Udo Albus

Protective effects of HOE642, a selective sodium-hydrogen exchange subtype 1 inhibitor, on cardiac ischaemia and reperfusion

OBJECTIVE The aim was to characterise the new compound HOE642 as a selective and cardioprotective Na+/H+ exchange inhibitor in various models. METHODS The effect of HOE642 was tested in the osmotically activated Na+/H+ exchange of rabbit erythrocytes and in propionate induced swelling of human thrombocytes. Recovery of pH after an NH4Cl prepulse and effects on other ion transport systems by patch clamp technique were investigated in rat cardiomyocytes. NHE subtype specifity of the compound was determined by 22Na+ uptake inhibition in a fibroblast cell line separately expressing subtype isoforms 1-3. Protective effects of HOE642 in cardiac ischaemia and reperfusion by ligation of coronary artery were investigated in isolated working rat hearts and in anaesthetised rats. RESULTS HOE642 concentration dependently inhibited the amiloride sensitive sodium influx in rabbit erythrocytes, reduced the swelling of human platelets induced by intracellular acidification, and delayed pH recovery in rat cardiomyocytes. In the isolated working rat heart subjected to ischaemia and reperfusion HOE642 dose dependently reduced the incidence and the duration of reperfusion arrhythmias. It also reduced the the release of lactate dehydrogenase and creatine kinase, and preserved the tissue content of glycogen, ATP, and creatine phosphate. In anaesthetised rats undergoing coronary artery ligation intravenous and oral pretreatment with HOE642 caused a dose dependent reduction or a complete prevention of ventricular premature beats, ventricular tachycardia, and ventricular fibrillation. The compound was well tolerated and neutral to circulatory variables. Other cardiovascular agents tested in this model were not, or were only partly, effective at doses showing marked cardiodepressive effects. CONCLUSIONS HOE642 is a very selective NHE subtype 1 inhibitor showing cardioprotective and antiarrhythmic effects in ischaemic and reperfused hearts. Further development of well tolerated compounds like HOE642 could lead to a new therapeutic approach in clinical indications related to cardiac ischaemia and reperfusion.

Hoe 694, a new Na+/H+ exchange inhibitor and its effects in cardiac ischaemia

1 The benzoylguanidine derivative Hoe 694 ((3‐methylsulphonyl‐4‐piperidino‐benzoyl) guanidine methanesulphonate) was characterized as an inhibitor of Na+/H+ exchange in rabbit erythrocytes, rat platelets and bovine endothelial cells. The potency of the compound was slightly lower or comparable to ethylisopropyl amiloride (EIPA). 2 To investigate a possible cardioprotective role of the Na+/H+ exchange inhibitor Hoe 694, rat isolated working hearts were subjected to ischaemia and reperfusion. In these experiments all untreated hearts suffered ventricular fibrillation on reperfusion. Addition of 10−7 m Hoe 694 to the perfusate almost abolished reperfusion arrhythmias in the rat isolated working hearts. 3 Hoe 694 reduced the release of lactate dehydrogenase (LDH) and creatine kinase (CK), which are indicators of cellular damage during ischaemia, into the venous effluent of the hearts by 60% and 54%, respectively. 4 The tissue content of glycogen at the end of the experiments was increased by 60% and the high energy phosphates ATP and creatine phosphate were increased by 240% and 270% respectively in the treated hearts as compared to control hearts. 5 Antiischaemic effects of the Na+/H+ exchange inhibitor, Hoe 694, were investigated in a second experiment in anaesthetized rats undergoing coronary artery ligation. In these animals, pretreatment with Hoe 694 caused a dose‐dependent reduction of ventricular premature beats and ventricular tachycardia as well as a complete suppression of ventricular fibrillation down to doses of 0.1 mg kg−1, i.v. Blood pressure and heart rate remained unchanged. 6 We conclude that the new Na+/H+ exchange inhibitor, Hoe 694, shows cardioprotective and antiarrhythmic effects in ischaemia and reperfusion in rat isolated hearts and in anaesthetized rats. In view of the role which Na+/H+ exchange seems to play in the pathophysiology of cardiac ischaemia these effects could probably be attributed to Na+/H+ exchange inhibition.

Ramipril prevents left ventricular hypertrophy with myocardial fibrosis without blood pressure reduction: a one year study in rats.

