Flufenamic acid, mefenamic acid and niflumic acid inhibit single nonselective cation channels in the rat exocrine pancreas.

The non-steroidal anti-inflammatory drugs, flufenamic acid, mefenamic acid and niflumic acid, block Ca2(+)-activated non-selective cation channels in inside-out patches from the basolateral membrane of rat exocrine pancreatic cells. Half-maximal inhibition was about 10 microM for flufenamic acid and mefenamic acid, whereas niflumic acid was less potent (IC50 about 50 microM). Indomethacin, aspirin, diltiazem and ibuprofen (100 microM) had not effect. It is concluded that the inhibitory effect of flufenamate, mefenamate and niflumate is dependent on the specific structure, consisting of two phenyl rings linked by an amino bridge.

Selective inhibitors of KCl cotransport in human red cells.

Two analogues of the loop diuretics furosemide and bumetanide have been identified as differential inhibitors of KCl and NaKCl cotransport systems, assayed by measuring K+ influx in 'young' human red cells. H25 inhibited both NaKCl and KCl cotransport, with I50% values of 0.03 and 30 microM respectively; H74 had no effect on NaKCl cotransport, even at 0.3 mM, but inhibited KCl cotransport with an I50% of 75 microM. These compounds are therefore useful for resolving the two transport systems.

Cardioselective K(ATP) channel blockers derived from a new series of m-anisamidoethylbenzenesulfonylthioureas.

Sulfonylthioureas exhibiting cardioselective blockade of ATP-sensitive potassium channels (K(ATP) channels) were discovered by stepwise structural variations of the antidiabetic sulfonylurea glibenclamide. As screening assays, reversal of rilmakalim-induced shortening of the cardiac action potential in guinea pig papillary muscles was used to probe for activity on cardiac K(ATP) channels as the target, and membrane depolarization in CHO cells stably transfected with hSUR1/hKir6.2 was used to probe for unwanted side effects on pancreatic K(ATP) channels. Changing glibenclamide's para-arrangement of substituents in the central aromatic ring to a meta-pattern associated with size reduction of the substituent at the terminal nitrogen atom of the sulfonylurea moiety was found to achieve cardioselectivity. An additional change from a sulfonylurea moiety to a sulfonylthiourea moiety along with an appropriate substituent in the ortho-position of the central aromatic system was a successful strategy to further improve potency on the cardiac K(ATP) channel. Among this series of sulfonylthioureas HMR1883, 1-[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl-3-methylthiourea, and its sodium salt HMR1098 were selected for development and represent a completely new therapeutic approach toward the prevention of life-threatening arrhythmias and sudden cardiac death in patients with coronary heart disease.

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.

Inhibitors of nonselective cation channels in cells of the blood-brain barrier.

In the antiluminal membrane of isolated capillaries of rat and porcine brain (blood-brain barrier) nonselective cation channels with g = 31 pS were observed in cell-excised membrane patches. The channel inactivated by decreasing cytosolic Ca2+ below 1 microM and was inhibited by 1 mM ATP on the intracellular side. Anions and divalent cations did not pass the channel, but Na+ and K+ were equally permeant. Like the nonselective cation channel of rat exocrine pancreatic cells, the channel in cerebral capillary endothelial cells was inhibited reversibly by derivatives of diphenylamine-2-carboxylate (DPC), like 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC, ki = 1 microM), and flufenamic acid (ki = 4.9 microM). 4'-methyldiphenylamine-2-carboxylic acid (4-MDPC), 5-chloro-2(3-trifluormethylphenylamino)-3-nitrobenzoic acid, and 5-nitro-2-(3-phenylpropylamino)-2-carboxylic acid (NPPB), as well as the antiinflammatory drug ((Z)-5-chloro2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-ox o-1H-indole-1- carboxamide (Tenidap)) had a relatively low blocking potency (ki > 10 microM). Gadolinium (10 microM), a blocker of stretch-activated channels, inhibited the nonselective cation channel potently.

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.

Effects of the cardioselective KATP channel blocker HMR 1098 on cardiac function in isolated perfused working rat hearts and in anesthetized rats during ischemia and reperfusion.

