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.

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.

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.

Conformationally constrained renin inhibitory peptides: cyclic (3-1)-1-(carboxymethyl)-L-prolyl-L-phenylalanyl-L-histidinamide as a conformational restriction at the P2-P4 tripeptide portion of the angiotensinogen template.

Interest in conformationally constrained peptides as potential inhibitors of renin led us to examine an N-terminal cycle of linear renin inhibitory peptides. A cyclic structure was prepared by joining the N-terminal proline at the P4 site to the imidazole ring of histidine at the P2 site via a carboxymethylene fragment. An efficient synthetic route to this 14-membered macrocycle was developed and this N-terminal cyclic tripeptide could be readily incorporated into renin inhibitory peptides. Monte Carlo molecular modeling methods were used to generate bound conformations of a representative inhibitor in a model of the renin active site, suggesting possible modes of binding of these inhibitors to renin. Two representative compounds that contain this 14-membered macrocycle were evaluated for their inhibitory activities against human plasma renin and they were found to exhibit very high binding affinity with IC50 values in the nanomolar and subnanomolar range.

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.

Renin inhibitory peptides. A beta-aspartyl residue as a replacement for the histidyl residue at the P-2 site.

In an effort to decrease the size and to increase the hydrophilicity of the previously prepared renin inhibitory peptides, it was postulated that one might be able to take advantage of the polar Thr-84 on the flap region of the enzyme renin by potential hydrogen bonding to polar functionality on the inhibitory peptide at the P-2 site. A beta-aspartyl residue with a carboxylic acid group was proposed to be a possible replacement for the histidyl residue at the P-2 site. A series of renin inhibitory peptides were prepared with the beta-aspartyl residue to probe the structure-activity relationship of the resulting peptides. Potent inhibitory peptides could be realized with activity in the subnanomolar range. Molecular modeling was also undertaken to investigate the interactions between the enzyme active site and the new inhibitors and to suggest a possible mode of binding of these ligands to the enzyme. From this modeling study, the role of Ser-229 at the active site in the bound conformer of the inhibitors was suggested. It was further noted in the analogue study that a malic acid residue, which is the oxygen analogue of the beta-aspartic acid residue, could lead to further enhancement of inhibitory potency of congeneric peptides. Small renin inhibitors, such as compound XII with molecular weight 535 and with no alpha-amino acid residue, could be prepared and exhibited renin inhibitory activity in the nanomolar range.

Renin inhibitory peptides: a study of structural modifications in the peptide backbone.

We studied the peptide backbone modifications that improve the metabolic stability of the resulting peptides and yet retain high inhibitory activity against human plasma renin. A systematic investigation of N-alpha-methyl and C-alpha-methyl modifications at the P2 and P3 sites of renin-inhibitory peptides that contain part of the human angiotensinogen sequence led to the discovery of N-alpha-methyl amino acids at the P2 site as a useful structural modification. U-71,038 (11) inhibited human plasma renin with an in vitro potency (IC50) of 2.6 x 10(-10) mol/l. It is highly selective for renin and, as anticipated, resistant to proteolytic degradation. Additional study based on molecular graphic modelling has led us to propose a gamma-lactam conformational constraint at the P2-P3 site. This pseudo-dipeptide has proved useful in the preparation of active renin inhibitors. Compound 18a inhibited human plasma renin with an in vitro potency (IC50) of 2.1 x 10(-9) mol/l. This class of compounds also offers structural features for the study of enzyme-bound conformers.

Systemic and renal haemodynamic effects of renin or angiotensin converting enzyme inhibition in non-human primates.