1 Angiotensin converting enzyme (ACE)‐inhibitors have been demonstrated to be effective in the treatment of cardiac hypertrophy when used in antihypertensive doses. The aim of our one year study with an ACE‐inhibitor in rats was to separate local cardiac effects produced by a non‐antihypertensive dose from those on systemic blood pressure when an antihypertensive dose was used. 2 Rats made hypertensive by aortic banding were subjected to chronic oral treatment for one year with an antihypertensive dose of the ACE inhibitor, ramipril 1 mg kg−1 daily, (RA 1 mg) or received a low dose of 10 μg kg−1 daily (RA 10 μg) which did not affect high blood pressure. 3 Chronic treatment with the ACE‐inhibitor prevented left ventricular hypertrophy in the antihypertensive rats as did the low dose which had no effects on blood pressure. Similar effects were observed on myocardial fibrosis. Plasma ACE activity was inhibited in the RA 1 mg but not in the RA 10 μg group although conversion of angiotensin (Ang) I to Ang II in isolated aortic strips was suppressed in both treated groups. Plasma catecholamines were increased in the untreated control group, but treatment with either dose of ramipril normalized the values. The myocardial phosphocreatine to ATP ratio (an indicator of the energy state in the heart) was reduced in the vehicle control group whereas the hearts from treated animals showed a normal ratio comparable to hearts from sham‐operated animals. 4 After one year, five animals were separated from each group, treatment withdrawn, and housed for additional six months. In the RA 1 mg group, blood pressure did not reach the value of the control vehicle group and surprisingly, left ventricular hypertrophy and myocardial fibrosis did not recur in animals during withdrawal of treatment. 5 These data show that long term ACE inhibitor treatment with ramipril in antihypertensive and non‐antihypertensive doses prevented cardiac hypertrophy and myocardial fibrosis. This protective effect was still present after 6 months treatment withdrawal.

Inhibition of endogenous nitric oxide synthase potentiates ischemia–reperfusion-induced myocardial apoptosis via a caspase-3 dependent pathway

OBJECTIVE Apoptosis of cardiomyocytes may contribute to ischemia-reperfusion injury. The role of nitric oxide (NO) in apoptosis is controversial. Therefore, we investigated the effect of NO synthase inhibition on apoptosis of cardiomyocytes during ischemia and reperfusion and elucidated the underlying mechanisms. METHODS AND RESULTS Isolated perfused rat hearts (n = 6/group) were subjected to ischemia (30 min) and reperfusion (30 min) in the presence or absence of the NO synthase inhibitor NG-mono-methyl-L-arginine. Reperfusion induced cardiomyocyte apoptosis as assessed by immunohistochemistry (TUNEL-staining) and the demonstration of the typical DNA laddering. Apoptosis during reperfusion was associated with the cleavage of caspase-3, the final down-stream executioner caspase, whereas the protein levels of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax were unchanged. Inhibition of the NO synthase drastically increased ischemia and reperfusion-induced apoptosis of cardiomyocytes. Moreover, the NO synthase inhibitor enhanced the activation of caspase-3, suggesting that NO interferes with the activation of caspases in ischemia-reperfusion. CONCLUSION The results of the present study demonstrate that inhibition of endogenous NO synthesis during ischemia and reperfusion leads to an enhanced induction of apoptosis, suggesting that the endogenous NO synthesis protects against apoptotic cell death. Inhibition of NO synthesis thereby activates the caspase cascade, whereas the Bcl-2/Bax protein levels remained unchanged.

Effects of the Na+/H+-exchange inhibitor Hoe 642 on intracellular pH, calcium and sodium in isolated rat ventricular myocytes.

Abstract The inhibitors of the Na+/H+-exchange (NHE1) system Hoe 694 and Hoe 642 possess cardioprotective effects in ischaemia/reperfusion. It is assumed that these effects are due to the prevention of intracellular sodium (Nai) and calcium (Cai) overload. The purpose of the present study was to investigate the effects of Hoe 642 on intracellular pH, Na+ and Ca2+ (pHi, Nai and Cai) in isolated rat ventricular myocytes under anoxic conditions or in cells in which oxidative phosphorylation had been inhibited by 1.5 mmol/l cyanide. In cells which were dually loaded with the fluorescent dyes 2,7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and Fura-2, anoxia caused acidification of the cells (from pHi 7.2 to pHi 6.8) and an increase in Cai from about 50 nmol/l to about 1 μmol/l. The decrease in pHi began before the cells underwent hypoxic (rigor) contracture, whereas Cai only began to rise after rigor shortening had taken place. After reoxygenation, pHi returned to its control value and Cai oscillated and then declined to resting levels. It was during this phase that the cells rounded up (hypercontracture). When 10 μmol/l Hoe 642 was present from the beginning of the experiment, pHi and Cai were not significantly different from control experiments. At reoxygenation, pHi did not recover, but Cai oscillated and returned to its resting level. To monitor Nai, the cells were loaded with the dye SBFI. After adding 1.5 mmol/l cyanide or 100 μmol/l ouabain, Nai increased from the initial 8 mmol/l to approximately 16 mmol/l. Hoe 642 or Hoe 694 (10 μmol/l) did not prevent the increase in Nai. In contrast, the blocker of the persistent Na+ current R56865 (10 μmol/l) attenuated the CN–-induced rise in Nai. The substance ethylisopropylamiloride was not used because it augmented considerably the intensity of the 380 nm wavelength of the cell’s autofluorescence. In conclusion, the specific NHE1 inhibitor Hoe 642 did not attenuate anoxia-induced Cai overload, nor CN–-induced Nai and Cai overload. Hoe 642 prevented the recovery of pHi from anoxic acidification. This low pHi maintained after reoxygenation may be cardioprotective. Other possible mechanisms of NHE1 inhibitors, such as prevention of Ca2+ overload in mitochondria, cannot be ruled out. The increase in Nai during anoxia is possibly due to an influx of Na+ via persistent Na+ channels.

