enalaprilat-anhydrous and ramiprilat

Increasing evidence suggests that angiotensin-converting enzyme (ACE) inhibitors can increase vascular nitric oxide (NO) production. Recent studies have found that combined inhibition of ACE and neutral endopeptidase (NEP) may have a greater beneficial effect in the treatment of heart failure than inhibition of ACE alone. Amlodipine, a calcium channel antagonist, has also been reported to have a favorable effect in the treatment of patients with cardiac dysfunction. The purpose of this study was to determine whether and the extent to which all of these agents used in the treatment of heart failure stimulate vascular NO production. Heart failure was induced by rapid ventricular pacing in conscious dogs. Coronary microvessels were isolated from normal and failing dog hearts. Nitrite, the stable metabolite of NO, was measured by the Griess reaction. ACE and NEP inhibitors and amlodipine significantly increased nitrite production from coronary microvessels in both normal and failing dog hearts. However, nitrite release was reduced after heart failure. For instance, the highest concentration of enalaprilat, thiorphan, and amlodipine increased nitrite release from 85 +/- 4 to 156 +/- 9, 82 +/- 7 to 139 +/- 8, and 74 +/- 4 to 134 +/-10 pmol/mg (all *p <.01 versus control), respectively, in normal dog hearts. Nitrite release in response to the highest concentration of these two inhibitors and amlodipine was reduced by 41% and 31% and 32% (all #p <.01 versus normal), respectively, in microvessels after heart failure. The increase in nitrite induced by either ACE or NEP inhibitors or amlodipine was entirely abolished by Nw-nitro-L-arginine methyl ester, HOE 140 (a B2-kinin receptor antagonist), and dichloroisocoumarin (a serine protease inhibitor) in both groups. Our results indicate that: 1) there is an impaired endothelial NO production after pacing-induced heart failure; 2) both ACE and NEP are largely responsible for the metabolism of kinins and modulate canine coronary NO production in normal and failing heart; and 3) amlodipine releases NO even after heart failure and this may be partly responsible for the favorable effect of amlodipine in the treatment of heart failure. Thus, the restoration of reduced coronary vascular NO production may contribute to the beneficial effects of these agents in the treatment of heart failure.

Topics: Amlodipine; Angiotensin-Converting Enzyme Inhibitors; Animals; Bradykinin Receptor Antagonists; Coronary Vessels; Cysteine Proteinase Inhibitors; Dogs; Enalaprilat; Glycopeptides; Heart Failure; Kininogens; Kinins; Nitric Oxide; Protease Inhibitors; Ramipril; Receptor, Bradykinin B2; Thiorphan; Vasodilator Agents

We have compared at the enzymological level pulmonary angiotensin I-converting enzymes (ACE) purified to electrophoretic homogeneity from four mammalians species: pig, rat, monkey and human. Using both substrates hippuryl-histidyl-leucine and furylacryloyl-phenylalanyl-glycyl-glycine in steady-state conditions, all the ACEs exhibited Michaelis kinetics with identical Michaelis constants, maximal velocities, optimal pH and optimal activating chloride-concentrations. The apparent inhibitory constant was higher for Captopril than for Enalaprilat and even more so for Ramiprilat irrespective of the origin of ACE and the substrate used. Although these inhibitors have been described as competitive inhibitors, Lineweaver-Burk plots were not in accordance with a simple competitive model; moreover, Dixon plots were rather characteristic of non-competitive inhibition. These data emphasize the hypothesis that ACE inhibitors act with mixed-type inhibition, which is consistent with their slow-tight binding to the ACE active center, also with binding of chloride on a critical lysine residue leading to a potential conformational change, and finally with the fact that ACE has two domains, each bearing one catalytic site. On the other hand, as identical kinetic parameters were obtained on the different ACE preparations, results from animal models should allow the extrapolation to humans, in particular for investigations on both renin-angiotensin and kallikrein-kinin systems, and on their inhibition.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Binding, Competitive; Captopril; Enalaprilat; Humans; Kinetics; Lung; Macaca fascicularis; Peptidyl-Dipeptidase A; Ramipril; Rats; Rats, Sprague-Dawley; Swine

