David John Carini

Identification of angiotensin II receptor subtypes

We have demonstrated the existence of two distinct subtypes of the angiotensin II receptor in the rat adrenal gland using radioligand binding and tissue section autoradiography. The identification of the subtypes was made possible by the discovery of two structurally dissimilar, nonpeptide compounds, DuP 753 and EXP655, that show reciprocal selectivity for the two subtypes. In the rat adrenal cortex, DuP 753 inhibited 80% of the total AII binding with an IC50 value on the sensitive sites of 2 x 10(-8) M, while EXP655 displaced only 20%. In the rat adrenal medulla, EXP655 gave 90% inhibition of AII binding with an IC50 value of 3.0 x 10(-8) M, while DuP 753 was essentially inactive. The combination of the two compounds completely inhibited AII binding in both tissues.

Hypotensive action of DuP 753, an angiotensin II antagonist, in spontaneously hypertensive rats. Nonpeptide angiotensin II receptor antagonists: X.

In conscious 18-21-week-old spontaneously hypertensive rats, DuP 753, a nonpeptide angiotensin II receptor antagonist, given orally at 3 and 10 mg/kg or intravenously at 3, 10, and 30 mg/kg, reduced blood pressure dose dependently. It did not alter heart rate at these doses. At 10 mg/kg i.v., DuP 753 decreased blood pressure significantly for at least 24 hours, suggesting a long duration of the antihypertensive effect. Unlike saralasin, DuP 753 did not cause a transient increase in blood pressure. The acute antihypertensive efficacy of DuP 753 was greater than that of captopril. Our data indicate that, for captopril to reduce blood pressure to a similar extent as that of DuP 753, it would need to be supplemented by a diuretic. DuP 753 did not have an acute diuretic effect. Bilateral nephrectomy, but not inhibition of prostaglandin synthesis, abolished the antihypertensive effect of DuP 753, suggesting that the antihypertensive effect of DuP 753 is dependent on an active renin-angiotensin system. Furthermore, DuP 753 inhibited the pressor response to angiotensin II but not the responses to norepinephrine, vasopressin, and Bay K 8644 (a calcium agonist). As neither DuP 753 nor captopril decreased blood pressure acutely in Wistar-Kyoto normotensive rats, our results suggest that the renin-angiotensin system plays a significant role in the control of blood pressure in spontaneously hypertensive rats.

Discrimination of angiotensin II receptor subtypes by dithiothreitol

It was of interest to evaluate whether DTT has a disciminatory effect on these two types of AII receptor in rat adrenal cortex. In this study, rat isolated adrenal cortical microsomes were treated for 20 min with either 5 mM DTT

Nonpeptide angiotensin II receptor antagonists. Studies with EXP9270 and DuP 753.

A series of nonpeptide angiotensin II (Ang II) receptor antagonists was evaluated in rat adrenal cortical microsomes for their inhibitory effects on the specific binding of [3H]Ang II, in the isolated rabbit aorta bioassay for their functional antagonism of contractile response to Ang II, and in high renin, renal-hypertensive rats for their intravenous antihypertensive effects, expressed as IC50, pA2, and intravenous ED30, respectively. Highly significant linear correlations were found between ICM and pAj (r = −0.88), between IC50, and intravenous ED30 (r = 0.79), and between pA2 and intravenous EDM (r = −0.93). In both in vitro and in vivo functional assays, none of these antagonists exhibited agonistic effects. The orally active nonpeptide Ang II receptor antagonists EXP9270 and DuP 753 (oral ED30=3.6 and 0.59 mg/kg, respectively) were selected for further characterization. These antagonists exhibited selective and competitive Ang II antagonism in rabbit aorta and guinea pig ileum. In conscious normotensive rats, DuP 753 abolished the pressor response to saralasin, suggesting that the pressor effect of saralasin is attributed to its Ang II-like activity. In addition, DuP 753 also blocked the Ang II-induced drinking response and aldosterone release in rats. These results suggest that Ang II receptor blockade is the primary mechanism of the antihypertensive effect of these nonpeptide Ang II receptor antagonists. Further, the specificity and lack of partial agonistic effects of these molecules make them potentially useful physiological probes and therapeutic agents.

Nonpeptide angiotensin II receptor antagonists.

Although the most direct way to interfere with the renin-angiotensin system (RAS) is at the level of the angiotensin II (AII) receptor, the currently available AII receptor antagonists are peptides still retaining significant agonistic properties with the obvious drawbacks of limited stability and lack of oral activity. We have characterized simple N-benzylimidazoles as weak, but selective AII receptor antagonists with a competitive mode of action. Chemical modification of these early leads led to EXP6155 and EXP6803, which show approximately 10- and 100-fold higher affinity. Oral activity was obtained for EXP7711, and in particular for EXP9654. This class of compounds displaces 3H-AII from its specific binding sites in various tissues. They competitively antagonize AII-induced responses in various in vitro and in vivo preparations, but do not influence AII-induced responses to KCl, norepinephrine, and vasopressin, nor do they affect converting enzyme or renin. In high renin models of elevated blood pressure, such as the renal hypertensive rat and sodium-depleted dog, these substances produce a sustained decrease in arterial pressure without changing heart rate after intravenous and oral (EXP7711 and EXP9654) administration. None of these compounds showed agonistic activity in any of the above test systems. In conclusion, the nonpeptide structures described herein are selective and competitive AII receptor antagonists and add another dimension to the arsenal of drugs manipulating the RAS.