Effects of Na+/H+ exchange inhibitors in cardiac ischemia

To investigate a possible protective role of Na+/H+ exchange inhibition under ischemic conditions isolated rat hearts were subjected to regional ischemia and reperfusion. In these experiments all 6 untreated hearts suffered ventricular fibrillation on reperfusion. Addition of 1 x 10(-5) mol/l amiloride or 3 x 10(-7) mol/l 5-(N-ethyl-N-isopropyl)amiloride (EIPA) markedly decreased the incidence and duration of ventricular fibrillation or even suppressed fibrillation completely as in the case of 1 x 10(-6) mol/l EIPA. Both compounds diminished the activities of lactate dehydrogenase and creatine kinase in the venous effluent of the hearts during ischemia. At the end of the experiments tissue contents of glycogen, ATP and creatine phosphate were increased in the treated hearts as compared to control hearts. In an additional experiment the beneficial effects of Na+/H+ exchange inhibition during ischemia was confirmed in vivo with anaesthetized rats undergoing coronary artery ligation. In these animals amiloride or EIPA pretreatment caused a marked reduction of ventricular premature beats and ventricular tachycardia as well as a complete suppression of ventricular fibrillation. The concentration dependent inhibition of Na+ influx via Na+/H+ exchange by amiloride and EIPA was investigated in erythrocytes from hypercholesterolemic rabbits with Na+/H+ exchange activated by exposure to hyperosmotic medium. Furthermore the inhibition of Na+ influx by EIPA after intracellular acidification was studied in cardiac myocytes of neonatal rats. Both agents were effective in the same order of potency in the ischemic isolated working rat heart as in the erythrocyte model in which they inhibited Na+/H+ exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac arrhythmias are ameliorated by local inhibition of angiotensin formation and bradykinin degradation with the converting-enzyme inhibitor ramipril

SummaryWe investigated the influence of the angiotensin-converting enzyme (ACE) inhibitor ramipril on cardiac arrhythmias in guinea pigs and rats. Ramiprilat, the active moiety of ramipril, did not influence action potentials of isolated guinea-pig papillary muscle or rabbit sinus node, thereby excluding cellular electrophysiological evidence of anti-arrhythmic properties. Ramipril protected against cardiac arrhythmias induced by digoxin infusion in guinea pigs. This effect was comparable with that of lidocaine. In isolated perfused ischemic working rat hearts, angiotensin (ANG) I (3×10−9 M/l) and ANG II (1×10−9 M/l) aggravated reperfusion arrhythmias, accompanied by deterioration of cardiodynamic and metabolic events. Bradykinin (BK) (1×10−10−1×10−8 M/l), in contrast, protected against reperfusion arrhythmias, which corresponded to an increase in energy-rich phosphates and glycogen stores and a decrease in lactate levels in myocardial tissue. Identical changes were seen in hearts from rats pretreated with ramipril (1 mg/kg PO) or perfused with ramiprilat (2.58 ×10−7−2.58×10−5 M/l). Local ACE inhibition in these ischemic hearts antagonized ANG I but not ANG II effects and enhanced BK effects. The BK antagonist D-Arg-(Hyp2, Thi5,8, D-Phe7) BK abolished the beneficial effects of BK, ramipril, and ramiprilat. Increased concentrations of BK or ramiprilat were able to reverse the antagonism.The antiarrhythmic agent nicainoprol, a fast-sodiumchannel blocking drug (class Ib), also protected isolated rat hearts against reperfusion arrhythmias, but was without beneficial effects on cardiac hemodynamics and biochemical parameters, in contrast to the ACE inhibitor.These results suggest that the beneficial effects of the ACE inhibitor ramipril on digoxin and reperfusion arrhythmias are not mediated by their direct actions on ionic channels in the cell membrane. It seems that other factors are responsible for its beneficial effects on reperfusion arrhythmias, cardiac function, and metabolism, which are associated with a reduction in ANG II generation and BK degradation by local ACE inhibition in the heart.

