Pretreatment with a blocking monoclonal antibody to P-selectin accelerates pharmacological thrombolysis in a primate model of arterial thrombosis.

It has been established that the fibrin content of a developing thrombus can be dramatically reduced with the use of the GA6 monoclonal antibody, which is directed against P-selectin (CD62p). This effect is probably related to diminished tissue factor activity on monocytes in the presence of P-selectin antagonism. Therefore, we hypothesized that an occlusive arterial thrombus formed in the presence of a P-selectin monoclonal antibody would be more susceptible to lysis with standard thrombolytic therapy. To test this hypothesis, 22 male cynomolgus monkeys were anesthetized and instrumented for induction of thrombosis of a femoral artery. Endothelial injury was induced by passing a 150-microA anodal current through a small electrode that was placed in the femoral artery. Blood flow through the artery was continuously monitored using an ultrasonic transit-time flowmeter. The GA6 monoclonal antibody (1 mg/kg) or control, isotype matched mouse IgG1 (P23 or P7) was administered i.v. 1 hr before electrolytic endothelial injury. In the P23 group (n = 11), an occlusive thrombus formed in 52.1 +/- 8.5 min, and in the GA6 group (n = 11), an occlusive thrombus formed in an average time of 52.0 +/- 8.1 min. After formation of an occlusive thrombus, the current was terminated and intravenous heparin (100 U/kg + 50 U/kg/hr) was administered to prevent clot extension.(ABSTRACT TRUNCATED AT 250 WORDS)

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 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.

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).

An orally active inhibitor of renin.

A potent renin inhibitor, U-71038 (Boc-Pro-Phe-N-MeHis-Leu psi[CHOHCH2]Val-Ile-Amp), was tested for oral effectiveness. Enzyme kinetic studies indicated that U-71038 was a competitive inhibitor of hog renin with an inhibitor constant (Ki) value of 12 nM. Intravenous as well as oral administration of U-71038 to anesthetized, ganglion-blocked rats infused with hog renin elicited dose-related hypotensive responses. Intravenous administration of U-71038 to conscious, sodium-depleted monkeys caused dose-related decreases of blood pressure and plasma renin activity without affecting heart rate. Similarly, the oral administration of U-71038 at 50 mg/kg to conscious, sodium-depleted monkeys elicited a pronounced hypotension and decrease in plasma renin activity that persisted for 5 hours. The hypotensive responses elicited by intravenous and oral administration of U-71038 to hog renin-infused rats and sodium-depleted monkeys were shown to be due entirely to inhibition of the renin-angiotensin system. A comparison of the results obtained after the intravenous administration of U-71038 with the results obtained after the oral administration of U-71038 implied that at least 10% of the orally administered U-71038 must have been absorbed to cause the observed effects in hog renin-infused rats and sodium-depleted monkeys. The studies demonstrated that an inhibitor of renin with a long duration of action and with oral effectiveness is a feasible entity.

Cardiovascular effects of losulazine hydrochloride, a peripheral norepinephrine-depleting agent, in nonhuman primates.

THE CARDIOVAscular actions of losulazine hydrochloride, a peripheral norepinephrine-depleting agent in rodents, have been determined in conscious cynomolgus monkeys. Acute oral administration of losulazine at 0.1, 1, 10 and 30 mg/kg evoked dose-related hypotensive responses in the absence of significant alterations of heart rate. Although variable, the acute hypotensive effects of losulazine were associated with both reductions of peripheral vascular resistance and reductions in cardiac output. Acute oral administration of doses of losulazine ranging from 0.1 to 30 mg/kg did not cause orthostatic hypotension. Blockade of prostaglandin synthesis and of cholinergic, beta adrenergic and histaminergic receptors did not abolish the hypotensive effect of losulazine at 1 mg/kg i.v. Ganglionic blockade abolished the hypotensive effect of losulazine (1 mg/kg i.v. and 10 mg/kg p.o.), indicating that the hypotensive activity of losulazine was dependent on the presence of sympathetic neuronal activity. Additive combination studies of losulazine and nitroprusside, phentolamine, reserpine or guanethidine implied that losulazine, reserpine and guanethidine had the same site of action, i.e., inhibition of adrenergic neuron function. A comparison of the profiles of effect of losulazine, guanethidine, guanadrel and reserpine on blood pressure, heart rate, blood pressure responses to norepinephrine and tyramine and blood pressure after desipramine pretreatment indicated that losulazine and reserpine had similar mechanisms of action. The data presented in this report are consistent with the conclusion that losulazine reduced arterial blood pressure in nonhuman primates via depletion of norepinephrine from postganglionic adrenergic neurons.