It has been argued that activation of KATP channels in the sarcolemmal membrane of heart muscle cells during ischemia provides an endogenous cardioprotective mechanism. In order to test whether the novel cardioselective KATP channel blocker HMR 1098 affects cardiac function during ischemia, experiments were performed in rat hearts during ischemia and reperfusion. Isolated perfused working rat hearts were subjected to 30 min of low-flow ischemia in which the coronary flow was reduced to 10% of its control value, followed by 30-min reperfusion. In the first set of experiments the hearts were electrically paced at 5 Hz throughout the entire protocol. At the end of the 30-min ischemic period the aortic flow had fallen to 44 +/- 2% (n=8) of its nonischemic value in vehicle-treated hearts, whereas in the presence of 0.3 micromol/l and 3 micromol/l HMR 1098 it had fallen to 29 +/- 7% (n=5, not significant) and 8 +/- 2% (n=12, P<0.05), respectively. Glibenclamide (3 micromol/l) reduced the aortic flow to 9.5 +/- 7% (n=4, P<0.05). In control hearts the QT interval in the electrocardiogram shortened from 63 +/- 6 ms to 36 +/- 4 ms (n=10, P<0.05) within 4-6 min of low-flow ischemia. This shortening was completely prevented by 3 micromol/l HMR 1098 (60 +/- 5 ms before ischemia, 67 +/- 6 ms during ischemia, n=9, not significant). When rat hearts were not paced, the heart rate fell spontaneously during ischemia, and HMR 1,098 (3 micromol/l) caused only a slight, statistically non-significant reduction in aortic flow during the ischemic period. In order to investigate whether HMR 1098 shows cardiodepressant effects in a more pathophysiological model, the left descending coronary artery was occluded for 30 min followed by reperfusion for 60 min in anesthetized rats. Treatment with HMR 1098 (10 mg/kg i.v.) had no statistically significant effects on mean arterial blood pressure and heart rate during the control, ischemia and reperfusion periods. At the end of the reperfusion period, aortic blood flow was slightly reduced by HMR 1098, without reaching statistical significance (two-way analysis of ANOVA, P=0.15). Myocardial infarct size as a percentage of area at risk was not affected by HMR 1098 (vehicle: 75 +/- 3%, HMR 1098: 72 +/- 2%, n=7 in each group). In conclusion, cardiodepressant effects of HMR 1098 were observed only in isolated perfused working rat hearts which were continuously paced during global low-flow ischemia. In the model of anesthetized rats subjected to regional ischemia, HMR 1098 had no significant effect on cardiac function or infarct size.

K(ATP) channel blocker HMR 1883 reduces monophasic action potential shortening during coronary ischemia in anesthetised pigs.

ATP-sensitive potassium channels (KATP) open during myocardial ischemia. The ensuing repolarising potassium efflux shortens the action potential. Accumulation of extracellular potassium is able to partially depolarise the membrane, reducing the upstroke velocity of the action potential and thereby impairing impulse conduction. Both mechanisms are believed to be involved in the development of reentrant arrhythmias during cardiac ischemia. The sulfonylthiourea HMR 1883 (1-[[5-[2-(5-chloro-O-anisamido)ethyl]-methoxyphenyl]sulfonyl]-3-m ethylthiourea) was designed as a cardioselective KATP channel blocker for the prevention of arrhythmic sudden death in patients with ischemic heart disease. The aim of this study was to show that this compound, which has already shown antifibrillatory efficacy in dogs and rats, is able to inhibit ischemic changes of the action potential induced by coronary artery occlusion in anesthetised pigs. Action potentials were taken in situ with the technique of monophasic action potential (MAP) recording. In a control group (n=7), three consecutive occlusions of a small branch of the left circumflex coronary artery resulted in reproducible reductions in MAP duration and a decrease in upstroke velocity. In a separate group (n=7), HMR 1883 (3 mg/kg i.v.) significantly (P<0.05) reduced the ischemia-induced shortening of the MAP: during the first and second control occlusion of the coronary artery in the HMR 1883-group, MAP50 duration shortened from 218.5 +/- 3.0 ms to 166.7 +/- 3.3 ms and from 219.7 +/- 4.5 ms to 164.9 +/- 1.8 ms, respectively. After HMR 1883, during the third occlusion, MAP duration decreased from 226.9 +/- 3.6 ms to 205.3 +/- 4.3 ms only corresponding to 59% inhibition. HMR 1883 also improved the upstroke velocity of the MAP, which was depressed by ischemia: in the two preceding control occlusions ischemia prolonged the time to peak of the MAP, an index for upstroke velocity, from 10.83 +/- 0.43 ms to 39.42 +/- 1.60 ms and from 12.97 +/- 0.40 ms to 37.17 +/- 2.98 ms, respectively. With HMR 1883, time to peak during ischemia rose from 12.42 +/- 0.51 ms to 25.53+/-2.51 ms only, corresponding to an average inhibitory effect of 53.4%. The irregular repolarisation contour of the ischemic MAP was also improved. In conclusion, the present results indicate that HMR 1883 effectively blocks myocardial KATP channels during coronary ischemia in anesthetised pigs, preventing an excessive shortening of the action potential and improving excitation propagation.

The K(ATP) channel blocker HMR 1883 does not abolish the benefit of ischemic preconditioning on myocardial infarct mass in anesthetized rabbits.

