Appraisal of a glycopeptide cloaking strategy for a therapeutic oligopeptide: glycopeptide analogs of the renin inhibitor ditekiren.

Among the limitations to the practical therapeutic oligopeptide are low oral availability, indifferent aqueous solubility, and an astonishing efficient sequestration and biliary elimination by a multi-capacity liver transporter. Given the purposed use of N- and O- linked saccharides as functional appendages of eukaryotic peptides and proteins, a strategy of glycopeptide mimicry was examined for the oligopeptide renin inhibitor, ditekiren. The anticipation was that the saccharide would impart significant aqueous solubility, and might impact beneficially on the remaining two limitations. Execution of this approach was achieved by the removal of the (dimethylethoxy)carbonyl amino terminus of ditekiren, and its substitution by Boc-L-asparagine N-linked mono- and disaccharides. Potent hypotensive activity, as measured by a human renin-infused rat assay, is observed for virtually all of these structures (N-linked beta-pyranose D-N-acetyglucosaminyl, D-glucosaminyl, D-N-acetylgalactosaminyl, D-mannosyl, D-galactosyl, D-maltosyl, D-cellobiosyl, D-chitobiosyl, but not L-fucosyl). The basis for this dramatic improvement (relative to ditekiren in the same assay) is the diversion of the peptide clearance from rapid liver biliary clearance to slower urinary clearance (Fisher, J. F.; Harrison, A. W.; Wilkinson, K. F.; Rush, B. R.; Ruwart, M. J. J. Med. Chem. 1991, 34, 3140). Guided by the human renin-infused rat hypertension assay, an evaluation of the linker-saccharide pairing was made. Loss of hypotensive activity is observed upon substitution of the Boc-L-asn by Boc-D-asn, and by removal of the Boc amino terminus of the glycopeptide. Potent hypotensive activity is preserved by replacement of the Boc-L-asn linker by succinate, malate, tartrate, and adipate linkers. With the longer adipate spacer, attachment of the saccharide to the P-3 phenylalanine--with omission of the P-4 proline--retains activity. These data suggest value to the glycopeptide guise for preserving the in vivo activity, and for the beneficial manipulation of pharmacodynamics, of this renin inhibitory oligopeptide. This strategy may have general applicability.

Hormonal and cardiovascular effects of losartan (DuP753), an angiotensin receptor antagonist, in nonhuman primates.

Experiments were carried out in conscious dexamethasone-treated cynomolgus monkeys in an attempt to determine if losartan (DuP753) inhibited endogenous as well as exogenous angiotensin II stimulation of angiotensin receptors in vivo. Arterial blood pressure and heart rate were monitored via a radiotelemetry system and blood samples were obtained via vascular access ports for determination of plasma renin activity and plasma aldosterone (PA). Intravenous infusions of angiotensin II elicited reproducible pressor responses and increases in PA. The elevations of blood pressure and PA were completely blocked by losartan (10 mg/kg i.v.). Studies with normal monkeys showed that losartan (10 mg/kg i.v.) elicited a modest hypotension and hyper-reninemia without significantly altering PA. In contrast, studies with sodium-depleted monkeys showed that losartan (1 and 10 mg/kg i.v.) elicited hypotension, a decrease in PA and hyper-reninemia in a dose-related manner. Losartan did not significantly alter heart rate in either the normal or the sodium-depleted monkeys. These results were consistent with the conclusion that losartan was an inhibitor of endogenous as well as exogenous angiotensin II stimulation of vascular smooth muscle and adrenal cortical receptors.

Renin release induced by losartan (DuP 753), an angiotensin II receptor antagonist.

The AT2 angiotensin receptor antagonist, PD123177, did not elicit plasma renin activity (PRA) or blood pressure effects in conscious unrestrained normal rats at a dose of 30 mg/kg iv. In contrast, losartan (DuP 753), a nonpeptide AT1 angiotensin receptor antagonist, elicited dose-dependent increases in PRA. PRA increased between five- and fifty-fold after intravenous administration of 1-10 mg/kg in the absence of changes in blood pressure. At 3 mg/kg iv, losartan induced a twenty-fold increase of PRA which was of renal origin inasmuch as bilateral nephrectomy blocked the effect. Cyclooxygenase blockade with indomethacin or meclofenamate did not alter losartan-induced renin release at 3 mg/kg iv and suggested that the hyperreninemia was not mediated by renal prostaglandins. The nonselective beta-blocker propranolol and the beta 1 selective blocker atenolol attenuated losartan-induced renin release approximately 70 and 80% respectively without altering blood pressure. These results were consistent with a modulation of renin release by sympathetic nerve activity via beta-adrenergic receptors. The findings suggest that losartan interferes with the ability of angiotensin II to suppress that renin release which is mediated by sympathetic nerve activity.