The cardiovascular actions of a renin inhibitor, U-71038 (Boc-Pro-Phe-N-MeHis-Leu psi [CHOHCH2]Val-Ile-Amp), and of an angiotensin converting enzyme (ACE) inhibitor, captopril, were determined in conscious sodium-depleted cynomolgus monkeys. Cardiac output was measured with a thermodilution technique. The hypotension induced by U-71038 was associated with a significant reduction in total peripheral resistance without alteration in cardiac output or the heart rate. A similar reduction in total peripheral resistance was observed after captopril at a dose which caused hypotension equivalent to that elicited by U-71038. The latter effects were not accompanied by significant alterations in cardiac output or the heart rate. The glomerular filtration rate was measured by the plasma disappearance of 125I-sodium iothalamate. Renin or ACE inhibition adequate to cause equivalent hypotensive responses did not change the glomerular filtration rate to a significantly different degree. The systemic and renal haemodynamic profiles of U-71038 and captopril appear to be similar, suggesting that renin and ACE inhibition elicit fundamentally similar cardiovascular effects in conscious sodium-depleted cynomolgus monkeys via a decreased formation of angiotensin II (Ang II).

Conformationally constrained renin inhibitory peptides: gamma-lactam-bridged dipeptide isostere as conformational restriction.

A model of the conformation of the enzyme-bound inhibitor of human renin suggested the possibility of a gamma-lactam conformational restriction at the P2-P3 site. Synthetic routes to these gamma-lactam dipeptide isosteres and their incorporation into potential renin inhibitors are described. Peptide VIa,b with a gamma-lactam conformational constraint and a hydroxyethylene isostere at the cleavage site inhibited human plasma renin with an IC50 value of 6.5 nM. The flexibility of these syntheses should make available a number of potential enzyme inhibitors with this structural feature for the study of enzyme-bound conformers.

Synthesis and renin inhibitory activity of angiotensinogen analogues having dehydrostatine, Leu psi [CH2S]Val, or Leu psi [CH2SO]Val at the P1-P1' cleavage site.

The synthesis and in vitro renin inhibitory potencies of angiotensinogen (ANG) analogues having amide (CONH) bond replacements at P1-P1', the Leu-Val cleavage site, corresponding to Leu psi[CH2SO]Val, and the trans olefinic analogue of statine (Sta), 4(S)-amino-6-methyl-2(E)-heptenoic acid (dehydrostatine, Dhs), are reported. These are compared to P1-P1' Leu psi[CH2NH]Val-, Sta-, or Phe-Phe-substituted analogues of the same template. The Dhs pseudodipeptide was found to be an adequate mimic of a trans CONH bond and gave a peptide, H-Pro-His-Pro-Phe-His-Dhs-Ile-His-D-Lys-OH, approximately equal in potency to a Phe-Phe-containing inhibitor, but 200-fold less potent than its Sta-substituted congener. That the enhanced potency of the Sta-containing peptide most likely depends on hydrogen bonding as well as tetrahedral geometry is indicated by the 50-100-fold lower potency of the tetrahedral Leu psi[CH2S]Val and Leu psi[CH2SO]Val analogues as compared to the Leu psi[CH2NH]Val-containing congener.

Renin inhibitors containing psi[CH2O] pseudopeptide inserts.

Renin inhibitors 2-4 with the D-Lys renin inhibitory peptide (RIP) sequence, but containing Leu psi[CH2O]Ala (2), Leu psi[CH2O]Val (3), and Leu psi[CH2O]Leu (4) at the P1-P1' site, were of a comparable potency to RIP. N-Terminal Boc-protected inhibitors containing Pro psi[CH2O]Phe in positions P4-P3 were potent inhibitors of renin, with Boc-Phe-Pro psi[CH2O]Phe-His-Leu psi[CH(OH)CH2]Val-Ile-(2-aminomethyl) pyridine (17) having an IC50 of 1.6 X 10(-9) M.

Design, structure-activity, and molecular modeling studies of potent renin inhibitory peptides having N-terminal Nin-For-Trp (Ftr): angiotensinogen congeners modified by P1-P1' Phe-Phe, Sta, Leu psi[CH(OH)CH2]Val or leu psi[CH2NH]Val substitutions.

