h), but a different onset of action (valsartan at 1 h, losartan between 2 h and 24 h). 9 These results demonstrate that valsartan is a potent, specific, highly selective antagonist of AII at the AT1‐receptor subtype and does not possess agonistic activity. Furthermore, it is an efficacious, orally active, blood pressure‐lowering agent in conscious renal hypertensive rats and in conscious normotensive, sodium‐depleted primates.

FOOD INTAKE IN FREE-FEEDING AND ENERGY-DEPRIVED LEAN RATS IS MEDIATED BY THE NEUROPEPTIDE Y5 RECEPTOR

The new neuropeptide Y (NPY) Y5 receptor antagonist CGP 71683A displayed high affinity for the cloned rat NPY Y5 subtype, but > 1, 000-fold lower affinity for the cloned rat NPY Y1, Y2, and Y4 subtypes. In LMTK cells transfected with the human NPY Y5 receptor, CGP 71683A was without intrinsic activity and antagonized NPY-induced Ca2+ transients. CGP 71683A was given intraperitoneally (dose range 1-100 mg/kg) to a series of animal models of high hypothalamic NPY levels. In lean satiated rats CGP 71683A significantly antagonized the increase in food intake induced by intracerebroventricular injection of NPY. In 24-h fasted and streptozotocin diabetic rats CGP 71683A dose-dependently inhibited food intake. During the dark phase, CGP 71683A dose-dependently inhibited food intake in free-feeding lean rats without affecting the normal pattern of food intake or inducing taste aversion. In free-feeding lean rats, intraperitoneal administration of CGP 71683A for 28 d inhibited food intake dose-dependently with a maximum reduction observed on days 3 and 4. Despite the return of food intake to control levels, body weight and the peripheral fat mass remained significantly reduced. The data demonstrate that the NPY Y5 receptor subtype plays a role in NPY-induced food intake, but also suggest that, with chronic blockade, counterregulatory mechanisms are induced to restore appetite.

Angiotensin Blockade or Calcium Antagonists Improve Endothelial Dysfunction in Hypertension: Studies in Perfused Mesenteric Resistance Arteries

Summary Endothelial regulation of peripheral vascular resistance is impaired in hypertension. We studied the effects of different antihypertensive therapies on endothelial function in perfused mesenteric resistance arteries. Spontaneously hypertensive rats (SHR) aged 7 weeks were treated with either the nonpeptidic angiotensin II (AH) receptor antagonist CGP 48369, the angiotensin-converting enzyme (ACE) inhibitor benazepril HC1, or the calcium antagonist nifedipine (each 10 mg/kg/day orally, p.o.) for 8 weeks. All forms of therapy inhibited the increase in systolic blood pressure (SBP) to a comparable degree (18–23 mm Hg) and reduced but did not normalize medial hypertrophy in SHR. Changes in intraluminal vascular diameter to acetylcholine (ACh), norepinephrine (NE), and endothelin-1 (ET-1) were measured. Impaired endothelium-dependent relaxations to intraluminal ACh improved or normalized with all therapies, whereas the response to extraluminal ACh (which was unimpaired in SHR) remained unaffected. The endothelium-dependent inhibition of contractions to NE was lost in untreated SHR and improved or restored by antihypertensive therapy. In SHR, the sensitivity but not the maximal response of vascular smooth muscle (VSM) to ET-1 was paradoxically decreased. Antihypertensive therapy with CGP 48369, nifedipine, or benazepril HC1 restored or increased the sensitivity to ET-1. Thus, chronic blockade of the renin-angiotensin system or voltage-operated calcium channels reduces BP and improves endothelial dysfunction in the resistance circulation of SHR. This may contribute to normalization of peripheral vascular resistance during antihypertensive treatment and improve local blood flow to vital organs.

Valsartan, a potent, orally active angiotensin II antagonist developed from the structurally new amino acid series

Abstract Starting from the structure of DuP-753 and a 3-dimensional model of the pentapeptide Tyr-Ile-His-Pro-Ile, a series of new and highly potent antagonists has been designed where the imidazole moiety of the Du Pont compound has been replaced by an N-acylated aminoacid residue. VALSARTAN (Ex. 4e CGP48933, (S)-N-Valeryl-N-[2′-(1H-tetrazol-5-yl-)biphenyl-4-yl]methyl-valine), has been selected for clinical investigation.

Alcohol suppresses endothelium-dependent relaxation in rat mesenteric vascular beds.