Recent studies suggest that amlodipine may reduce mortality in patients with heart failure, especially those with dilated cardiomyopathy. In general, drugs that release NO, such as organic nitrates and ACE inhibitors, have been shown to be of substantial benefit in the treatment of heart failure.. We hypothesized that a portion of the beneficial actions of amlodipine may involve the release or action of NO. Coronary microvessels were isolated from the heart of normal dogs and incubated with increasing doses of the calcium channel blockers nifedipine, diltiazem, and amlodipine or the ACE inhibitors enalaprilat and ramiprilat. Neither nifedipine nor diltiazem increased nitrite production at any dose studied. In marked contrast, amlodipine caused a dose-dependent increase in nitrite production from 74+/-5 to 130+/-8 pmol/mg (by 85+/-21%,10(-5) mol/L, P<.05) that was similar in magnitude to that of either of the ACE inhibitors. Amlodipine also increased nitrite production in large coronary arteries and in aorta. N(omega)-Nitro-L-arginine methyl ester, HOE-140, and dichloroisocoumarin essentially abolished the increase in nitrite production, indicating that (1) nitrite production reflected NO formation, (2) nitrite production was dependent on stimulation of the kinin2 receptor, and (3) nitrite production is most likely secondary to the formation of local kinins.. Thus, unlike nifedipine and diltiazem, amlodipine releases NO from blood vessels.

Topics: Amlodipine; Analysis of Variance; Angiotensin-Converting Enzyme Inhibitors; Animals; Calcium Channel Blockers; Coronary Vessels; Diltiazem; Dogs; Dose-Response Relationship, Drug; Enalaprilat; In Vitro Techniques; Male; Nitric Oxide; Ramipril; Regression Analysis

ACE inhibitors potentiate kinin-nitric oxide (NO)-dependent coronary vascular dilation, and NO can modulate myocardial oxygen consumption. Whether ACE inhibitors also affect myocardial O2 consumption has not been established.. Production of nitrite, a metabolite of NO in aqueous solution, in coronary microvessels and O2 consumption in myocardium were quantified with the use of in vitro tissue preparations, the Greiss reaction, and a Clark-type O2 electrode. In coronary microvessels, kininogen (the precursor of kinin; 10 micrograms/mL) and three ACE inhibitors (captopril, enalaprilat, or ramiprilat; 10(-8) mol/L) increased nitrite production from 76 +/- 6 to 173 +/- 15, 123 +/- 12, 125 +/- 12, and 153 +/- 12 pmol/mg, respectively (all P < .05). In myocardium, kininogen (10 micrograms/mL) and captopril, enalaprilat, or ramiprilat (10(-4) mol/L) reduced cardiac O2 consumption by 41 +/- 2%, 19 +/- 3%, 25 +/- 2%, and 35 +/- 2%, respectively. The changes in both nitrite release and O2 consumption in vitro were blocked by N omega-nitro-L-arginine methyl ester or N omega-nitro-L-arginine, inhibitors of endogenous NO formation. The effects were also blocked by HOE 140, which blocks the bradykinin B2-kinin receptor, and serine protease inhibitors, which inhibit local kinin formation.. Our data indicate that stimulation of local kinin formation by use of a precursor for kinin formation or inhibition of kinin degradation by use of ACE inhibitors increases NO formation and is important in the control of cardiac O2 consumption. Vasodilation and control of myocardial O2 consumption by NO may contribute importantly to the therapeutic actions of ACE inhibitors in cardiac disease states.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Bradykinin; Bradykinin Receptor Antagonists; Captopril; Coronary Vessels; Culture Techniques; Dogs; Enalaprilat; Kallikrein-Kinin System; Kininogens; Male; Microcirculation; Myocardium; Nitric Oxide; Nitrites; Oxygen Consumption; Ramipril

