Pharmacological characterization of SR 47436, a new nonpeptide AT1 subtype angiotensin II receptor antagonist.

SR 47436, 2-n-butyl-3-[(2'-(1H-tetrazol-5-yl)-biphenyl-4-yl) methyl]-1,3-diaza-spiro[4,4]non-1-en-4-one, is a new potent and selective AT1 angiotensin II (AII) receptor antagonist. It competitively inhibited [125I]AII binding to AT1 subtype receptors in rat liver membranes (IC50 = 1.7 nM) and did not interact with AT2 subtypes in rat adrenal cortical membranes. In rabbit aorta, SR 47436 inhibited contractions induced by 10 nM AII (IC50 = 4.0 nM) and shifted AII contractile response curves to the right in a parallel fashion, without total recovery of the maximal response. The potency of SR 47436 was higher than that of the lead compound, 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5- yl)biphenyl-4-yl)methyl]imidazole (DuP 753) (rat liver binding: IC50 = 16 nM; rabbit aorta: IC50 = 26 nM), and equivalent to saralasin (IC50 = 1.8 and 2.7 nM, respectively). The high specificity of SR 47436 was demonstrated by its lack of activity (IC50 > 10 microM) on various other receptors, ionic channels and antiports and rabbit aorta contracted by norepinephrine and KCl, and its lack of inhibition of renin and converting enzyme. In conscious rats, SR 47436 as well as DuP 753 (0.1 to 3 mg/kg, i.v., and 0.3 to 30 mg/kg, p.o.) antagonized the AII-pressor response in a dose-related manner. In conscious dogs, SR 47436 (1-10 mg/kg, p.o.) was a more potent antagonist of the AII pressor response than DuP 753. In conscious chronically implanted cynomolgus monkeys, SR 47436 antagonized the AII-pressor response at 1 mg/kg (89% i.v. and 66% p.o.) much more strongly than DuP 753 at 10 mg/kg (83% i.v. and 20% p.o.).(ABSTRACT TRUNCATED AT 250 WORDS)

SR 33557, a novel calcium-antagonist: interaction with [3H]-(+/-)-nitrendipine and [3H]-(-)-desmethoxy-verapamil binding sites in cerebral membranes.

We investigated the pharmacological properties of SR 33557, a novel compound with calcium-antagonist properties, in both functional tests in vitro and radioligand binding studies. SR 33557 potently antagonized calcium-induced contraction of potassium-depolarized rat aorta in vitro with an IC50 value of 5.6 +/- 0.9 nM, but was a much weaker inhibitor of noradrenaline-induced contraction of the same tissue (IC50 = 96 +/- 22 nM). SR 33557 totally inhibited [3H]-(+/-)-nitrendipine binding to rat brain membranes with a Ki value of 0.19 +/- 0.03 nM. Diltiazem, which used alone increases [3H]-(+/-)-nitrendipine binding, reversed this inhibition indicating that SR 33557 allosterically regulates [3H]-(+/-)-nitrendipine binding. SR 33557 also fully inhibited [3H]-(-)-desmethoxyverapamil binding to cerebral membranes, but inhibition curves were biphasic. IC50 value calculated for that part of the curve which reflects the high affinity binding site of SR 33557 (IC50 = 0.20 +/- 0.02 nM) was very similar to the Ki value determined for inhibition of [3H]-(+/-)-nitrendipine binding. Kinetic evidences indicate that SR 33557 binds to a site which is distinct from the 1,4-dihydropyridine or the phenylalkylamine binding sites associated with the calcium channel. To test the pharmacological specificity of these interactions, the ability of SR 33557 to interact with eight other receptors in cerebral or heart membranes was assessed by binding assays. No high-affinity interaction was observed between SR 33557 and any of the receptors investigated. We conclude that SR 33557 binds specifically and with a high affinity to a site closely associated with the voltage-operated calcium channel in cerebral membranes.

[Increase in plasma levels of active and inactive renin after inhibition of renin activity by SR 42128 in conscious Macaca monkeys]. / Augmentation des taux plasmatiques de rénine active et inactive après inhibition de l'activité rénine par le SR 42128 chez le macaque vigile.