The effects of prolonged infusions of ethanol on endothelium-dependent vasorelaxation induced by acetylcholine and adenosine triphosphate (ATP) and on endothelium-independent relaxation induced by papaverine were studied and compared in isolated perfused rat mesenteric artery preparations. Infusion of ethanol over 60 minutes at concentrations of 1.6, 4.7, 6.3, and 7.9 mg/ml caused concentration-related inhibition of norepinephrine-induced vasoconstriction. In preparations infused with 6.3 and 7.9 mg/ml, this effect reached a maximum after 10–20 minutes but had vanished by the end of the infusion; 1 hour after the end of the infusion, the effects of norepinephrine were potentiated by 71% and 108%, respectively. Acetylcholineinduced vasorelaxation (EC50 3.0 ng/ml in controls) was significantly reduced after 6.3 mg/ ml ethanol infusion and totally abolished after 7.9 mg/ml ethanol infusion. ATP-induced vasorelaxation (EC50 180 ng/ml in controls) was also abolished after 7.9 mg/ml of ethanol infusion. By contrast, the vasorelaxant effects of papaverine were not affected by 7.9 mg/ml ethanol infusion. Light-microscopic examination revealed that the endothelial cells were present in ethanol-treated and in control mesenteric arterial beds. These observations indicate that ethanol suppresses endothelium-dependent vasorelaxation without apparent removal of the endothelial cells. The compromised relaxant capacity of the endothelium after ethanol and the resultant intensification of the vasoconstrictor response to norepinephrine may contribute to the development of vascular diseases such as hypertension and stroke.

Binding characteristics and vascular effects of various angiotensin II antagonists.

Subtypes of angiotensin II (Ang II) receptors have been recently identified using specific ligands (see Whitebread et al. Biochem Biophys Res Comm 1989; 163:284-291). The present study compares the binding characteristics of different structural classes of Ang II receptor ligands in rat aortic smooth muscle cells (Ang IIB subtype) and in human uterus (Ang IIA subtype) and their effects on the constrictor response to Ang II in isolated rabbit aortic rings. Saralasin and [Sar1 Ile8]-Ang II displayed similar affinity for the two subtypes. In contrast, CGP 42112A bound with high affinity to the uterus (Ki 0.24 nM), but showed a low affinity for the aortic receptor (Ki 1,760 nM). Compound 89 displayed affinity for the aortic receptor only (Ki 26 nM) whereas Ex 169 recognized specifically the uterus receptor (Ki 310 nM). In rabbit aortic rings, saralasin, [Sar1 Ile8]Ang II, CGP42112A and compound 89 inhibited Ang II-induced contractions at concentrations similar to those required to bind to the Ang IIB receptor subtype. IC50s were 3, 0.7, 1,850, and 23 nM respectively. Ex 169 was ineffective in concentrations up to 100 microM. There was a highly significant correlation between inhibition of Ang II-induced contraction in aortic rings and binding to smooth muscle cells. This correlation does not exist with human uterus. Our results indicate that the Ang IIB receptor subtype is responsible for vascular contractions. Antagonists of this vascular receptor subtype are potential antihypertensive agents.

Renovascular hypertension impairs formation of endothelium-derived relaxing factors and sensitivity to endothelin-1 in resistance arteries

1 Endothelium‐dependent vascular regulation was investigated in mesenteric resistance arteries of Goldblatt two kidney‐one clip (2K1C) renovascular hypertensive rats. 2 Third order branches of mesenteric arteries were dissected free and mounted on glass cannulae in an organ chamber. Changes in vascular diameter were measured in pressurized and perfused arteries with a video dimension analyzer. 3 Acetylcholine evoked endothelium‐dependent relaxations that were much more pronounced with intraluminal than with extraluminal application. In 2K1C rats the relaxation induced by intraluminal, but not extraluminal acetylcholine was decreased compared to normotensive Wistar Kyoto rats (WKY). Increased duration of hypertension further decreased the response to intraluminal but not extraluminal acetylcholine. 4 Endothelin‐1 and noradrenaline caused contractions which were augmented by removal of the endothelium. This augmentation was reduced in 2K1C rats compared to WKY; the difference was small with noradrenaline but more pronounced with endothelin‐1. 5 In arteries without endothelium the sensitivity, but not the maximal contraction to endothelin‐1 was lower in 2K1C rats, while the response to noradrenaline was not different in 2K1C rats and WKY. The sensitivity to the peptide was not further affected by increasing the duration of hypertension. 6 Thus, renovascular hypertension leads to an impaired intraluminal, but not extraluminal activation of the release of endothelium‐derived relaxing factor and a decreased inhibitory effect of the endothelium against endothelin‐1‐ and noradrenaline‐induced contractions in mesenteric resistance arteries. Furthermore, the sensitivity, but not the maximal response of vascular smooth muscle to endothelin‐1 was reduced.