Development of the Na+/H+ exchange inhibitor cariporide as a cardioprotective drug: from the laboratory to the GUARDIAN trial.

In the late 1980 and the beginning of the 1990s, it became clear that Na1/H1 exchange subtype 1 (NHE1) plays a signi~cant role in the pathophysiology of cardiac ischemia/reperfusion. Excessive activation of NHE1 leads to energy depletion and intracellular sodium and calcium overload, which can cause many types of injuries, including arrhythmias, contracture, stunning, and necrosis, in the endangered myocardium. By reducing sodium in_ux during ischemia/reperfusion, NHE inhibitors preserve high-energy phosphates, prevent calcium overload, and signi~cantly delay the process that leads from the ischemic state in cardiac tissue to necrosis. Cardioprotective effects were ~rst demonstrated with amiloride-type NHE inhibitors such as amiloride itself and several derivatives, which have been used in a multitude of preclinical studies. Benzoylguanidinetype NHE inhibitors were later synthesized. Some of these compounds, especially HOE 694 and its successor HOE 642 (Cariporide), showed superior potency and selectivity for the NHE system. The structure of the benzoylguanidine compound cariporide (4-isopropyl-3-methylsulfonyl-benzoylguanidine methanesulfonate) is shown in Figure 1. Cariporide has been characterized as a potent and speci~c inhibitor of Na1/H1 exchanger subtype 1 (NHE1) in different cell types from various species. Protective effects in ischemia/reperfusion have been demonstrated with the NHE inhibitor cariporide in isolated heart cells and in isolated hearts. Unique and consistent cardioprotection has been shown with the NHE inhibitor in numerous animal models of cardiac ischemia/ reperfusion. Cariporide has been found to reduce related injuries such as arrhythmias, contracture, stunning, and necrosis, and to prevent mortality in animals. In preclinical studies, the highly speci~c NHE1 inhibitor cariporide was well tolerated and showed a favorable kinetics/metabolism pro~le as well as excellent bioavailability. Cariporide was thus selected over other benzoylguanidine compounds for development as a cardioprotective drug.

Airway pharmacology of the potassium channel opener, HOE 234, in guinea pigs: in vitro and in vivo studies

The smooth muscle relaxant effects of the novel potassium channel opener, HOE 234, were investigated in guinea pig airways and compared with those of lemakalim (BRL 38227). Both agents evoked concentration-related reduction in spontaneous tracheal tone or in the tone induced by histamine, prostaglandin E2 or carbachol. HOE 234 was more potent, particularly against carbachol, and was considerably longer acting than lemakalim in a wash-out experiment. On testing for preventive efficacy against histamine-induced bronchoconstriction in anaesthetized animals a dose-related decrease of pulmonary resistance (RL) was observed. HOE 234 given either intravenously (i.v.) or by inhalation was longer acting and 3 and 6 times more potent than lemakalim. Administration of 30 micrograms/kg i.v. HOE 234 during continuous bronchoconstriction maintained by infusion of histamine decreased RL for more than 20 min whereas the effect of 100 micrograms/kg i.v. lemakalin disappeared within 4 min. These results show that HOE 234 is effective against contractile response induced by asthma mediators in guinea pig airways and compares favourably with lemakalim. Moreover it acts on acute existing bronchospasm and therefore has the potential to act against asthma attacks.

Atrial natriuretic factor protects the isolated working ischaemic rat heart against the action of angiotensin II

The interaction between atrial natriuretic factor [synthetic human ANF-(103–126)] and angiotensin II (Ang II) and its influence on reperfusion arrhythmias, cardiodynamics, enzyme loss and metabolic changes were investigated in isolated ischaemic working rat hearts. Acute regional myocardial ischaemia was induced by coronary artery occlusion which was associated with ventricular fibrillation. Perfusion with 1 x 10-9 mol/l Ang II markedly aggravated these arrhythmias. Perfusion with 1 x 107 mol/l ANF, in contrast, gave protection against ventricular fibrillation and prevented Ang II-induced aggravation of ventricular fibrillation. Atrial natriuretic factor improved cardiodynamics, in particular, during reperfusion, whereas Ang II impaired cardiodynamics and increased the release of creatine kinase and lactate dehydrogenase. These adverse effects of Ang II were absent when ANF was simultaneously perfused. Compared with control hearts, myocardial tissue levels of glycogen, ATP and creatine phosphate were increased in hearts perfused with either ANF or ANF plus Ang II, whereas lactate levels decreased. Perfusion with Ang II alone led to deterioration in these metabolic parameters. These results in isolated working rat hearts suggest that ANF protects against the consequences of ischaemia and reperfusion and that functional antagonism between ANF and Ang II may contribute to this.