Rochus Jonas

The Two Mechanisms of Action of Racemic Cardiotonic Emd 53998, Calcium Sensitization and Phosphodiesterase Inhibition, Reside in Different Enantiomers

The novel cardiotonic EMD 53 998 increases contractile force in vitro through both inhibition of phosphodiesterase III (PDE III) activity and increase in the responsiveness of the contractile proteins to calcium (“calcium sensitization”). Because EMD 53 998 is a racemate, the possibility arose that the two modes of action do not reside equally in the enantiomers. Therefore, the effects of the racemate and its two enantiomers [(+)EMD 57 033 and (-)EMD 57 439] were analyzed in guinea pig and rat cardiac tissue with respect to Ca2+ sensitization (Ca2+-induced force development in skinned cardiac myofibers and myofibrillar ATPase activity) and PDE III inhibition (isolated PDE isoenzymes and cyclic AMP level in isolated cardiac myocytes). In addition, the positive inotropic effects were compared in isometrically contracting papillary muscles. Enhancement of force of contraction (Fc) in submaximally activated skinned fibers showed a selectivity for the (+)enantiomer with EC50 = 1.7, 4.8, and <100 μM for EMD 57 033, EMD 53 998, and EMD 57 439, respectively. Ca2+ concentration for half-maximal activation was decreased by 0.5 log units, and Cmax was increased by 15% at 10 μM EMD 57 033. Similarly, myofibrillar ATPase activity was most potently enhanced by the (+)enantiomer, with EC50 values of 1.8, 2.5, and <30 μM for EMD 57 033, EMD 53 998, and EMD 57 439. respectively. PDE III activity was inhibited with greater potency by the (-)enantiomer, with IC50 values of 0.05, 0.06, and 1.94 μM for EMD 57 439, EMD 53 998, and EMD 57 033, respectively. The cyclic AMP content of isoprenaline-stimulated rat cardiac myocytes was increased by 50% at 13.6 and 0.71 μM for EMD 57 033 and EMD 57 439, respectively. In intact guinea pig papillary muscle, the positive inotropic effect of the (+)enantiomer was insensitive to isoprenaline pretreatment; in contrast, the (-)enantiomer showed only a weak positive inotropic action which was strongly enhanced in the presence of isoprenaline. We conclude that one of the two different mechanisms underlying the overall positive inotropic activity of EMD 53 998 can be assigned, almost exclusively, to one of the two enantiomers. Thus, the (-)enantiomer EMD 57 439 is a “pure” PDE III inhibitor with almost no Ca2+ sensitizing activity; the (+)enantiomer EMD 57 033 is a potent Ca2+ sensitizer with only a weak PDE III inhibitory activity as compared with the racemate. In contrast to other compounds with mixed activity, EMD 57 033 is unique in possessing both a high absolute potency at the level of the contractile elements and a favorable relation of Ca2+ sensitization to PDE inhibition.

The novel cardiotonic agent EMD 53 998 is a potent calcium sensitizer

EMD 53 998, a novel thiadiazinone derivative, increases the contractile force of cardiac tissue in vitro through both an inhibition of phosphodiesterase III (PDE III) and a sensitization of cardiac contractile proteins to Ca2+. Guinea pig ventricular PDE III is selectively inhibited by EMD 53 998 (IC50 = 60 nM) without major effects on other PDE isoenzymes. Consonant with this is an increase in cAMP content of rat ventricular cells and a potentiation by EMD 53 998 of the cAMP-elevating action of isoprenaline (increase by 50% at 1.3 μM). Sensitization to Ca2+ by EMD 53 998 (3–30 μM) finds its expression in a leftward shift of the Ca2+ response curve for force generation in skinned fibers from porcine ventricular muscle and failing human heart as well as for activation of bovine cardiac myofibrillar actomyosin ATPase. Interestingly, EMD 53 998 elevates the maximum of the Ca2+ -response curve for both parameters. Pimobendan studied under identical conditions was 100 times less potent than EMD 53 998. EMD 53 998 increases force development of guinea pig papillary muscle in a concentration-dependent manner with an EC50 of 3.6 μM. thus being 10 times more potent than pimobendan. In contrast to pimobendan, the positive inotropic effect of EMD 53 998 is barely affected by carbachol. Further evidence for a Ca2+ -sensitizing effect of EMD 53 998 is provided by an additional increase in force generation in the presence of supramaximal isoprenaline concentrations. It is concluded that the positive inotropic action of EMD 53 998 is mediated through both cAMP-independent and cAMP-dependent mechanisms, with the former probably prevailing. We are not aware of other compounds with a similarly high Ca2+ -sensitizing potency. On these grounds, EMD 53 998 appears to be a promising inotropic agent.