Renin inhibitory peptides. Incorporation of polar, hydrophilic end groups into an active renin inhibitory peptide template and their evaluation in a human renin infused rat model and in conscious sodium-depleted monkeys.

We previously reported that Boc-Pro-Phe-N-MeHis-Leu psi [CHOHCH2]-Ile-Amp (1) is a potent and specific inhibitor of human renin in vitro. It was shown to resist degradation by selected proteases and a rat liver homogenate. It was shown to inhibit plasma renin activity and to reduce blood pressure in renin-dependent-animal models both by the intravenous and by the oral routes using dilute citric acid as vehicle. In an effort to discover compounds with improved pharmacological efficacy, we set out to modify the physical characteristics of this highly lipophilic renin inhibitor by incorporation of hydrophilic end groups. We report here a variety of water-solubilizing groups and the resulting structure-activity relationship of these compounds. They all maintain an extremely high level of enzyme inhibitory activity in vitro. Evaluation of these potent renin inhibitors in a human renin infused rat model suggests that some of these compounds exhibit improved pharmacological efficacy in vivo. This observation was further confirmed in the conscious sodium-depleted cynomolgus monkey. Importantly, the oral efficacy was demonstrated in a water vehicle in the absence of citric acid.

Additive combination studies of captopril and ditekiren, a renin inhibitor, in nonhuman primates.

Additive combination studies of an angiotensin converting enzyme (ACE) inhibitor, captopril, and a renin inhibitor, ditekiren (U-71038), were carried out in conscious sodium-depleted and sodium replete cynomolgus monkeys. The agents elicited dose-additive hypotensive responses regardless of the order of drug administration in sodium-depleted monkeys. A dose-additive blood pressure response was also observed when the administration of captopril was preceded by ditekiren in conscious sodium replete monkeys. None of the animals in these groups exhibited significant alterations of heart rate. An apparent over-additive hypotensive response, accompanied by tachycardia, occurred in sodium replete monkeys when ditekiren was administered after captopril. It was proposed that the captopril-induced hyperreninemia may have allowed the blood pressure to become partially renin-dependent and therefore susceptible to the inhibitory action of ditekiren. The results of these studies suggested that both ditekiren and captopril elicited cardiovascular effects in conscious cynomolgus monkeys via a decreased formation of angiotensin II.

Potent renin inhibitory peptides containing hydrophilic end groups.

A previously reported renin inhibitor, Boc-Pro-Phe-N(Me)His-Leu psi [CHOHCH2]Val-Ile-Amp (U-71038), was altered by the incorporation of polar, hydrophilic moieties at either end, e.g., tris(hydroxymethyl)aminomethane (THAM) or glucosamine urea groups at the N-terminus, and the THAM amide or aminomethylpyridine N-oxide at the C-terminus. These modified analogues, with dramatically improved water solubility, all retained the potent renin inhibitory activity of U-71038 in vitro. The fact that good activity was maintained in these new analogues, which possess hydrophilicity and steric bulk considerably different from the parent compound, suggests that neither end of these molecules is critical for recognition and binding of the inhibitors by renin. These modified analogues were evaluated in a rat model, and several exhibited hypotensive activity after both oral and iv administration which was greater in magnitude and longer in duration than that caused by equimolar doses of U-71038. Furthermore, unlike U-71038, the oral activity of these analogues was not dependent upon administration in a citric acid vehicle.

Rat model for evaluating inhibitors of human renin.

A rat model that provides a rapid method for the in vivo evaluation of potential inhibitors of human renin has been developed and validated. Recombinant human renin was infused intravenously into anesthetized, nephrectomized, ganglion-blocked rats. The resulting blood pressure had an approximate 60 mm Hg human renin-dependent component. The angiotensin I to angiotensin II converting enzyme inhibitor, captopril, and the renin inhibitor, ditekiren (U-71038), were capable of abolishing this component after oral administration. Oral administration of ditekiren to rats receiving human renin infusions evoked dose-dependent hypotensive responses that were greater in magnitude and longer in duration than those elicited in rats receiving hog renin infusions. Observations made in the renin-infused rats reflected the results of in vitro kinetic studies that had indicated a greater binding affinity of ditekiren for human renin than for hog renin.