A structure-conformation-activity investigation of several angiotensinogen (ANG) based inhibitors of human renin modified by either Phe-Phe, Sta, Leu psi[CH2NH]Val, or Leu psi[CH(OH)CH2]Val at the P1-P1' clevage site and P5 Trp(Nin-For) (Ftr) was performed. Specifically, Ac-Ftr-Pro-Phe-His-Phe-Phe-Val-Ftr-NH2 (1) provided a potent (KI = 2.7 X 10(-8) M) P1-P1' Phe-Phe substituted renin inhibitor to initiate these studies. Substitution of the P1-P1' Phe-Phe in compound 1 by Sta effected a 1,000-fold increase in biological potency for the resultant octapeptide Ac-Ftr-Pro-Phe-His-Sta-Val-Ftr-NH2 (10; KI = 6.7 X 10(-11) M). Kinetic analysis of compound 10 showed it to be a competitive inhibitor of human renin catalyzed proteolysis of human ANG. Chemical modifications of the compounds 1 and 10 were evaluated on the basis of comparative human plasma renin inhibitory activities (IC50 values) in vitro. Carboxy-terminal truncation studies on compound 10 showed that the P2' Val and P3' Ftr residues could both be eliminated without significant loss (ca. 10-fold) in renin inhibitory activity as exemplified by the pentapeptide Ac-Ftr-Pro-Phe-His-Sta-NH2 (12; IC50 = 3.8 X 10(-9) M). In addition, the corresponding P1-P1' Leu psi[CH(OH)CH2]Val and Leu psi[CH2NH]Val derivatives of compound 12 were potent renin inhibitors: Ac-Ftr-Pro-Phe-His-Leu psi[CH(OH)CH2]Val-NH2 (13; IC50 = 3.1 X 10(-10) M) and Ac-Ftr-Pro-Phe-His-Leu psi[CH2NH]Val-NH2 (14; IC50 = 2.1 X 10(-8) M). The structure-conformation-activity properties of the N-terminal Ftr substitution of these human renin inhibitors was examined by (1) comparative analysis of several analogues of 1 and Ac-Ftr-Pro-Phe-His-Sta-Ile-NH2 (17; IC50 = 1.0 X 10(-10) M) having P5 site modifications by Trp, His, D-Ftr, and D-His, (2) deletion of the N-terminal Ftr residue from compounds 12 and 17, to provide Ac-Pro-Phe-His-Sta-Ile-NH2 (16; IC50 = 3.1 X 10(-8) M) and Ac-Pro-Phe-His-Sta-NH2 (15; IC50 = 5.6 X 10(-6) M), and (3) computer modeling and dynamics studies of compounds 1 and 17 bound to CKH-RENIN, a simulated human renin model, which were focused on identifying potential intermolecular interactions of their common P5-P2 sequence, Ac-Ftr-Pro-Phe-His, at the enzyme active site.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetics of the inhibition of human renin by an inhibitor containing a hydroxyethylene dipeptide isostere.

We have studied the inhibition of both human and hog renins by compound 1 [Boc-Pro-Phe-N alpha-MeHis-Leu psi(CHOHCH2)Val-Ile-(aminomethyl)pyridine] using kinetics. The inhibition of human renin was shown to be time dependent and followed a minimal two-step mechanism. A loosely bound EI complex was formed rapidly with a dissociation constant, KI, of 12 nM. A second EI complex was slowly formed and was found to be 64-fold more strongly bound with an overall KI of 0.19 nM. The inhibition of human renin was shown to be competitive by both initial and final steady-state velocities. Compound 1 was also shown to be a competitive inhibitor of hog renin with a KI of 12 nM, but no evidence for time-dependent inhibition was detected. The differences in overall KI and inhibition kinetics may be a consequence of the similarities in structure between 1 and human angiotensinogen.

alpha,alpha-Difluoro-beta-aminodeoxystatine-containing renin inhibitory peptides.

