Uniqueness of Pilsicainide in Class Ic Antiarrhythmics Takeshi

Uniqueness of Pilsicainide in Class Ic Antiarrhythmics

Takeshi YAMASHITA, MD, Yuji MURAKAWA, MD,

Kazunori SEZAKI, MD, Noriyuki HAYAMI, MD,

Masashi INOUE, MD, Ei-ichi FUKUI, MD,

and Masao OMATA, MD

SUMMARY Pilsicainide, a class Ic agent, is known to be an effective drug particularly for treating atrial tachyarrhythmias. However, its electrophysiological effects on the atrium have not been well studied. To characterize the electrophysiologic effects of pilsicainide on atrial myocytes in class Ic drugs, we examined the

effects of this drug on membrane currents in single rabbit atrial myocytes using the tight-seal whole cell voltage-clamp technique. Under the current-clamp condition, pilsicainide did not affect the action potential duration at therapeutic ranges (3ƒÊM) and slightly shortened it at higher concentrations (10ƒÊM). These observations were quite different from those with other class Ic agents including flecainide and propafenone which prolong the atrial action potential

duration. The drug did not affect the resting membrane potential. Under the voltage-clamp condition, pilsicainide inhibited the transient outward current

(Ito) that is more prominent in the atrium than in the ventricle in a concentration-dependent manner. However, in contrast to other class Ic agents, the inhibition of Ito by pilsicainide was observed only at much higher concentra-

tions (IC50-300ƒÊM) and did not affect the inactivation time-course of Ito. Moreover, the drug (10ƒÊM) did not significantly affect the Ca2+, delayed rectifier K+, inward rectifying K+, acetylcholine-induced K+ or ATP-sensitive K+ currents. From these results, pilsicainide could be differentiated as a pure Na+ channel blocker from other class Ic agents with diverse effects on membrane currents and should be recognized accordingly in clinical situations. (Jpn Heart

J 1998; 39: 389-397)

Key words: Pilsicainide, Potassium channel, Antiarrhythmics, Electrophysi- ology, Atrium

ILSICAINIDE, a class Ic agent according to the Vaughan Williams

classification,1,2) is known to be an effective antiarrhythmic drug, particularly for atrial tachyarrhythmia.3,4) Clinically, a single oral dose of 150mg

From the Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan. Address for correspondence: Takeshi Yamashita, MD, The Second Department of Internal Medicine, Fac- ulty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan. Received for publication February 3, 1998. Accepted March 25, 1998.

389 Jpn Heart J 390 YAMASHITAET AL May 1998

pilsicainide has been reported to be effective in terminating paroxysmal atrial fibrillation in approximately half of the patients.3) Class Ic drugs including pilsicainide, flecainide, and propafenone which are clinically used in Japan are characterized by their potent inhibitory effect on the conduction of excitation, which is ascribed to the use-dependent blocking action on Na+ inward current with slow onset and offset kinetics.5) However, class Ic drugs have been shown to have many other significant effects on membrane currents, thereby exerting their antiarrhythmic action.6-12) Actually, flecainide and propafenone are shown to prolong the atrial (but not ventricular) action potential duration .6,12) This effect can result from their inhibitory effect on the Ca2+-independent transient outward current (Ito) that is more prominent in the atrium than in the ventricle.6,12) In addition, both drugs are also reported to inhibit Ca2+ and delayed rectifier K+ currents .6,7,11) These additional effects might lead to their antiarrhythmic action, but at the same time could result in their adverse effects (proarrhythmic and negative inotropic action). Although pilsicainide is classified in the same group as these class Ic agents,1,2) the effects of this drug on other membrane currents have not been studied. Because this drug is frequently used in treating atrial tachyarrhythmia, its effects on Ito, which is important in determining the atrial action potential, should be clarified. The present study was undertaken to exam- ine whether pilsicainide has any effects on atrial myocyte membrane currents other than Na+ current with special reference to Ito.

METHODS

Preparations: New Zealand White rabbits weighing about 1.0kg were anes- thetized with pentobarbital (-50mg, i.v.) and the hearts were rapidly removed and mounted on a Langendorff apparatus. Single atrial cells were enzymatically isolated as reported previously.13) Briefly, the heart was perfused for about 10 min with nominally Ca2+-free bathing solution containing 0.6mg/ml type II collag- enase (Worthington, Lakewood, USA) at a perfusion pressure of 80cmH2O.

Thereafter, the enzyme-containing solution was flushed with the high-K/low-Cl solution. After the atrium was separated, single atrial cells were obtained by gentle shaking, kept in the high-K/low-Cl solution at 4•Ž for at least 1 hour before use, and dispersed in a recording chamber with the control bathing solution.