Non-peptide angiotensin II receptor antagonists. II . Pharmacology of S-8308 ☆

2-Butyl-4-chloro-1-(2-nitrobenzyl)imidazole-5-acetic acid, sodium salt (S-8308), inhibited the specific binding of labeled angiotensin II (AII) to its receptor sites in rat adrenal cortical microsomes and in cultured aortic smooth muscle cells with IC50S of 15 and 4.5 microM, respectively. In the presence of S-8308 (15 microM) the dissociation constant for AII was increased 2-fold and the total number of binding sites was unaltered. In a concentration-dependent manner S-8308 blocked the 45Ca2+ influx induced by AII (3 X 10(-8) M) in rat aortic rings (IC50 7 microM) and the contractile response in rabbit aorta was competitively inhibited (pA2 = 5.74). This agent was highly specific for AII: it showed no affinity for alpha 1-adrenoceptors or Ca2+ channels and in addition, it did not alter the contractile responses to norepinephrine (10(-7) M) or KCl (55 mM). In conscious renal artery-ligated rats, S-8308 (30 mg/kg i.v.) elicited a rapid decrease of mean arterial pressure with a duration of about 30 min. The results demonstrate that S-8308 is a weak, but specific and competitive, non-peptide antagonist of AII exerting its inhibitory action at the receptor level.

DuP 532: a second generation of nonpeptide angiotensin II receptor antagonists.

DuP 532 is a novel nonpeptide angiotensin II (AII) receptor antagonist under development for the treatment of hypertension. DuP 532 is a more potent antihypertensive agent in renal hypertensive rats (ED30 = 0.042 mg/kg, i.v.) and displays a similar or longer duration of action than the previously described AII antagonist, DuP 753. DuP 532, in contrast to DuP 753, is a noncompetitive antagonist of AII-induced contractions of rabbit aortic strips (KB = 1.1 x 10(-10) M). However, the inhibition of AII binding by DuP 532 in rat adrenal cortex does not correlate with either the aortic contractile response or with the hypotensive response. Assay conditions were evaluated and the presence or absence of BSA was shown to markedly affect the apparent binding affinity of DuP 532 and other 5-carboxylic acid derivatives. DuP 753 and other compounds were much less affected. The IC50 for DuP 532 was 4.7 x 10(-6) M with and 3 x 10(-9) M without BSA. The IC50s for DuP 753 were 1.7 x 10(-8) M with and 5 x -9 M without BSA. Both compounds with or without BSA did not completely inhibit AII binding which is characteristic of AT1 selectivity. BSA also reduced the effect of DuP 532 on the AII-induced contractions of rat main pulmonary artery preparations and the AII-induced Ca2+ mobilization in rat aortic smooth muscle cells. DuP 532 was very specific for AT1 receptors and did not interfere with receptors associated with neurotensin, prazosin, bradykinin, nitrendipine, or vasopressin. It is concluded that DuP 532 represents a new class of specific, but noncompetitive. AII receptor antagonists whose binding characteristics may provide new insight into AII receptor function.

Potent imidazole angiotensinII antagonists: acyl sulfonamides and acyl sulfamides as tetrazole replacements

Abstract Acyl sulfonamides and acyl sulfamides were synthesized and their in vitro and in vivo biological properties evaluated. AT1 binding affinities for these potent AII antagonists were similar to their tetrazole analogs. An enhancement in AT2 potencies was observed, particularly with acyl sulfonamides or sulfamides bearing hydrophobic substituents.

The Discovery and Development of Angiotensin II Antagonists

During the development of losartan, various issues arose that could have stopped losartan and hampered the successful development of the angiotensin II antagonists in general. Some of these issues were real problems that had to be solved if losartan was to advance, such as the need for a commercially viable synthesis. Other issues, such as whether EXP3174 would form in humans, were anticipated as potential problems that should be addressed. Because of the combined efforts of discovery and development groups at DuPont, and later at Merck and DuPont Merck, the development of losartan was rapid. While some people were working to answer questions such as the antihypertensive efficacy of losartan in humans as quickly as possible, other people were searching for potential backups to losartan. Without an efficient integration of Discovery and Development efforts, the commercially successful development of any drug would be threatened, and it helped to give losartan a critical 2-year lead on the most advanced competitive Ang II antagonists.

The preparation of (perfluoroalkyl)imidazoles as nonpeptide angiotensin II receptor antagonists

Abstract A series of (perfluoroalkyl)imidazoles have been prepared and are potent angiotensin II antagonists. One of these compounds, DuP 532, 4-pentafluoroethyl-2-propyl-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole-5-carboxylic acid, is a selective antagonist of angiotensin II which causes a marked and long-lasting drop in blood pressure when administered orally to a renal hypertensive rat.