Design, synthesis and SAR of a series of 2-substituted 4-amino-quinazoline neuropeptide Y Y5 receptor antagonists.

The design of a novel series of NPY-Y5 receptor antagonists is described. Key elements for the design were the identification of weak Y5 hits from a Y1 program, results from a combinatorial approach and database mining. This led to the discovery of the quinazoline 4 and the aryl-sulphonamide moiety as major components of the pharmacophore for Y5 affinity. The synthesis and SAR towards CGP71683A is described.

Reactivity and sensitivity of mesenteric vascular beds and aortic rings of spontaneously hypertensive rats to endothelin: effects of calcium entry blockers

1 The vasoconstrictor effects of endothelin‐1 were studied in perfused mesenteric vascular beds (MVB) and aortic rings of 14–16 week‐old spontaneously hypertensive rats (SHR) and age‐matched Wistar Kyoto rats (WKY). 2 Reactivity to endothelin‐1 was increased in MVBs of SHR, as indicated by the maximum perfusion pressure obtained (264 ± 8 and 141 ± 9 mmHg respectively) (P < 0.001), whereas sensitivity was not significantly different between the two strains (EC50 171 ± 21 and 102 ± 19, respectively). 3 In aortic rings, in constrast, reactivity to endothelin‐1 was reduced in SHR as compared to WKY, whereas sensitivity was similar (EC50 0.78 ± 0.08 and 0.87 ± 0.09 nm). 4 As with endothelin‐1, reactivity to noradrenaline and potassium chloride was increased in MVBs, but not in aortic rings of SHR. Endothelin‐1 was 30 times more potent than noradrenaline in MVBs of SHR, and 15 times more potent than noradrenaline in aortic rings. 5 In both strains, nifedipine and nitrendipine almost completely blocked potassium‐induced contractions in MVB and aortic rings, respectively, whereas contractions induced by endothelin‐1 or noradrenaline were only partially inhibited. 6 It is concluded that calcium influx via the voltage‐operated calcium channel is only partially responsible for the vasoconstrictor action of endothelin‐1 in MVBs and aortic rings of SHR and WKY rats. The increased reactivity of the MVB of SHR to endothelin‐1 at this stage of the hypertensive process is most likely to be the result of a change in vascular structure rather than due to a primary hypertensive mechanism.

In vitro comparative studies of the calcium-entry activators YC-170, CGP 28392, and BAY K 8644.

Recently, the novel dihydropyridine derivates YC-170, CGP 28392, and BAY K 8644 have been reported to act in the opposite way to Ca2+ -entry blockers. We have found that these compounds inhibit the binding of [3H]nitrendipine on guinea pig heart membranes (Ki: 6 nM BAY K 8644, 115 nMCGP 28392 and 690 nM YC-170). Like those of nifedipine (Ki 1 nM), the curves had slopes close to unity, and. unlike those of some nondihydropryridine Ca2+ antagonists, were not altered in the presence of diltiazem, indicating a competitive interaction at dihydropyridine-sensitive sites. In isolated guinea pig atria, these agents exerted positively inotropic effects similar in their potency ratio to those observed in the binding experiments (BAY K 8644 1, CGP 28392 1:17, YC-170 1:600). The maximum inotropic effects of BAY K 8644 and CGP 28392, and of YC-170 corresponded respectively to two-thirds and one-third of those induced by isoprenaline or extracellular Ca2+ In the isolated rat mesenteric artery, perfused with a depolarizing solution, vasoconstrictor Ca2+ dose-response curves are shifted to the right by nifedipine. By contrast, BAY K 8644 and CGP 28392 caused a distinct leftward shift of the Ca2+ dose-response curves, at concentrations of 3–300 nM and 30–300 nM, respectively, and YC-170 a marginal shift at concentrations of 200–2000 nM, i.e., similar ranges to their inhibitory effects on [3H]nitrendipine binding. At higher concentrations, all three compounds produced Ca2+ -antagonistic effects. These results indicate that the compounds act at dihydropyidine-sensitive sites and exert partial agonistic activities in vascular and myocardial tissue.