Electrophysiologic measurements: The patch-clamp technique was used in the whole cell configuration.14) A heat-polished patch pipette, filled with an arti- ficial internal solution, had a tip resistance of 1 to 4Mƒ¶. Membrane currents and potentials were measured through a patch-clamp amplifier (EPC-7, List Electron- ics, Darmstadt, Germany) and monitored with a high-gain oscilloscope. The data Vol 39 No 3 PILSICAINIDEIN CLASSIC ANTIARRHYTHMICS 391 were stored on video tapes using a PCM converter system (VR-10B, Instrutech

Corp., New York, USA), reproduced, low-pass-filtered at 1kHz by a filter with

Bessel characteristics, sampled at 5kHz and analysed off-line on a personal computer. Results are expressed as means•}SD, and significant differences between values were assessed using a paired t-test.

Solutions and drugs: The control bathing solution contained (in mM) NaCl

136.5, KCl 5.4, HEPES 5.5, Na2HPO4 0.33, glucose 5.5, CaCl2 1.8, MgCl2 0.53, pH 7.4. The high-K/low-Cl solution contained (in mM): glutamic acid 70, KCl 25, KH2PO4 10, taurine 10, oxalic acid 10, glucose 11, EGTA 0.5, HEPES 10,

pH 7.4. The internal pipette solution contained (in mM) KCl 130, ATPK2 5, HEPES 5, MgCl2 1, EGTA 1, pH 7.2. When measuring Ca2+-independent tran-

sient outward current (Ito), the concentration of EGTA in the internal solution

was raised to 5mM to block Ca2+-dependent transient outward current, and 0.2

mM Cd2+ was added to the bathing solution to block Ca2+ current. Na+ current

was blocked by 10ƒÊM TTX in the bathing solution. Pilsicainide, a gift from

Suntory, was used from stock solution (10mM).

RESULTS

The effects of pilsicainide on the atrial action potential duration: Figure 1

illustrates the effects of pilsicainide (1, 3, 10, 30ƒÊM) on the action potential of

isolated rabbit atrial cells. Action potentials were elicited by passing currents

through the patch pipette, which caused artifacts at the beginning of each action

potential. In contrast to other class Ic drugs (flecainide, propafenone), pilsicainide did not affect the action potential duration significantly at therapeutic ranges

(_??_3ƒÊM). At higher concentrations, the drug significantly shortened the atrial

Figure 1. A representative example showing atrial action potentials before and after application of pilsicainide (1-30ƒÊM at 0.5Hz). Pilsicainide did not affect the action

potential duration at therapeutic ranges (_??_3ƒÊM) but shortened it at higher concentrations. Action potentials at control and after application of 1ƒÊM pilsicainide are com-

pletely overlapped. Jpn Heart J 392 YAMASHITAET AL May 1998

Table. % Changes in Action Potential Duration by Pilsicainide

*p<0 .05 vs before pilsicainide

Figure 2. Effects of pilsicainide (3-1000ƒÊM) on Ito. A: Ito was elicited by the command pulses at +40mV for 300ms from the holding potential of -80mV. Na+ current

was blocked by 10ƒÊM TTX in the bathing solution. Ca2+ current and Ca2+-dependent transient outward current were blocked by 0.2mM Cd2+ in the bathing solution and 5

mM EGTA in the pipette solution. Pilsicainide (_??_10ƒÊM) depressed the peak outward

current in a concentration-dependent manner and also suppressed the steady-state current at a concentration of_??_300ƒÊM. However, in contrast to other class Ic drugs,

the inhibition of Ito was observed only at concentrations higher than the therapeutic range (_??_3ƒÊM) and did not accelerate the inactivation time-course of Ito. B: Dose-

response relationships of the inhibition of Ito by pilsicainide. Ito was estimated as the

difference between the initial peak and steady-state currents.

action potential duration in a concentration-dependent manner (Table), and this observation contrasted with those in flecainide and propafenone which prolonged the atrial action potential duration in a concentration-dependent manner.6,2) Pilsicainide did not affect the resting membrane potential, a finding similar to that with flecainide and propafenone.6,12) Vol 39 No 3 PILSICAINIDEIN CLASSIC ANTIARRHYTHMICS 393

Figure 3. Effects of pilsicainide (10ƒÊM) on voltage-dependent Ca2+ current, delayed

rectifier K+ current, inward rectifying K+ current, and acetylcholine (Ach)-induced

current. A: Pilsicainide (10ƒÊM) did not affect voltage-dependent Ca2+ current, delayed rectifier K+ current, or inward rectifying K+ current. Ito was blocked by 2mM 4-

aminopyridine in the bathing solution. Currents elicited by command pulses at -100, 0, and +40mV from a holding potential of -40mV are shown. Zero current level is shown by the bold bar. B: Pilsicainide (10ƒÊM) slightly suppressed the Ach-induced

current. The current was induced by application of 1 ƒÊM Ach in the bathing solution.

