Assays to measure nanomolar levels of the renin inhibitor CGP 38 560 in plasma.

Abstract

A radioinhibitor binding assay and an enzyme inhibition assay have been developed to measure plasma levels of CGP 38 560, a potent human renin inhibitor. The detection limit of the assays was between 0.5 and 1 pmol/ml. There was a good correlation (r=0.989) between the two assays for the measurement of human plasma spiked with CGP 38 560 in concentrations from 1.9 nM to 12 fiM. Intra-assay variability was 6.1-17.3% and 4.4-27.2% for the radioinhibitor binding assay and the enzyme inhibition assay, respectively. Interassay variability was 6.0-28.2% and 3.8-28.4% for the radioinhibitor binding assay and the enzyme inhibition assay, respectively. Blood samples were collected during a pharmacological study performed in normotensive human volunteers on an unrestricted diet who were infused during a 30-minute period with T he renin-angiotensin system plays a major role in the regulation of blood pressure and in the maintenance of sodium and volume homeostasis. The enzymatic cleavage of angiotensinogen by human renin (EC 3.4.23.15) generates a decapeptide, angiotensin I, which is immediately hydrolyzed by a carboxydipeptidyl hydrolase, the angiotensin converting enzyme (EC 3.4.15.1), to the octapeptide angiotensin II, a potent vasoconstrictor. Renin has only one known substrate, angiotensinogen, and therefore provides an interesting target for the design of specific blockers of the renin-angiotensin system. During recent years, several specific and potent inhibitors of primate renin have been synthesized and characterized in vitro and in vivo. The in vitro studies have demonstrated the potency and specificity of the compounds against renin from different species and other aspartic proteinases, 1 and the in vivo studies have demonstrated the effects of renin inhibitors on blood

Structural Modification of the P2‘ Position of 2,7-Dialkyl-Substituted 5(S)-Amino-4(S)-hydroxy-8-phenyl-octanecarboxamides: The Discovery of Aliskiren, a Potent Nonpeptide Human Renin Inhibitor Active after Once Daily Dosing in Marmosets

“…This inhibitor also revealed a very prolonged activity, with MAP being still lowered by −10 mmHg at the 24 h time point and, hence, appeared to be one of the longest acting inhibitors in this primate model. The absolute oral bioavailability of 46 in non-Na + -depleted marmosets, dog, and rat was determined to be 16, 32, and 2.4%, respectively, as determined from plasma concentrations using an enzymatic assay. − All analogues bearing an acidic functionality at P2‘ (cf. Table ) were completely inactive in vivo, suggesting a poor bioavailability profile in marmosets.…”

“…The absolute oral bioavailability of 46 in non-Na + -depleted marmosets, dog, and rat was determined to be 16, 32, and 2.4%, respectively, as determined from plasma concentrations using an enzymatic assay. [45][46][47] All analogues bearing an acidic functionality at P2′ (cf. Table 3) were completely inactive in vivo, suggesting a poor bioavailability profile in marmosets.…”

Novel 2,7-Dialkyl-Substituted 5(S)-Amino-4(S)-hydroxy-8-phenyl-octanecarboxamide Transition State Peptidomimetics Are Potent and Orally Active Inhibitors of Human Renin

“…The absolute oral bioavailability for 38a in marmosets was 16%, as estimated from plasma inhibitor concentrations (and possibly of any circulating potent in vivo metabolites) after 3 mg/kg intravenous and 10 mg/kg oral administration by using a renin activity assay . The terminal elimination half-life t 0.5 for 38a was 2.0 ± 0.06 h, with biphasic elimination kinetics (first distribution−elimination phase followed by a second elimination phase).…”

Renin release regulation during acute renin inhibition in normal volunteers.

“…15 For total renin, 3E8 was the immobilized antibody, and the labeled antibody was 3-16-16 (CIBA-GEIGY), which recognizes total renin. 8 Circulating levels of Ro 42-5892 were determined by the radioinhibitor binding assay of Cumin et al, 16 using [ 100 /iM MK 422. 17 The tubes were centrifuged at 4°C, and 2.2-ml plasma aliquots were immediately extracted on phenylsilylsilica columns (Bondelut PH, Analytichem) according to Nussberger et al 18 The dried extracts containing angiotensins were diluted in 220 Ail of 0.1 M (pH 7.5) Tris HC1 buffer containing 2 g/1 bovine serum albumin and kept frozen at -80°C.…”