Cardiovascular effects of a renin inhibitor in relation to posture in nonhuman primates.

Orthostatic cardiovascular reflexes were evaluated in conscious cynomolgus monkeys during interruption of the renin-angiotensin system with the renin inhibitor: RIP (Pro-His-Pro-Phe-His-Phe-Phe-Val-Tyr-Lys-OH). RIP was synthesized via solid phase techniques and purified to homogeneity. In vitro studies indicated that it exhibited classical competitive inhibition of renin with a KI of 2.3 microM. In vivo, RIP at 2 mg/kg per min inhibited renin and angiotensin I pressor responses indicating that it was not a specific renin inhibitor at this dose. However, in spite of the nonspecificity, RIP did not affect the supine blood pressure of sodium-replete monkeys, but did evoke hypotension in supine sodium depleted monkeys. RIP did not elicit significant orthostatic hypotension in either sodium-replete or sodium depleted monkeys. The cardiovascular effects of RIP described in this study appear to be due to inhibition of the renin-angiotensin system.

Bronchodilator effects of prostacyclin (PGI2) in dogs and guinea pigs.

prostacyclin (PGI2), a recently discovered unstable product in the biosynthetic conversion of prostaglandin endoperoxides, was examined for bronchopulmonary actions. in anesthetized dogs, PGI2 given i.v. (0.3-30.0 microgram/kg) and by aerosol (0.002-0.2%) inhibited significantly PGF2 alpha-induced increases in pulmonary resistance and decreases in dynamic lung compliance in a dose-related fashion. Intrinsically, PGI2 affected resting bronchopulmonary and cardiac functions minimally, but decreased peripheral and pulmonary vascular pressures. PGI2 (0.1-10 mg/kg, i.p.) afforded protection against histamine-induced asphyxial collapse in normal guinea pigs and ovalbumin-induced anaphylaxis in sensitized animals. Cumulative concentrations of PGI2 (1.0 x 10(-9)--3.0 x 10(-4) M) relaxed contractions of the isolated guine pig trachea produced by carbachol. These bronchodilator and hemodynamic effects could not be ascribed to the stable metabolic product of PGI2, because 6-keto-PGF1 alpha was inactive or markedly less active than PGI2 in these test systems. The results of this investigation suggest that PGI2 possesses considerable bronchodilator and vasodilator activity in experimental animal systems.

Bronchopulmonary and cardiovascular effects of prostaglandin D2 in the dog.

The effects of intravenously administered prostaglandin D2 (PGD2) on bronchopulmonary and cardiovascular functions were examined in the dog. PGD2 (0.03-1.0 microng/kg) was shown to be more active than PGF2alpha, a known bronchoconstrictor, in decreasing dynamic lung compliance, tidal volume, and expiratory airflow rate, as well as in elevating lung resistance. PGD2 demonstrated a potency approximately 4-6 times that of PGF2alpha on pulmonary mechanics. Atropine sulfate infusions reduced significantly the resistance and compliance responses to PGF2alpha, but only the resistance responses to PGD2, thereby suggesting that part of the bronchoconstrictor activities of these agents involved a cholinergic component. In another series of anesthetized dogs, PGD2 (0.1-10.0 microng/kg) increased pulmonary arterial pressure (comparable to PGF2alpha) and heart rate (greater than PGF2alpha, but less than PGE2), while concomitantly decreasing systemic arterial pressure in a dose-related manner (1/10 that of PGE2). Qualitatively similar alterations in cardiovascular parameters were obtained for PGD2 in conscious dogs. Therefore, potent biologic activity of PGD2 has been shown in the dog. No physiologic or pathologic role for PGD2 has yet been demonstrated, but nonetheless, since it is a naturally occurring PG derived from arachidonic acid, further studies are warranted.