The circulating and urinary bladder tissue concentrations of angiotensin I (ANG I) and angiotensin II [ANG-(1-8)] were examined in anesthetized Sprague-Dawley male rats given an intravenous bolus infusion of either ANG I, the angiotensin-converting enzyme (ACE) inhibitors enalaprilat or ramiprilat, or saline. The mean concentrations of ANG I and ANG-(1-8) were markedly higher in the urinary bladder tissue than in whole blood. There was a significant increase in the concentration of ANG I and ANG-(1-8), both in the urinary bladder tissue and the circulation, after the ANG I infusion. Both ACE inhibitors were associated with an increase in the concentration of whole blood ANG I; however, tissue ANG I levels were significantly increased only following ACE inhibition with ramiprilat but not with enalaprilat. Both plasma and urinary bladder tissue ANG-(1-8) levels decreased significantly following ACE inhibition, but only with ramiprilat. The elevated urinary bladder tissue levels of ANG I and ANG-(1-8) at baseline, compared with circulating levels, and the maintenance of ANG-(1-8) in bladder tissue in the face of inhibition of the circulatory renin-angiotensin system with enalaprilat support the presence of an autocrine/paracrine renin-angiotensin system in the urinary bladder. Under the current experimental conditions, ramiprilat appears to have enhanced bladder activity compared with enalaprilat.

Topics: Anesthesia; Angiotensin I; Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Animals; Blood Pressure; Chromatography, High Pressure Liquid; Enalaprilat; Male; Ramipril; Rats; Rats, Sprague-Dawley; Urinary Bladder

Topics: Amino Acid Sequence; Aminopeptidases; Angiotensin-Converting Enzyme Inhibitors; Animals; Bradykinin; Cilazapril; Enalaprilat; Kidney; Kinetics; Molecular Sequence Data; Oligopeptides; Ramipril; Substance P; Substrate Specificity; Swine

Polymorphonuclear neutrophils (PMN) participate in the development of myocardial injury during ischaemia/reperfusion and granules released by human neutrophils contain proteases capable of activating prorenin in human plasma and can cleave angiotensin II directly from angiotensin I and angiotensinogen. The purpose of the present study was to investigate whether angiotensin converting enzyme (ACE)-inhibitors exert an in vitro effect on PMN degranulation. Isolated neutrophils were incubated with captopril, lisinopril, enalaprilat or ramiprilat and release of lysozyme and myeloperoxidase was measured from unstimulated and opsonised zymosan stimulated cells. All ACE inhibitors increased neutrophil myeloperoxidase release and lysozyme release by both unstimulated and stimulated cells. In the presence of saline unstimulated PMN released 4.48 +/- 0.68% and zymosan-stimulated cells released 7.28 +/- 0.76% of myeloperoxidase content and the enzyme release increased after incubation with captopril (5.55 +/- 0.71 and 8.74 +/- 0.72%), lisinopril (5.43 +/- 0.57 and 9.02 +/- 0.7%), enalaprilat (6.05 +/- 0.67 and 9.20 +/- 0.82%) and ramiprilat (5.82 +/- 0.69 and 9.26 +/- 0.74%), respectively. In the presence of saline unstimulated PMN released 16.71 +/- 1.28% and zymosanstimulated PMN released 34.42 +/- 1.71% of lysozyme content and the release increased after incubation with captopril (21.15 +/- 1.36 and 42.75 +/- 1.95%), lisinopril (23.95 +/- 1.26 and 39.23 +/- 1.94%), enalaprilat (21.34 +/- 1.32 and 41.59 +/- 1.99%) and ramiprilat (20.88 +/- 1.35 and 37.53 +/- 1.95%) by unstimulated PMN, respectively. The ACE-inhibitory effect of these drugs may therefore be decreased by stimulation of PMN degranulation and neutrophil-dependent angiotensin II forming pathway.

