Rational design, synthesis, and X-ray structure of selective noncovalent thrombin inhibitors.

We have designed, synthesized, and tested in vitro a novel class of noncovalent thrombin inhibitors. The main feature of these inhibitors is a 6,5-fused bicyclic core structure that fills the S2 pocket of the active site of thrombin. The bicycle introduces conformational constraint into the ligand and locks the Xaa-Pro amide bond into the desired trans configuration. Among the known ring systems, we selected by molecular modeling the 7-thiaindolizidinones (BTD) as our basic template. The influence of several structural features was analyzed: the length of the argininal side chain, the stereochemistry at C6, and the importance of making optimal use of the S3 pocket. Finally, an X-ray crystal structure of inhibitor 15 bound to thrombin was obtained at a resolution of 2.3 A. These designed thrombin inhibitors, which were prepared by an efficient synthesis, showed high selectivity over trypsin and other serine proteases. Further derivation based on the information obtained by X-ray crystallography should certainly allow to improve the potency.

Time course of spirapril-induced structural and functional changes after myocardial infarction in rats followed with magnetic resonance imaging.

Structural alterations after myocardial infarction (MI) in rats are usually examined only after death of the experimental animal. Magnetic resonance imaging (MRI) allows repeated and noninvasive measurements of important structural [left ventricular (LV) mass, LV wall thickness, LV chamber radius] as well as function [LV end-systolic and LV end-diastolic volume, stroke volume (SV), ejection fraction (EF)] parameters for a prolonged period. We describe our experience in a series of experiments in rats. Three weeks after MI, infarct size (IS) was determined by MRI and the rats were divided into two groups with equal IS. Three weeks later, treatment with the angiotensin-converting enzyme (ACE) inhibitor spirapril (10 mg/kg in food) or placebo was started. In both groups, the first MRI scan taken before the treatment showed moderately dilated left ventricles and signs of impaired LV function, i.e., an increase in LV end-systolic and end-diastolic volume and decreased EF. After 3-week treatment, no significant differences with respect to heart structure and function were detected as compared with those of untreated animals. Prolonged treatment for 10 weeks with spirapril resulted in significant reduction of LV dilatation, LV mass, and LV end-systolic and end-diastolic volume, which was accompanied by improved EF. Hemodynamic examinations after treatment for 6 months showed, in contrast to control animals, no increase in right ventricular systolic pressure in animals receiving spirapril. Furthermore, histologic examination of perfusion-fixed hearts at the end of the study demonstrated more pronounced LV dilatation in control animals, thus confirming the in vivo MRI data. Delayed treatment with spirapril proved to have beneficial effects on structure and function of infarcted hearts within 10 weeks. Spirapril limited LV dilatation, reduced LV weight and LV end-systolic and end-diastolic volumes, and improved EF.

Assessment of right ventricle dimensions with microsonometry in anesthetized rabbits.

Dimension measurements of the right ventricle are difficult to obtain because of its complex geometry, thus we evaluated a method of right ventricular dimension measurements. Crystals were placed on the ventral and dorsal side of the right ventricular free wall, approximately one-fourth of the right ventricular semicircle away from the septum, in the middle of a cranio-caudal axis of the ventricles. The effects of an increased (infusion of 20 mL/kg of 5% glucose for 3 min into seven rabbits), as well as decreased preload (nitroglycerin, 5 micrograms/kg/min i.v. n = 6) were measured and compared with changes during a placebo infusion (n = 6). The change in shortening of the right ventricle wall segment correlated with changes in both atrial natriuretic factor (ANF) plasma concentration (r = 0.89, p < 0.05) and central venous pressure (CVP) (r = 0.94, p < 0.05), respectively. Both these variables are influenced by right ventricular function and dimensions in healthy animals. Dimension measurements obtained across the free wall of the right ventricle appear to reflect right ventricular function well and should be useful in assessing the effects of drugs intended for the treatment of angina pectoris or heart failure.

Autonomic nervous system dysfunction alters drug effects: implications for testing drugs for the treatment of heart failure.

Blunted cardiac responses to sympathetic and vagal activation are key features of heart failure. Since the modulation of drug effects by a selective autonomic dysfunction is little known, we developed an acute rabbit model imitating these defects. Anesthetized rabbits were subject to cervical vagotomy and propranolol (1 mg/kg i.v.) pretreatment, thus eliminating vagally and sympathetically mediated cardiac responses, while maintaining the responsiveness of the peripheral circulation to these reflexes ("V-B" animals). Responses to drugs were altered in V-B compared with normal animals: Ouabain (5-50 micrograms/kg) increased myocardial contractile force more and milrinone (30-300 micrograms/kg) less, yet it increased the heart rate more; the reflex tachycardia to nitroprusside (1-10 micrograms/kg/min) was blunted and spirapril (0.1 and 1 mg/kg, all i.v.) decreased the central venous pressure only in V-B animals. Several drug effects were thus strongly modulated by autonomic dysfunction and responses of V-B animals were closer to those of heart failure patients than the responses of the normal animals, especially for milrinone.

Effects of isradipine on plasma renin activity in sodium-loaded and -depleted conscious rabbits.

