Pharmacological profile of GR117289 in vitro: a novel, potent and specific non‐peptide angiotensin AT₁ receptor antagonist

Abstract

1 This paper describes the effects of GRI17289 (1-[[3-bromo-2-[2-(lH-tetrazol-5-yl)phenyl]-5-benzofuranyl]methyl]-2-butyl-4-chloro-lH-imidazole-5-carboxylic acid) at angiotensin receptors and binding sites in rabbit aorta, rat liver and bovine cerebellum preparations in vitro. 2 In rabbit isolated aortic strips, GRI 17289 (0.3, 1 and 3 nM) caused a concentration-related, insurmountable suppression of the concentration-response curve to angiotensin II (All). When the contact time was increased, a greater degree of antagonism of All was observed, suggesting that GRi17289 is slow to reach equilibrium. A pKB of 9.8±0.1 was calculated for GRI17289 after 3h incubation.GR1 17289 (1 tiM) did not affect contractile responses to phenylephrine or 5-hydroxytryptamine (5-HT) in the rabbit aorta. 3 GRI 17289 (1 nM) alone caused a marked suppression and a slight rightward displacement of the All concentration-response curve. Co-incubation with the competitive, surmountable AT, receptor antagonist, losartan (10 nM, 100 nm and 1 ,LM), resulted in a concentration-related upward and rightward displacement of the concentration-response curve to subsequently administered All. In separate experiments in which preparations were pre-incubated with GR1 17289 (1 nM), subsequent addition of losartan (1 JLM) for 2, 15 or 45 min caused a further, but similar, rightward displacement of the concentration-response curve to subsequently administered All with a time-dependent increase in the maximum response. 4 Suppression of All-induced contractile responses, caused by superfusion with GRI17289 (0.3, 1 or 3 nM) was not reversed by continuously washing the tissues for 3 h; in fact, the potency of GRI 17289 was slightly enhanced after this period.5 In rat liver membranes, GRI17289 was a potent competitor with [3H]-AII for AT, binding sites (pKi = 8.7 ± 0.1) but in bovine cerebellum membranes, it was a very weak competitor for AT2 binding sites (pKi<6). Pre-incubation of rat liver membranes with GRI17289 had little effect on its affinity (pKi = 9.1 ± 0.21), but increasing the concentration of bovine serum albumen in the assay buffer from 0.001% to 0.1% w/v decreased affinity (pKi= 7.5 ± 0.1). 6In saturation binding experiments in rat liver membranes, GRI 17289 (12 nM) increased the Kd of[3H]-AII from 0.28 ± 0.06 nM to 0.37 ± 0.02 nM, and decreased Bm. from 10.0 ± 0.1 to 5.6 ± 0.3 fmol mg' tissue. In other experiments, GR1 17289 (1 jIM) did not alter the rate of dissociation of[3H]-AII from AT1 binding sites, following addition of excess unlabelled All.7 In rabbit aorta vascular smooth muscle membranes, GR1 17289 competed with ['25I]-Sar'1le8 All for binding to AT, binding sites. In the presence of 0.1% w/v bovine serum albumen, a pIC50 of 7.6 ± 0.1 was calculated. Under the same conditions, but with rat liver membranes, a pIC50 of 7.8 ± 0.1 was determined.8 Taken together, these results show that GRI17289 is a potent, specific, selective and insurmountable antagonist at angiotensin AT, receptors. Its profile in the rabbit aorta is consistent with ...

Distinction between surmountable and insurmountable selective AT1 receptor antagonists by use of CHO‐K1 cells expressing human angiotensin II AT1 receptors

“…This suggests that the equilibrium binding between this antagonist and the receptors is already attained after a very short incubation time. In agreement, the slope of the Schild regression analysis for losartan is close to unity (0.92+0.05) and the pK B -value (7.90+0.06) is comparable to those (8.2 ± 8.5) reported in earlier contraction studies (Robertson et al, 1992;Renzetti et al, 1995;Mochizuki et al, 1995).…”

“…The molecular basis for insurmountable antagonism is still a matter of debate. Although the longevity of the antagonist-receptor complex is retained as the cause in many studies, insurmountable antagonism has also been explained by the presence of allosteric binding sites on the receptor (Timmermans et al, 1991); slowly interconverting receptor conformations (de Cha oy de Courcelles et al, 1986;Robertson et al, 1994); slow removal of the antagonist from tissue compartments, cells or matrix surrounding the receptor (Robertson et al, 1992;Panek et al, 1995) and even by the ability of the antagonist to modulate the amount of internalized receptors (Liu et al, 1992).…”

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

“…Repeated oral administration On the first day of administration of doses of 3 or 10mgkg-' (Figure 1la), the blood pressure-lowering effect was similar to that seen in the singledose experiment (Figure 9a) (Wong et al, 1990c;Robertson et al, 1992), the rightward displacement of the AII concentration-response curve induced by valsartan was accompanied by a reduction in the maximal response. In contrast, no reduction in the maximum contractions to All has been observed in rabbit aorta treated with losartan (Chiu et al, 1991b).…”

“…In the isolated aortic rings, the conventional Schild analysis for estimation of potency could not be used because valsartan reduced the maximum response to All. Instead, apparent pKB values were derived by using a double-reciprocal regression plot as described by Kenakin (1984) and Robertson et al (1992) (Figure 2). …”

Cardiac and gastric effects of histamine H2 receptor antagonists: no evidence for a correlation between lipophilicity and receptor affinity

“…In accordance with this finding, a significantly longer time was required for full development of the histamine responses in the presence of these drugs. A slow dissociation from receptors has been proposed to explain the insurmountable antagonism induced by angiotensin non-peptide antagonists at AT, receptors (Robertson et al, 1992), whereas an allosteric interaction might explain the insurmountable antagonism by methysergide at vascular 5-HT receptors (Kaumann & Frenken, 1985). Moreover, conformational changes in the receptor induced by the insurmountable antagonist could reduce the efficiency of stimulus-response coupling, according to the allosteric model proposed by de Chaffoy de Courcelles et al (1986).…”