Topics: Adult; Angiotensin-Converting Enzyme Inhibitors; Captopril; Cell Degranulation; Enalaprilat; Female; Humans; In Vitro Techniques; Lisinopril; Male; Middle Aged; Muramidase; Neutrophils; Peroxidase; Ramipril; Spectrophotometry; Zymosan

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Cilazapril; Enalaprilat; Erythema; Female; Guinea Pigs; Inflammation; Ramipril

We have investigated in human endothelial cells in culture the effects of angiotensin-converting enzyme (ACE) inhibitors on the concentration of intracellular free Ca2+ ([Ca2+]i) and the formation of nitric oxide (NO) and prostacyclin (PGI2). Enalaprilat, moexiprilat and ramiprilat similarly potentiated the increase in [Ca2+]i elicited by bradykinin and caused an increase in resting [Ca2+]i when given alone. The latter effect was long-lasting and accompanied by an increased formation of NO and PGI2. All of these effects were inhibited by the B2-kinin receptor antagonist Hoe 140, suggesting that the endogenous synthesis/release of bradykinin represents an autocrine mechanism for the stimulation of endothelial autacoid formation. Thus these findings strongly support the concept that ACE inhibitors promote local vasodilation by increasing the level of bradykinin generated in subthreshold concentrations by the endothelium.

Topics: 1-Methyl-3-isobutylxanthine; Angiotensin-Converting Enzyme Inhibitors; Bradykinin; Calcium; Cells, Cultured; Cyclic GMP; Enalaprilat; Endothelium, Vascular; Epoprostenol; Humans; Isoquinolines; Kinetics; Peptidyl-Dipeptidase A; Ramipril; Tetrahydroisoquinolines; Umbilical Veins

The interaction of angiotensin-converting enzyme (ACE) inhibitors and bradykinin was investigated in isolated bovine and human coronary arteries. Rings with and without endothelium were mounted in organ chambers for measurement of isometric force. The effects of the ACE inhibitors lisinopril, enalaprilat, fosinoprilat, ramiprilat, and captopril were determined during submaximal stimulation with bradykinin or other vasodilators. Lisinopril and captopril alone did not affect vascular tone; however, in rings with endothelium partially relaxed with bradykinin (> or = 10(-10) M), all ACE inhibitors caused further relaxations. Lisinopril did not affect bradykinin concentrations in the incubation medium. Mechanical removal of the endothelium or incubation with nitro-L-arginine or the bradykinin2-receptor antagonist Hoe 140 prevented the relaxations to bradykinin and lisinopril. Other vasodilators including acetylcholine, adenosine diphosphate, substance P, or SIN-1 did not prime the rings to respond to ACE inhibitors. Endothelium-dependent relaxations to lisinopril were also observed in human coronary arteries treated with bradykinin (> or = 10(-7) M). Thus, ACE inhibitors potentiate endothelium-dependent relaxations to submaximal concentrations of bradykinin in bovine and human coronary arteries. This local mechanism occurs regardless of elevated bradykinin concentrations in the blood and reduced angiotensin II generation.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Arginine; Bradykinin; Captopril; Cattle; Coronary Vessels; Dipeptides; Enalaprilat; Endothelium, Vascular; Fosinopril; Humans; In Vitro Techniques; Lisinopril; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroarginine; Ramipril; Receptors, Bradykinin; Receptors, Neurotransmitter; Vasodilation; Vasodilator Agents

We determined the relationship between cardiac angiotensin-converting enzyme (ACE) inhibition and anti-ischemic efficacy of several structurally different ACE inhibitors or their prodrug esters perfused through the isolated rat heart. Seven ACE inhibitors inhibited cardiac ACE to varying degrees due to differences in uptake during perfusion through nonischemic rat hearts. Zofenopril-sulfhydryl and fosinoprilic acid were the most effective of the free inhibitors. Among the prodrugs, zofenopril and S-benzoylcaptopril, hydrolyzed rapidly by cardiac esterase, were more effective than their component ACE-inhibitors, whereas fosinopril, ramipril and enalapril were poorly active. For studies in ischemic rat hearts, vehicle or drug treatment was initiated 10 min before a 25-min period of global ischemia and during a 30-min reperfusion period. Of five unesterified ACE inhibitors studied for anti-ischemic activity, only captopril and zofenopril-sulfhydryl were found to improve postischemic contractile function and reduce cell death in the isolated rat hearts. Fosinoprilic acid, ramiprilat and enalaprilat were not cardioprotective at high perfusion concentrations, despite the fact that nearly complete inhibition of cardiac ACE was achieved with all of the compounds studied. The S-benzoyl prodrugs of zofenopril-sulfhydryl and captopril were at least as potent as their component ACE inhibitors in reducing ischemic-reperfusion damage in the same model. Neither zofenopril nor captopril, however, had any effect on coronary flow before or after ischemia. Thus, it appears that the cardioprotective effects of zofenopril and captopril are independent of cardiac ACE inhibition or, at least, that ACE inhibition alone is not sufficient. Both captopril and zofenopril are sulfhydryl-containing compounds whereas the inactive compounds are not; and, thus, this group appears to be important in mediating their cardioprotective actions.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Captopril; Coronary Disease; Drug Stability; Enalaprilat; Fosinopril; Heart; In Vitro Techniques; Male; Myocardium; Organophosphorus Compounds; Prodrugs; Proline; Pyrroles; Ramipril; Rats; Rats, Inbred Strains; Sulfhydryl Compounds