The membrane potential was maintained at -40mV.

Effects on Ito: When treating atrial tachyarrhythmias with antiarrhythmic drugs, their effects on Ito would greatly influence their antiarrhythmic action because Ito is much more developed in the atrium than in the ventricle.15) In class

Ic drugs, flecainide and propafenone significantly prolong the atrial action potential duration by their inhibitory action on Ito.6,12) Although pilsicainide did not prolong the atrial action potential duration, its effects on Ito, if any, could not be denied, because the effects of Ito inhibition on the action potential duration might be canceled by its inhibitory action on inward currents, including Na+ current.

To determine this possibility, we examined the effects of this drug on the Ito of atrial myocytes. Under the voltage-clamp condition, Ito was elicited by a voltage- step to +40mV from a holding potential of -80mV. Na+ current was blocked by 10ƒÊM TTX in the bathing solution. Ca2+ current and Cat2+-dependent transient outward current were blocked by 0.2mM Cd2+ in the bathing solution and high EGTA (5mM) in the internal pipette solution. Therefore, the amplitude of Jpn Heart J 394 YAMASHITA ET AL May 1998 the Ca2+-independent transient outward current (Ito) could be estimated from the difference between the peak outward and steady-state currents.13) Pilsicainide suppressed Ito in response to the voltage-step in a concentration-dependent manner (Figure 2A & B). At a concentration of more than 100ƒÊM, the drug also

suppressed the steady-state current. However, the inhibition of Ito. by this drug was quite different from that observed in flecainide and propafenone, both of which accelerate the inactivation of Ito as open channel blockers.6,12) Pilsicainide

did not affect the inactivation of Ito, significantly. Moreover, the inhibition of ho by

this drug was observed only at high concentrations (_??_10ƒÊM, IC50-300ƒÊM). At

therapeutic concentrations (_??_3ƒÊM), the drug did not affect Ito significantly. This

inhibitory effect of the drug on Ito at high concentrations was considered to be a

non-specific effect compared with its blocking action on Na+ current at low con-

centrations (>1ƒÊM).1)

Effects on other membrane currents: We also examined the effects of

pilsicainide on atrial myocyte membrane currents other than Ito. A representative recording showing the effects of pilsicainide (10ƒÊM) on Ca2+ current, inward

rectifying K+ current and delayed rectifier K+ current are presented in Figure 3A.

To eliminate Ito, 2mM 4-aminopyridine was added to the bathing solution.

Membrane currents in response to a series of test pulses for 200ms from a

holding potential of -40mV are shown before (Figure 3A, left) and after 10ƒÊM

pilsicainide application (Figure 3A, right). Pilsicainide (10ƒÊM) at a concentration higher than therapeutic ranges did not affect Ca2+ current, inwardly rectifying K+

current or delayed rectifier K+ current. Figure 3B showed the effects of

pilsicainide (10ƒÊM) on acetylcholine (Ach)-induced K+ current. The membrane

potential was held at -40mV, and the current was induced by application of 1ƒÊ M ACh in the bathing solution. Pilsicainide (10ƒÊM) suppressed the ACh-in-

duced current only slightly (6•}2%, n=5). Also, pilsicainide (10ƒÊM) did not

affect the ATP-sensitive K+ current evoked by application of 10ƒÊM pinacidil

(data not shown).

DISCUSSION

The present study describes the effects of pilsicainide on non-Na+ mem-

brane currents of atrial myocytes to characterize the drug as a class Ic agent. At

therapeutic concentrations (_??_3ƒÊM), pilsicainide did not significantly affect Ito,

voltage-dependent-Ca2+, delayed rectifier K+, inwardly-rectifying K+, ACh-in-

duced K+ or ATP-sensitive K+ currents. Thus, pilsicainide could be regarded as a

pure Na+ channel blocker at therapeutic ranges, and therefore quite unique among class Ic drugs on the basis that other class Ic drugs (flecainide and

propafenone) have many significant effects on membrane currents other than Vol 39 No 3 PILSICAINIDEIN CLASSIC ANTIARRHYTHMICS 395

Na+ current.6-12)