Previous experimental studies showed that the benefit of ischemic preconditioning (IPC) is abolished by K(ATP) channel blockade with glibenclamide. However, the newly discovered K(ATP) channel blocker HMR 1883 (1-[[5-[2-(5-chloro-o-anisamido)ethyl]-methoxyphenyl]sulfonyl]-3-m ethylthiourea) shows marked antifibrillatory activity in the dose range of 3 mg/kg to 10 mg/kg i.v. in various experimental models without affecting blood glucose levels. In order to investigate in a head to head comparison glibenclamide and HMR 1883 with respect to their influence on IPC, experiments were performed in rabbits with ischemia-reperfusion using myocardial infarct mass as final read out. Male New Zealand White rabbits (2.6-3.0 kg) were subjected to 30-min occlusion of a branch of the left descending coronary artery (LAD) followed by 2-h reperfusion. For IPC experiments the LAD was additionally occluded for two periods of 5 min, each followed by 10-min reperfusion, before the long-term ischemia. Infarct mass was evaluated by TTC staining and expressed as a percentage of area at risk. Rabbits (n=7/group) were randomly selected to receive (i.v.) saline vehicle 5 min prior to the 30-min occlusion period in infarct studies without IPC or to receive glibenclamide (0.3 mg/kg) or HMR 1883 (3 mg/kg) in IPC experiments, these substances being given 5 min prior to the first preconditioning or 5 min prior to the long-term ischemia of 30 min. Myocardial risk mass as a percentage of left ventricular mass did not differ between groups. The same was true for the ratio of left ventricular mass to 100 g body weight. Myocardial infarct mass as a percentage of the area at risk in the saline vehicle group without IPC was 41+/-3%. Whereas glibenclamide significantly increased infarct mass (from 41+/-3% to 55+/-4%), HMR 1883 did not affect it. IPC reduced infarct mass from 41+/-3% to 21+/-4% (P<0.05 vs. control without IPC). Glibenclamide given prior to IPC or prior to the long-term ischemia totally abolished the IPC effect (42+/-2% and 55+/-4%, respectively; P<0.05 vs. control). In contrast, HMR 1883 under the same conditions did not affect infarct size when given prior to IPC or prior to the long-term ischemia (21+/-3% and 26+/-2%, respectively). The monophasic action potential duration (MAP50) was reduced from 103+/-3 ms under normoxic conditions to 82+/-2 ms, 5 min after ischemia in the absence of drugs. This ischemia-induced shortening of the MAP was prevented by both HMR 1883 (MAP50 103+/-3 ms) and glibenclamide (MAP50 106+/-3 ms). In conclusion, although both K(ATP) channel blockers prevented ischemia-induced shortening of MAP, HMR 1883 did not abolish the beneficial effects of IPC on myocardial infarct mass in rabbits, whereas glibenclamide totally reversed this cardioprotective effect of IPC. This suggests that the sarcolemmal ATP-sensitive potassium channels are not involved in the mechanism of IPC.

HMR 1883, a novel cardioselective inhibitor of the ATP-sensitive potassium channel. Part II: effects on susceptibility to ventricular fibrillation induced by myocardial ischemia in conscious dogs.

The activation of the ATP-sensitive potassium channel (KATP) during myocardial ischemia leads to potassium efflux, reductions in action potential duration and the formation of ventricular fibrillation (VF). Drugs that inactivate KATP should prevent these changes and thereby prevent VF. However, most KATP antagonists also alter pancreatic channels, which promote insulin release and hypoglycemia. Recently, a cardioselective KATP antagonist, HMR 1883, has been developed that may offer cardioprotection without the untoward side effects of existing compounds. Therefore, VF was induced in 13 mongrel dogs with healed myocardial infarctions by a 2-min coronary artery occlusion during the last minute of a submaximal exercise test. On subsequent days, the exercise-plus-ischemia test was repeated after pretreatment with HMR 1883 (3.0 mg/kg i.v., n = 13) or glibenclamide (1.0 mg/kg i.v., n = 7). HMR 1883 (P < .001) and glibenclamide (P < .01) prevented VF in 11 of 13 and 6 of 7 animals, respectively. Glibenclamide, but not HMR 1883, elicited increases in plasma insulin and reductions in blood glucose. Glibenclamide also reduced (P < .01) both mean coronary blood flow and left ventricular dP/dt maximum as well as the reactive hyperemia induced by 15-sec coronary occlusions (-30.3 +/- 11%), whereas HMR 1883 did not alter this increase in coronary flow (-3.0 +/- 4.7%). Finally, myocardial ischemia (n = 10) significantly (P < .01) reduced refractory period (control, 121 +/- 2 msec; occlusion, 115 +/- 2 msec), which was prevented by either glibenclamide or HMR 1883. Thus, the cardioselective KATP antagonist HMR 1883 can prevent ischemically induced reductions in refractory period and VF without major hemodynamic effects or alterations in blood glucose levels. These data further suggest that the activation of KATPs may play a particularly important role in both the reductions in refractory period and lethal arrhythmia formation associated with myocardial ischemia.