The preparations of sodium 4(S)-[(tert-butyloxycarbonyl)amino]-2,2-difluoro-3(S)- and -3(R)-[(4-methoxyphenyl)amino]-6-methylheptanoates (7a and 7b) from sodium 4(S)-[(tert-butyloxycarbonyl)amino]-2,2-difluoro-3(R)- and -3(S)-hydroxy-6-methylheptanoates (1a and 1b) are described. The key step involves the stereospecific intramolecular displacement via a Mitsunobu reaction for the conversion of a beta-hydroxy hydroxamate to a beta-lactam ring. Compounds 7a and 7b are useful as synthetic intermediates for the preparation of enzyme inhibitors that contain 3(S),4(S)- and 3(R),4(S)-diamino-2,2-difluoro-6-methylheptanoic acid inserts. Angiotensinogen analogues VII and VIII that contain these novel amino analogues of difluorostatine were shown to be inhibitors of the enzyme renin. The alpha,alpha-difluoro-beta-aminodeoxystatine-containing compounds were shown to be weaker inhibitors than the corresponding difluorostatine-containing congeners.

Renin inhibitors. Design of angiotensinogen transition-state analogues containing novel. (2R,3R,4R,5S)-5-amino-3,4-dihydroxy-2-isopropyl-7-methyloctanoic acid.

A highly stereoselective synthesis of 2(R)-[5(R)-[1(S)-[(tert-butyloxycarbonyl)amino]-3-methylbutyl]-2,2- dimethyl-4(R)-dioxolanyl]-3-methylbutanoic acid is described. This is a suitably protected carboxylic acid useful as an intermediate for the preparation of renin inhibitory peptides. Angiotensinogen analogues such as peptides IX and X that contain the dipeptide isostere (2R,3R,4R,5S)-5-amino-3,4-dihydroxy-2-isopropyl-7-methyloctanoic acid residue at the scissile site are shown to be potent inhibitors of human plasma renin. The glycol moiety in this novel acid, dihydroxyethylene isostere, is suggested to act as a transition-state analogue and mimics the tetrahedral intermediate formed during the enzyme-catalyzed hydrolysis of the peptidic bond.

alpha-Methylproline-containing renin inhibitory peptides: in vivo evaluation in an anesthetized, ganglion-blocked, hog renin infused rat model.

A structure-activity analysis of peptides containing backbone C alpha-methyl modification at the P4 site of the angiotensinogen sequence led to the discovery of potent renin inhibitors with apparent in vitro metabolic stability. Boc-alpha-MePro-Phe-His-Leu psi[CHOHCH2]Val-Ile-Amp dicitrate (Va) is a potent inhibitor of human plasma renin with an IC50 value of 1.8 nM. This peptide was shown not to be degraded in vitro by chymotrypsin, elastase, pepsin, and a rat liver homogenate preparation. It is also a potent inhibitor of hog renin with an IC50 value of 1.6 nM and was shown to elicit in vivo activity and cause dose-dependent hypotensive responses when given intravenously to anesthetized ganglion-blocked, hog renin infused rats.

Significant depletion of NPY in the innervation of the rat mesenteric, renal arteries and kidneys in experimentally (aorta coarctation) induced hypertension.

The distribution and concentrations of neuropeptide Y (NPY) in kidneys, renal arteries, heart, aorta, mesenteric artery and adrenal glands from aorta-ligated hypertensive rats were studied by immunocytochemistry and radioimmunoassay. Immunocytochemistry showed that in the hypertensive animals NPY-immunoreactive fibres were decreased in both kidney and renal artery, above and below the ligation, and in mesenteric arteries. The depletion of NPY-containing nerves in the kidney was more pronounced around the juxtaglomerular apparatus than in other areas of the organ. By radioimmunoassay, the concentrations of NPY immunoreactivity were significantly lower in the hypertensive animals when compared with the controls, (kidney: hypertensive 1.0 +/- 0.1; controls 2.0 +/- 0.2 pmol/g, mean +/- SEM; p less than 0.05 renal artery: hypertensive 5.0 +/- 0.8; controls 12.1 +/- 2.0; p less than 0.05 and mesenteric artery: hypertensive 8.6 +/- 1.9; 17.6 +/- 3.0; p less than 0.01). While there were no statistically significant changes in the levels of NPY immunoreactivity in the other areas studied, there was a general trend for the level to fall in the renal artery below the ligation (hypertensive 10.6 +/- 1.5; control 15.3 +/- 2.4; p greater than 0.05). It is of interest that changes were observed in the vasoconstrictor peptide NPY in this commonly used model of hypertension.