In the widely accepted Vaughan Williams classification, class I drugs are characterized by their direct membrane action of Na+ channel blockade.5) Ac- cordingly, class Ic drugs, a subclassification of class I drugs, are classified as those with a marked slowing of conduction and slight effect on repolarization.5) How- ever, because recent studies have demonstrated diverse effects of many class Ic drugs on membrane currents and consequent various effects on repolarization,6- 12) the classification of antiarrhythmic drugs is not easily interpreted . In fact, although flecainide slightly shortens the action potential duration of ventricular myocytes and Purkinje fibers and is thereby classified as a class Ic drug,16) it also prolongs the atrial action potential duration.6) This effect on the atrial action potential duration could be derived from its diverse effects on non-Na+ membrane currents. The drug inhibits the Ca2+-independent transient outward current (Ito), Ca2+ current, and delayed rectifier K+ current.6 8) Because Ito is more prominent in the atrium than in the ventricle, the blockade of Ito by this drug leads to prolongation of the action potential duration. Similar results have been reported for another class Ic drug, propafenone.9-12) As with propafenone, the drug also inhibits ACh-induced K+ current and ATP-sensitive K+ current, and moreover has a beta-blocking action.17-19) From these viewpoints, the present results have differentiated pilsicainide from these other class Ic drugs: the drug almost had no effects on membrane currents other than Na+ channels, and therefore should be recognized as a unique and rather pure class Ic drug.

Recently, a new classification of antiarrhythmic drugs, The Sicilian Gambit approach, has been proposed.20) In this classification, the classification of antiarrhythmic drugs is based on their action on membrane currents, receptors, and pumps. In contrast to other antiarrhythmic drugs, pilsicainide is now clinically used only in Japan. Therefore, the effects of pilsicainide on membrane currents of myocytes have not been extensively studied. Only its effects on Na+ channels, ACh-induced K+ current and ATP-sensitive K+ current have been reported.1,17-19) Apart from its potent inhibitory action on Na+ channels, pilsicainide has been reported to inhibit ACh-induced K+ current (IC50-30ƒÊM) and ATP-sensitive K+ current (IC50-500ƒÊM), but the inhibitory effects were observed at concentrations higher than therapeutic ranges.17 19) The present results are consistent with these reports. The present study was conducted to gain information on the effects of the drug on voltage-dependent K+ and Ca2+ currents, and inward rectifying K+ current, which would assist in classifying pilsicainide in the new classification of antiarrhythmic drugs. Clinically, pilsicainide has been used particularly for atrial tachyarrhythmias including paroxysmal atrial fibrillation, as a class Ic drug simi- larly to flecainide and propafenone.21) However, the present results suggest that Jpn Heart J 396 YAMASHITAET AL May 1998 its clinical use should be distinguished from that of flecainide and propafenone. The blockade of Ca2+ current with these 2 drugs may lead to negative inotropic effects and blockade of Ito and delayed rectifier K+ current may lead to proarrhythmic effects through prolongation of action potential duration. Al- though the recent Cardiac Arrhythmia Suppression Trial (CAST)22)study has reported a higher incidence of life-threatening arrhythmia in postmyocardial infarction patients treated with class Ic drugs, this result might be associated with the diverse effects of class Ic agents on membrane currents. However, class Ic drugs are still useful for supraventricular arrhythmias23)and a strategy for using these drugs is required. The results of the present study indicate pilsicainide could serve as a first choice among class Ic drugs because the antiarrhythmic action of pure Na+ channel blockade can be evaluated with this drug. It is conceivable that flecainide and propafenone, with various electrophysiological effects, should be used as drugs of second choice when pure Na+ channel blockade without prolongation of action potential duration fails to exert appropriate antiarrhythmic action. Although still hypothetical, the present study should promote clinical studies of this drug in comparison with other class Ic drugs from these points of view.

