BISARAMIL AND ANTIARRHYTHMICS AS INHIBITORS OF FREE RADICAL GENERATION

Pharmacological Research, Vol. 35, No. 4, 1997

BISARAMIL AND ANTIARRHYTHMICS AS INHIBITORS OF FREE RADICAL GENERATION ´ ¨ ´ ´ MARGIT PAROCZAI*, ELIZABETH RO TH† and EGON KARPATI Pharmacological Research Institute, Chemical Works of Gedeon Richter Ltd, Budapest, Hungary and †Institute of ´ Experimental Surgery, Medical School of Pe cs, Hungary Accepted 23 December 1996 The aim of this study was to investigate the effect of bisaramil—an antiarrhythmic drug under clinical trials—on free radical generation of isolated polymorph neutrophil granulocytes (PMN) and furthermore to compare its activity to that of well-known antiarrhythmics which have different modes of action. PMNs were isolated from healthy beagle dogs, and superoxide radical generation was induced by phorbol-myristate-acetate. Stimulated free radical generation capacity of PMNs and the time lag necessary for the initiation of free radical production were measured. All compounds were used at the concentrations of 10, 25, 50, 75, 100 µg ml−1. None of the antiarrhythmics stimulated by itself the free radical generation. Bisaramil exerted concentration dependent inhibitory effect on PMA-stimulated free radical generation and prolonged the time lag concentration dependently. At the investigated concentration range of antiarrhythmics only propafenon, mexiletine and diltiazem showed similar activity to bisaramil, but clear concentration dependency could not be seen in any of the cases. According to the results of this study inhibition of the stimulated free radical production of isolated PMNs cannot be closely connected merely to either membrane stabilizing or Caantagonistic activity of drugs. In vitro and earlier measured in vivo inhibitory action of bisaramil on free radical generation indicate a possible cardioprotective effect existing independently from its antiarrhythmic one. This observation may be important in outlining of the clinical indication field of bisaramil, and may be useful in the treatment of reperfusional damage. 1997 The Italian Pharmacological Society KEY WORDS: free radicals, polymorph neutrophil granulocytes, bisaramil.

INTRODUCTION The role of leukocytes in inflammatory process, phagocytosis, and removal of decaying cells has been well known for decades [1–3]. It was also recognised that the process of myocardial infarction with reperfusion of the previously ischemic myocardium involves components of a typical inflammatory reaction. This acute inflammatory response can be characterized by enhanced protein efflux [4], release of inflammatory mediators [5, 6] as well as infiltration of blood cells such as polymorphonuclear neutrophils (PMN) [7, 8]. During the early acute inflammatory reaction PMNs undergo a complex series of functional and biochemical alterations, and in this activated state they are involved in several pathobiochemical reac-

´ *Correspondence to: M. Paroczai, Pharmacological Research Centre, Chemical Works of Gedeon Richter Ltd., H-1475 Budapest, 10. P.O.B. 27. Hungary.

1043–6618/97/040279–07/$25·00/0/fr970128

tions. PMNs, sticking to the vascular endothelium and aggregating with each other increase vascular resistance and can induce ‘no flow’ phenomenon in smaller vessels [9, 10]. In addition, activated neutrophils release lysosomal enzymes capable of proteolytic disruption of viable as well as irreversibly injured tissue [11, 12]. Stimulated neutrophils trigger membrane phospholipids to release arachidonic acid, which is converted by specific lipoxygenases to potent chemotactic hydroxy-eicosa teraenoic acids (HETEs), which promote the further recruitment of neutrophils in the acute inflammatory response at the site of tissue injury [13]. Finally, stimulated neutrophils release highly reactive and cytotoxic activated oxygen species such as superoxide anion, hydroxyl radical, hydrogen peroxide and singlet oxygen [14, 15]. The lipidperoxidation induced by these radicals influences the lipid metabolism of cells and the movement of myocardial Ca ions, therefore they have arrhythmogenic effect [16, 17]. Numerous trials have been performed to decrease the number or the activity of leukocytes during exper1997 The Italian Pharmacological Society

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imental coronary occlusion-reperfusion models. The results have demonstrated the beneficial effect of the treatment with antiinflammatory drugs [18, 19], or of using monoclonal antibody [20] or filters to remove neutrophil granulocytes [21]. The results of the mentioned treatments manifested as a significant reduction in the size of the infarcted area and in the incidence of arrhythmic events. The realization of arrhythmogenic effect of free radicals produced by activated neutrophils raised the question whether antiarrhythmic drugs used in clinical practice possess inhibitory effect on free radical production of activated neutrophils or on the progression of the damaged area. Bisaramil is an antiarrhythmic drug under Phase II clinical studies. Its antiarrhythmic activity and mode of action has been already investigated in detail [22–24]. Recently, it was also shown, that this compound favourably influences the biochemical parameters related to free radical reactions [25]. The aim of the present study was to investigate free radical generating capacity of PMNs isolated from the blood of beagle dogs with and without the stimulator phorbol-myristate-acetate (PMA) administration, and furthermore to investigate and to compare the effect of bisaramil and other antiarrhythmic drugs on the capacity and the time lag necessary to start PMAinduced free radical generation of neutrophil granulocytes.