The antihypertensive effect of calcium antagonists is altered little by high salt intake or concomitant diuretic treatment. We therefore investigated whether modest changes in the salt balance might alter the acute response of the renin-angiotensin system to the calcium antagonist isradipine in conscious rabbits. Mongrel rabbits with indwelling arterial and venous catheters were pretreated with either a single subcutaneous injection of 20 mg/kg furosemide 24 h before the experiment [sodium depletion (SD)] or the addition of 0.45% NaCl to the drinking water, which was available ad libitum for 24 h [sodium loading (SL)]. Compared with SL, SD pretreatment modestly increased plasma renin activity and lowered body weight; mean arterial pressure and heart rate were unchanged. Isradipine (10, 30, and 100 micrograms/kg) infused into the femoral vein catheter decreased blood pressure and increased heart rate similarly in both groups. Increases in plasma renin activity plotted as a function of the decreases in blood pressure showed a significantly steeper slope in SD than in SL animals. When blood pressure started to recover 15 min after the end of drug infusion, plasma renin activity decreased in SL animals only. Therefore, SL or SD strongly influences the responsiveness of the renin-angiotensin system to calcium antagonists, and this may be one reason why high salt intake does not diminish the antihypertensive effect of calcium antagonists and a calcium antagonist/diuretic combination may not yield optimal therapeutic results.

Binding of a Bolton-Hunter substituted homostatine analog to affinity-immobilized human renin.

The binding of a Bolton-Hunter reagent substituted homostatine analog, SDZ 213-776, to human renin was investigated at pH 6.5 and 7.4. At both pH values, SDZ 213-776 bound to human renin in a reversible and saturable manner. The binding characteristics conformed to a one-site binding model. The dissociation constant Kd, obtained at equilibrium, was four-fold lower at pH 6.5 that at pH 7.4 (0.94 nM vs 3.7 nM). Under non-equilibrium conditions, only the association kinetic constant k+1 was affected by pH. The results of the binding assay at pH 6.5 correlated well with those obtained in enzymatic assay at the same pH.

Hemodynamic effects of a new angiotensin converting enzyme inhibitor, spirapril, in sodium-loaded and sodium-depleted rabbits.

The new angiotensin converting enzyme inhibitor, spirapril, was found to inhibit the pressor effects of angiotensin I about 10 times more potently than captopril in conscious rabbits. Its effects on systemic hemodynamics and regional blood flows were studied in two groups of 8 normotensive anaesthetized rabbits pretreated 24 h before the acute experiment either by substituting 0.45% NaCl for drinking water or with a subcutaneous injection of 20 mg kg-1 furosemide. Under barbiturate anaesthesia, regional blood flows were then measured with microspheres before and after i.v. administration of two doses of spirapril. With anaesthesia and surgery, plasma renin activity (PRA) was elevated in the salt-replete group and very high in the salt-depleted group (37 and 152 ng ml-1 h-1, respectively). Spirapril lowered blood pressure more in salt-depleted animals and increased only cardiac output and renal blood flow in salt-replete ones. Weak cardiodepression, associated with a small fall of cardiac output, was found in salt-depleted rabbits, indicating that very high angiotensin II levels may contribute to maintain cardiac function in volume-depleted animals with an activated sympathetic system. Coronary and skin blood flow decreased, and pancreatic and hepatic artery flow increased only in this group. A change in the salt balance can thus cause major differences in the responses of the peripheral circulation to angiotensin converting enzyme inhibition.

Similar increase in circulating renin after equihypotensive doses of nitroprusside, dihydralazine or isradipine in conscious rabbits.

Calcium entry blockers but not nitroprusside sodium (NPS) or dihydralazine are known to enhance renin release in vitro. Isradipine (PN200-110), NPS or dihydralazine were infused at 15 min intervals into groups of 6 conscious, chronically instrumented rabbits each, at 3 doses carefully matched to elicit comparable falls in blood pressure. Plasma renin activity increased similarly with all treatments. In intact animals, the magnitude of the pressure decrease appears to be more important for renin release than the mechanism of vasodilation and a typical calcium entry blocker was indistinguishable from other vasodilators.

Characterization of beta-adrenoceptor subtypes in rat kidney with new highly selective beta 1 blockers and their role in renin release.

Highly selective beta-adrenoceptor blocking agents with a beta 1: beta 2-selectivity ratio of 0.015 to 3400 were used to characterize the beta-adrenoceptors present in rat kidney and to identify those mediating renin release. The results obtained with ICYP binding to kidney membranes revealed the presence of both beta 1- and beta 2-adrenoceptors in a ratio of 1:1. The pKD beta 1- and pKD beta 2-values of selective beta-antagonists obtained in rat kidney membranes correlated well with those found in guinea pig left ventricle (beta 1) and lung (beta 2), indicating that kidney receptor subtypes are pharmacologically identical with those in the ventricle and lung, respectively. In the isolated perfused rat kidney, the apparent pA2 values of beta 1-selective blockers for inhibition of isoprenaline-stimulated renin release correlated well with pKD beta 1, but not with pKD beta 2 values. These results clearly show that the beta 1-adrenoceptor subtype mediates renin release in the rat kidney.