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To determine the concentration of renin inhibitors in plasma or other biological fluids, we developed an original approach to the already existing methods, such as HPLC or radio-inhibitor binding and enzyme inhibitor assays. We made an antigen-antibody complex by linking human renin (0.8 nM) to a specific monoclonal antibody fixed to magnetic spheres (3E8-magnogel). A binding technique with 3H-43845 (4 nM) was applied to this preparation to assess the association and dissociation kinetics of SR 43845, a highly potent and specific renin inhibitor. Standard curves were built and CI50 was 0.53 +/- 0.07 nM (n = 5). One of the application of this method is the biochemical characterization of renin inhibitors. We close several various chemical structure renin inhibitors having the inhibiting activity of 0.01 to 10,000 nM (CI50 human PRA). Preliminary results show a good correlation (r = 0.97; n = 12) with those obtained in plasma. In other respects, this new assay was applied to determine SR 43845 plasma concentrations in human pharmacokinetic studies and in primate pharmacological studies. The limit of detection was 0.09 ng/ml. Finally, it is now possible to perform a relationship between plasma concentrations of renin inhibitor and blood pressure changes or other biochemical parameters changes to allow a better understanding to the renin-angiotension system.

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Pepstatin analogues corresponding to the general formula A-X-Y-Sta-Ala-Sta-R were synthesized in solution phase. Various changes in the nature of the A, X, and Y groups were made to improve the inhibitory potency against human plasma renin activity. The results were interpreted by use of the active-site model based on the sequence of human angiotensinogen. The tert-butyloxycarbonyl group and the isovaleryl group were found to be the most effective acyl groups (A). The analogues having a Phe residue in place of Val1 (X) and His or amino acid with an aliphatic side chain such as norleucine or norvaline in the Y position showed the highest inhibition of human plasma renin activity with IC50 values of about 10(-8)M. Esterification or amidification of the carboxyl group of the C-terminal statine did not change the inhibitory potency. The selectivity for rat, dog, pig, and monkey plasma renin of the most interesting compounds was studied.

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A dose-response relationship was involved after an intravenous bolus of a human antirenin monoclonal antibody (4G1D8), in sodium depleted marmosets. The sodium depletion (furosemide: 30 mg/kg/d for 2 days) was used to potentiate the contribution of the renin-angiotensin system in the blood pressure (BP) control. To record BP and inject the antibody, 2 catheters were implanted the day before the experiment. The plasma renin activity (PRA) was measured by the RIA of angiotensin I after an incubation of plasmas for 1 hour at pH 7.4. The sodium depletion induced a dramatic increase of PRA (63.68 +/- 20.03 ng/ml/h of angiotensin I compared to 2.96 +/- 1.03; p less than or equal to 0.01; n = 13). The basal BP was 102.6 +/- 2.4 mmHg (n = 17). The maximal fall in BP was noted at about 30 min for the three groups of animals treated by 4G1D8; it was -7.5 +/- 4.3 mmHg at the dose of 0.01 mg/kg (n = 4), -21.3 +/- 3.8 mmHg (p less than or equal to 0.01) at 0.10 mg/kg (n = 4), and -27.5 +/- 1.4 mmHg (p = 0.10) at 0.24 mg/kg (n = 4). At the 0.10 and 0.24 mg/kg doses, the hypotension was lasting (greater than 3 h). PRA was strongly inhibited and HR was little modified. A dose-response relationship with a human antirenin monoclonal antibody, 4G1D8, provides a very interesting pharmacological model for a comparative study of renin inhibitors.

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We studied the haemodynamic effects of captopril [3 mg/kg intravenously (i.v.)] and SR 42128 (8 mg/kg in a 30-min perfusion) in the conscious baboon after sodium depletion by furosemide. The evolution of the following parameters was studied: plasma renin activity (PRA), heart rate (HR), mean arterial pressure (MAP), cardiac output (CO), first derivative of the left intraventricular pressure (dP/dt) and total peripheral resistance (TPR). Captopril (n = 5) increased PRA twofold and decreased MAP and TPR. Heart rate, CO and dP/dtmax were not significantly modified. As compared with a control group (n = 5), SR 42128 (n = 5) decreased PRA to almost undetectable levels for at least 2 h and decreased MAP, CO and TPR. It did not alter HR or dP/dtmax. Thus we can conclude that SR 42128 is a long-acting inhibitor of circulating renin in baboons and, in our experimental conditions, SR 42128, like captopril, induced a decrease in blood pressure without detrimental effect on cardiac function.

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