Michael Klockow

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.

A novel positive inotropic substance enhances contractility without increasing the Ca2+ transient in rat myocardium

We have investigated the mechanism of action of a novel positive inotropic agent, the thiadiazinone derivative 5-(1-(3,4-dimethoxybenzoyl)-1,2,3,4-tetrahydrochinolin-6-yl)-6- methyl-3,6-dihydro-2H-1,3,4-thiadiazin-2-on (EMD 53998). This substance inhibits phosphodiesterase III and, in skinned myocardial fibers, it increases myofilament sensitivity to Ca2+. However the effects of EMD 53998 on intact myocardial preparations are still undefined. In isolated rat hearts EMD 53998 (0.5 to 5 microM) had a dose-dependent effect to increase left ventricular systolic pressure. In isolated left ventricular myocytes loaded with the ester derivative of the Ca2+ probe indo-1, EMD 53998 (0.5 to 5 microM) enhanced twitch amplitude without increasing the associated indo-1 transient. The myofilament responsiveness to Ca2+ was assessed as the relationship between twitch and the indo-1 transient amplitudes as the latter is varied by altering the bathing [Ca2+], or stimulation pattern. EMD 53998 reversibly shifted this relationship to the left which indicates that for indo-1 transients of the same amplitude in the absence and presence of the drug, twitch amplitude was enhanced by EMD 53998. In isolated myocytes studied in the absence of electrical stimulation, EMD 53998. (1.5 to 5 microM) had a concentration-dependent effect to markedly and reversibly decrease cell length without increasing indo-1 fluorescence ratio. Thus, the cellular basis for the positive inotropic action of EMD 53998 in rat myocardium is related to the unique effect of this substance to enhance myofilament responsiveness to Ca2+ and not to an increase in the indo-1 transient amplitude.

Stereoselectivity of actions of the calcium sensitizer [+]-EMD 60263 and its enantiomer [-]-EMD 60264.

The thiadiazinone derivative [+]-EMD 60263 ((+)-5-(1-(α-ethylimino-3,4-dimethoxybenzyl)-1,2,3,4- tetrahydroquinoline-6-yl)-6-methyl-3,6-dihydro-2H-1,3,4 -thiadiazine-2-on) is a Ca2+-sensitizing agent with only minor phosphodiesterase inhibitory activity. Our aim was to characterize the inotropic and electrophysiological effects of [+]-EMD 60263 and its enantiomer [-]-EMD 60264 in several cardiac muscle preparations. The Ca2+-sensitizing activity resided in the [+]-enantiomer only. [+]-EMD 60263 (3 µM) shifted the EC50 of Ca2+ for contractile activation of skinned fibers of pig heart from 2.41 µM to 0.73 µM, whereas [-]-EMD 60264 (30 µM) was ineffective. In Langendorff-perfused guinea pig hearts, [+]-EMD 60263 and [-]-EMD 60264 induced concentration-dependent positive and negative inotropic effects, respectively; both enantiomers reduced spontaneous heart rate but did not influence perfusion pressure. The maximum increase in force of human atrial trabeculae was 35 % of pre-drug control with [+]-EMD 60263 in comparison to 113 % with forskolin. In guinea-pig papillary muscles, [+]-EMD 60263 and [-]-EMD 60264 had opposite inotropic responses, however, both agents similarly prolonged action potential duration. Both enantiomers concentration-dependently blocked the rapidly activating component IKr of the delayed rectifier in guinea-pig myocytes. The block saturated at potentials positive to +30 mV, closely resembling the effects of the antiarrhythmic agent E-4031 which had been originally used to define IKr. It is concluded, that the positive inotropic action of [+]-EMD 60263 can be explained by prevalence of the Ca2+-sensitizing effect. The accompanying prolongation in action potential duration is caused by block of the IKr component of the delayed rectifier. While the inotropic effects are stereoselective, most of the electrophysiological actions are clearly independent of sterical configuration. The combination of Ca2+-sensitizing with class-III antiarrhythmic action may provide an interesting pharmacological profile of potential therapeutic use.