Synthesis and biological activities of meribendan and related heterocyclic benzimidazolo-pyridazinones

Abstract The synthesis of new heterocyclic benzimidazolo-pyridazinones is described. The new compounds were evaluated as inotropic agents with ‘calcium-sensitizing’ effects. 5-Methyl-6-[2-(3-pyrazolyl)-5-benzimidazolyl]-2,3,4,5-tetrahydro-pyridazin-3-one hydrochloride (meribendan) turned out to be the most interesting compound and was chosen for development as a positive inotrope.

In vivo evidence of positive inotropism of EMD 57 033 through calcium sensitization

The previous separation of the racemic cardiotonic thiadiazinone derivative EMD 53998 yielded two enantiomers with different pharmacologic properties: EMD 57,033, a potent Ca2+ sensitizer with some residual phosphodiesterase III (PDE III) inhibition, and EMD 57,439, a pure PDE III inhibitor. Although numerous in vitro studies demonstrated the ability of EMD 57,033 to increase the responsiveness of cardiac contractile proteins to Ca2+, in vivo evidence for such an action is lacking. Because there is no possibility of directly proving Ca2+ sensitization in vivo, we attempted to exclude PDE III inhibition as a major contributing component of the positive inotropic action of EMD 57,033. In anesthetized rats, EMD 57,033 increased left ventricular (LV) first derivative of change in systolic pressure over time (dP/dt max) without affecting blood pressure. In contrast, the PDE III-inhibitory enantiomer EMD 57,439 decreased blood pressure. The pattern of hemodynamic effects in anesthetized dogs revealed similar differences between EMD 57,033 and PDE inhibitors. Thus the increase in LV dP/dt max in response to EMD 57,033 was not accompanied by changes of heart rate and blood pressure. As expected for PDE inhibitors, pimobendan and milrinone increased cardiac contractile force in dogs, concomitant, however, with tachycardia, hypotension, and a decrease in total peripheral resistance. When regional contractility was measured separately in two different areas of the dog myocardium, the positive inotropic action of the PDE inhibitors pimobendan and milrinone was antagonized by local coronary infusion of acetylcholine. The cardiotonic effect of the Ca2+ sensitizer EMD 57,033 was entirely resistant to inhibition by acetylcholine. In conscious dogs, beta-blockade markedly attenuated the increase in LV dP/dt max produced by two different doses of the PDE III inhibitor EMD 57,439. In contrast, a dose of EMD 57,033 equieffective in positive inotropic action with the lower dose of EMD 57,439 remained unaffected by < b tau-blockade. We concluded (a) that EMD 57,033 increases cardiac contractile force in two species in vivo, (b) that this action is independent of the cardiac cyclic adenosine monophosphate (AMP) system, (c) that EMD 57,033 does not reduce blood pressure and increase heart rate, an action indicative of PDE inhibition, and (d) that, on the basis of numerous previous in vitro findings, the mechanism of action of EMD 57,033, also in vivo, is consistent with sensitization of the cardiac myofibrils to Ca2+. Of special importance is the finding that this Ca2+ sensitizer at appropriate doses may be able to improve systolic function without adverse effects on diastolic function, as indicated by a slight decrease in left ventricular end-diastolic pressure.

Preparation of the enantiomers of the novel CA-sensitizer EMD 53 998

Abstract Both enantiomers of EMD 53 998 (1), a novel cardiotonic with Ca-sensitizing activity, were readily prepared by kinetic resolution of the N-acylthiadiazinone 2. The Ca-sensitizing effect is highly stereospecific and resides in the (+)-enantiomer. The (−)-entantiomer is a pure PDE inhibitor devoid of any Ca-sensitizing activity.

STEREOSELECTIVITY OF THE POSITIVE INOTROPIC EFFECTS OF NEWER DIAZINONE-CARDIOTONICS

Abstract In 5-methyl-dihydropyridazinones, a stereoselectivity is observed only with respect to the phosphodiesterase III-inhibition. The (−)-enantiomers are very strong inhibitors whereas their (+)-counterparts exhibit only a weak activity. In the case of the thiadiazinone EMD 53998 a pronounced stereoselectivity regarding Ca-sensitivity and phosphodiesterase inhibition is apparent. It is concluded that Levosimendan exerts its positive inotropism almost exclusively through its potent phosphodiesterase III-inhibitory effect.