REFERENCES I. InomataN, IshiharaT, AkaikeN. Differenttime course of the blockadeof sodiumcurrent by lignocaineand SUN 1165 in singlemyocytes isolated from guinea-pig atrium. Br J Pharmacol1989; 98: 149-54. 2. KuharaY, Inoko M,Hatakeyama N, MomoseY, SasayamaS. Mechanismsof negative inotropic effectsof class Ic antiarrhythmicagents: comparative study of the effects of flecainide and pilsicainide on intracellularcalcium handling in dogventricular myocardium. J Cardiovasc Pharmacol 1996; 27: 42- 51. 3. AtarashiH, InoueH, HiejimaK, HayakawaH, for thePSTAF Investigators. Conversion ofrecent- onsetatrial fibrillation by a singleoral dose of pilsicainide(Pilsicainide suppression trial on atrial fibrillation).Am JCardiol 1996; 78: 694-7. 4. TerazawaT, Suzuki M,Goto T, et al. Suppressiveeffect of SUN1165 on supraventricular tachycardia.Am Heart J1991; 121: 1437-44. 5. HarrisonDC. Antiarrhythmicdrug classification: new science and practicalapplications. Am J Cardiol1985; 56: 185-7. 6. YamashitaT, NakajimaHamada T, E, HazamaH, OmataM, KurachiY. Flecainideinhibits the transientoutward current in atrialmyocytes isolated from the rabbit heart. J PharmacolExp Ther 1995; 274:315-21. 7. ScampsF, UndrovinasA, VassortG. Inhibitionof ICa,in singlefrog cardiac cells by quinidine, flecainide,ethmozin, and ethacizin. Am JPhysiol 1989; 257: H1773-81. 8. FollmerCH, Cullinan CA, Colatsky TJ. Differentialblock of cardiac delayed rectifier current by class Icantiarrhythmic drugs: evidence for open channel block and unblock. Cardiovasc Res 1992; 26: 1121- 30. 9. DelponE, Valenzuela C,Perez O,Casis O,Tamargo J. Propafenonepreferentially blocks the rapidly activatingcomponent of delayedrectifier K+ current in guineapig ventricular myocytes: voltage-inde- pendentand time-dependent block of the slowly activating component. Circ Res 1995; 76: 223-35. 10. SlawskyMT, CastleNA. K+channel blocking action of flecainidecompared with those of propafenoneand quinidine in adultrat ventricular myocytes. J Pharmacol Exp Ther 1994; 269: 66-74. Vol 39 PILSICAINIDEIN CLASSIC ANTIARRHYTHMICS 397 No 3

11. Delgado C, Tamargo J, Henzel D, Lorente P. Effects of propafenone on calcium current in guinea-pig ventricular myocytes. Br J Pharmacol 1993; 108: 721-7. 12. Duan D, Fermini B, Nattel S. Potassium channel blocking properties of propafenone in rabbit atrial myocytes. J Pharmacol Exp Ther 1993; 264: 1113-23. 13. Yamashita T, Nakajima T, Hamada E, Hazama H, Murakawa Y, Omata M. Regional differences in the transient outward current density and repolarization inhomogeneities in the rabbit right atrium. Circulation 1995; 92: 3061-9. 14. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high- resolution current recording from cells and cell free patches. Pflugers Arch 1981; 391: 85-100. 15. Giles WR, Imaizumi Y. Comparison of potassium currents in rabbit atrial and ventricular cells. J Physiol 1988; 405: 123-45. 16. Winslow E, Campbell JK. Comparative frequency-dependent effects of three class Ic agents, Org 7797, flecainide, and propafenone, on ventricular action potential duration. J Cardiovasc Pharmacol 1991; 18: 911-7. 17. Inomata N, Ohno T, Ishihara T, Akaike N. Antiarrhythmic agents act differently on the activation phase of the Ach-response in guinea-pig atrial myocytes. Br J Pharmacol 1993; 108: 111-5. 18. Sakuta H, Okamoto K, Watanabe Y. Blockade by antiarrhythmic drugs of glibenclamide-sensitivc K+ channels in Xenopus oocytes. Br J Pharmacol 1992; 107: 1061-70. 19.Wu B, Sato T, Kiyosue T, Arita M. Blockade of 2, 4-dinitrophenol induced ATP sensitive potassium current in guinea pig ventricular myocytes by class I antiarrhythmic drugs. Cardiovasc Res 1992; 26: 1095-101. 20. Task Force of the Working Group on Arrhythmia of European Society of Cardiology. The Sicilian Gambit-new approach to the classification of antiarrhythmic drugs based on their actions on arrhythmogenic mechanisms. Eur Heart J 1991; 12: 1112-31. 21. Fujito T, Takayanagi K, Shimizu M, et al. Days required for 75% suppression of ventricular prema- ture contractions by antiarrhythmic agents obtained from continuous in-hospital ECG monitoring. Jpn Heart J 1994; 35: 125-40. 22. The Cardiac Arrhythmia Suppression Trial (CAST) Investigators. Preliminary report: effect of encainide and flecainide on mortality in a randomized trial of arrhythmia suppression after myocardial infarction. N Engl J Med 1989; 321: 406-12. 23. Inoue M, Inoue D, Ishibashi K, et al. Effects of pilsicainide on the atrial fibrillation in guinea pig atria. A comparative study with disopyramide, lidocaine and flecainide. Jpn Heart J 1993; 34: 301-12.