The rate of conversion of ramipril (Hoe 498), a new angiotensin converting enzyme (ACE) inhibitor, to its active metabolite was compared with that of enalapril. After intravenous administration to rats, ramipril was very rapidly deesterified to its active moiety, ramiprilat. The ratio of the active metabolite level to the prodrug level in plasma at 5 min after administration was 10.7 for ramipril, which was about 5 times the ratio for enalapril. The in vitro conversion rates of ramipril were higher than those of enalapril in all rat tissue homogenates examined, including the liver, a main site of metabolism. The apparent Km values of ramipril and enalapril in the liver were 190 and 710 mumol/l, respectively, suggesting that ramipril has a higher affinity for esterase than enalapril. In conclusion, ramipril was superior to enalapril in efficiency of conversion to the active metabolite.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Aorta, Thoracic; Biotransformation; Bridged Bicyclo Compounds; Chromatography, Gas; Enalapril; Enalaprilat; In Vitro Techniques; Kidney; Liver; Lung; Male; Muscle, Smooth, Vascular; Prodrugs; Pyrroles; Ramipril; Rats; Rats, Inbred Strains

The kinetics of the steady-state inhibition of angiotension I-converting enzyme (EC 3.4.15.1) at 25 degrees C and 37 degrees C with enalaprilat and ramiprilat can be simulated, assuming only one inhibitor-binding site, consistent with a 1:1 stoichiometry if the protein concentration was determined by amino acid analysis. In this temperature range the apparent inhibition constants for ramiprilat and enalaprilat were roughly doubled by a decrease in the chloride concentration from 0.300 M to 0.120 M.

Topics: Angiotensin-Converting Enzyme Inhibitors; Animals; Chlorides; Enalaprilat; Kinetics; Lung; Mathematics; Molecular Structure; Pyrroles; Rabbits; Ramipril; Sodium Chloride; Thermodynamics

Evidence for effects of angiotensin converting enzyme (ACE) on isolated human glomeruli was provided using specific binding of tritium-labeled ramiprilat, a potent inhibitor of ACE. [3H]ramiprilat bound to isolated glomeruli, depending on time and temperature, displaying a KD of 3.8 nmol/L and a Bmax of 853 fmol/mg protein. Specific binding represented more than 90% of total binding. Dissociation occurred rapidly after dilution of the sample with incubation buffer or after addition of an excess of unlabeled inhibitor. Binding of [3H]ramiprilat was also inhibited by increasing concentrations of enalaprilat, another ACE inhibitor. ACE is a zinc-containing enzyme. Addition of EGTA to the assay, which chelates zinc ions, completely prevented binding. This was reversed by divalent Zn2+ and Ca2+ ions, but not by magnesium. Binding of [3H]ramiprilat to isolated glomeruli was maximal at pH 8, which also is optimal for ACE activity. The binding of [3H]ramiprilat to isolated human glomeruli is specific, and resembles the characteristics which have been found earlier for enzyme activity of ACE. Thus, binding of [3H]ramiprilat to isolated glomeruli can be assumed to be directed to ACE.

Topics: Adult; Angiotensin-Converting Enzyme Inhibitors; Enalapril; Enalaprilat; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Kidney Glomerulus; Protein Binding; Pyrroles; Ramipril