Plasma active renin (PAR), plasma inactive renin (PIR), and plasma renin activity (PRA) were determined after intravenous bolus injection of the renin inhibitor SR 42128, in sodium repleted and sodium depleted macacas. The kit renin of Pasteur Diagnostics allows determination of PAR after renin inhibition by SR 42128. PAR and total plasmatic renin (TPR) were determined before and after treatment of plasma using trypsin. IR = TPR-PAR. ARP was measured by RIA of angiotensin I. Sodium depletion induced a dramatic increase of PAR (1,678 + 11.5 pg/ml compared to 94.4 + 11.5, n = 6). PIR rose from 322.1 + 34.3 pg/ml to 1,137 + 206 (n = 6). In sodium repleted macacas, SR 42128 (3 mg/kg and 9 mg/kg) induced a PRA inhibition of 90 to 100 p. 100, for 4 h post-injection. PAR increased to reach maximal level after 90 min and remained constant up to 4 h post-injection (increase of 420 p. 100 at 3 mg/kg and 620 p. 100 at 9 mg/kg). PIR increased more slowly for 4 h (maximum increase of 250 p. 100). PRA was also inhibited in sodium depleted macacas by SR 42128 at the doses of 3 mg/kg and 9 mg/kg ARP was inhibited. PIR increased more slowly, but significantly at 9 mg/kg. We conclude that the activity of SR 42128 on PAR and PIR levels is the sole consequence of the inhibition of the Renin Angiotensin system.

A solid-phase radioimmunoassay using digitonin-permeabilized cells to screen surface or intracellular membrane-bound antigens.

Membrane-bound receptor or enzyme distribution between cell surface and cell interior can be determined using the non-ionic detergent digitonin. A solid-phase radioimmunoassay using myocardial cells from newborn rats was performed to screen hybridoma culture supernatants and the cells were rendered permeable with increasing concentrations of digitonin (0-0.1%). This was achieved in 5 min at 37 degrees C and did not require the continuous presence of detergent. A characteristic amount of cytoplasmic protein (approximately equal to 45%) was released with subunit molecular weights of up to 200,000. This allowed exogenous molecules such as immunoglobulin G to gain access to the intracellular targets. The binding to rat myocardial cells of 36 monoclonal antibodies was examined by this procedure. The binding assays were carried out for 30 min at 37 degrees C using cells previously treated for 10 min with 0.05% of digitonin. This appears to be a simple and rapid method of screening and selecting the hybridoma culture supernatants.

Inhibition of platelet phospholipase-A2 as a mechanism for the anti-aggregating effect of linoleic acid.

Linoleic acid was incorporated into platelet phospholipids and then released after activation of phospholipase A2 (PL-A2) with thrombin or ionophore A23187. The rate of this release was tenfold lower for linoleic than for arachidonic acid. This observation strongly suggests that incorporation of linoleic acid in platelet phospholipids might inhibit platelet PL-A2 and might explain the anti-aggregating effect of linoelic acid. In fact it has also been shown that linoleic acid inhibits PL-A2 activity at concentrations which antagonize platelet aggregation. Moreover, in experimental conditions where platelet cyclo-oxygenase is totally inhibited, aggregation does occur and can still be blocked by linoleic acid. This latter observation led to the conclusion that the anti-aggregating effect of linoleic acid is independent from prostaglandin pathway and is probably related to the phospholipid metabolism.

Anti-inflammatory and platelet anti-aggregant activity of phospholipase-A2 inhibitors.

Mepacrine, papaverine, p-bromophenacyl bromide and 2,3-dibromo(4'-cyclohexyl-3'-chloro)-phenyl-4-oxo-butyric acid (CB 874) inhibit the hydrolysis of phospholipids induced by thrombin in dog platelets. They also exhibit anti-inflammatory and anti-aggregant properties. These biological activities may be explained by a direct or indirect inhibitory action on phospholipase A2. Phospholipase A2 inhibitors may block not only the release of arachidonic acid and its subsequent conversion into prostaglandins but also the formation of lysophospholipids involved in inflammation and/or platelet aggregation.