MATERIALS AND METHODS

Isolation of neutrophil granulocytes from blood [26] Nine millilitres of peripheral blood withdrawn from healthy beagle dogs was given into a plastic tube containing 1 ml EDTA of 0·1 M concentration. Three millilitres Dextrane (SIGMA, mol. weight: 16,200) of 6% concentration was added to this anticoagulated blood, and the tubes were incubated in a water bath of 37°C temperature for 1 h to promote the sedimentation of erythrocytes. The supernatant was drawn off and centrifuged at low speed at 4°C for 8–10 minutes. To remove the erythrocytes from the pellet 5 ml NH4Cl in 0·83% concentration was used for hypotonic lysis. The tubes were then centrifuged at 700 rpm for 10 minutes. This process was repeated until the pellet became completely white. Gradient centrifuging was performed on a FicollPaque (Pharmacia Lkb, Biotechnology AB, Uppsala, Sweden) medium. Three millilitres of Ficoll was measured into a plastic tube, and 2 ml of the cell suspension in Dulbeco buffer enriched with glucose was cautiously layered on top of it. The mixture was centrifuged on 1900 rpm for 30 minutes. The supernatant was drawn off carefully, mixed with Dulbeco buffer centrifuged once again on 700 rpm for 10 minutes. The pellet was then resuspended with 0·2 ml Dulbeco + glucose buffer, and this final suspension contained

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polymorph neutrophil granulocytes in 98% concentration.

Determination of the viability of isolated cells

After the isolation procedure 10 µl of the cell suspension was mixed with 490 µl staining mixture, containing four parts of Trypane blue solution of 0·2% and one part of NaCl solution of 4·25%. The ratio of living and dead cells and the absolute number of isolated cells was determined in Buerker chamber. Practically only a low number of blue-stained cells could be observed, showing that the cell membrane very scarcely became permeable for the Tripane blue, meaning the cell had lost its viability.

Measurement of the free radical producing activity of isolated PMN-s The suspension was diluted with Dulbeco buffer containing glucose if necessary to produce the leukocyte concentration of 1·5–2×106. The superoxide radical production of isolated leukocytes was determined by kinetic spectrophotometric method on 550 nm, using a Specord M-40 photometer. The essence of the method was that the superoxide radicals produce ferro irons from the ferri irons in the presence of ferricytochrome c and this change was accompanied by a color reaction (pink discoloration). The intensity of the color reaction linearly correlates with the quantity of radicals produced. The test mixture contains 30 µl PMN, 20 µl cytochrome c (SIGMA from horse heart, purity 99%), and 950 µl Dulbeco buffer (containing glucose) (pH 7·0–7·4). This reaction mixture was incubated in water bath of 37°C temperature for 3 minutes, then photometry was performed against a blank (which contained the same components as the test tube without isolated PMN-s) to determine spontaneous radical production. (The practical significance of this procedure was that some leukocytes can become activated during the isolation if it was not performed properly.) If spontaneous radical production was not observed within 3 minutes, the stimulator phorbol-12-myristate-13-acetate (PMA, SIGMA) was added in a volume of 20 µl. Depending on the free radical producing capacity of the isolated cells, the extinction values started to rise immediately or after 0·5–1 minute, showing the initiation of reduction and free radical production. The reaction was followed for 4–6 minutes to record a sufficiently long linear line for subsequent calculations. The slope of the kinetic curve was in direct correlation with the superoxide radical producing capacity of activated leukocytes. The quantity of superoxide radicals produced was referred to the cell number of the cell suspension (1·5 ×106 PMN). Protein content was determined with Bradford’s method. To determine the effect of the compound on the free radical production of isolated leukocytes different quantities of the drug were measured into the test cuvette containing the cell suspension and the original

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3.0 **

2.5 15

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nmol O2 1.5 × 10 PMN

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* **

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50 20 µg ml–1

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Fig. 1. Effect of bisaramil on superoxide generating activity of PMNs. *P<0·01, **P<0·001.

950 µl volume of the buffer was decreased accordingly. A basic solutions of 1 mg ml−1 concentration were prepared from the test compounds, and 10, 20, 50, 75 or 100 µl of these solutions were used for 1 ml total volume (thus the cuvettes contained 10, 20, 50, 100 µg of the compounds). The test cuvette was incubated in a water bath of 37°C temperature for 5 minutes, then the spontaneous and PMA-induced radical producing capacity of PMN-s was determined according to the method described. The following antiarrhythmic compounds were tested: Disopyramide, lidocaine, procainamide, mexiletine, propafenon, sotalol, amiodarone, verapamil, nifedipine, diltiazem. Free radical generating capacity of PMN-s in presence of various concentration of drugs was given as a percentage of the values of stimulated radical production value obtained at the initial, control measurement. The effect of various concentrations of the drugs on the time lag (the time between the PMAadministration and the beginning of free radical production) was expressed as a percentage of the time lag that was observed without any drug administration. In case of bisaramil, an EC50 value was also calculated from the concentration-effect relationship using probit analysis of Litchfield and Wilcoxon [27].

RESULTS Figure 1 displays the concentrations-dependent inhibitory effect of bisaramil on production of superoxide radicals. The results were calculated from values obtained in 10 separate experiments. Bisaramil significantly inhibited the superoxide radical producing capacity of PMN-s at concentrations of 50, 75, and 100 µg ml−1. Its IC50 value—the concentration of bisaramil which inhibit free radical generation by 50% —was found to be 43·1 µg ml−1 (0·12 mM). Figure 2 shows the lengthening of time lag by bisaramil. It can be seen that the values were significantly higher than

0.0

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50 20 µg ml–1

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Fig. 2. Effect of bisaramil on the time lag necessary for starting free radical generation. *P<0·05, **P<0·01.

the initial, control values especially after concentrations of 50 µg ml−1 and above. Figures 3 and 4 summarize the results obtained with well-known antiarrhythmics. In each case, the data of one representative experiment are shown. PMA-stimulated activity or time lag necessary for the initiation of stimulated free radical production measured at the beginning of experiment were considered as 100%. It is worth mentioning, that none of the investigated compounds—including bisaramil—stimulated spontaneous free radical generation, henceforward the effect of drugs on PMAstimulated free radical generation was studied and discussed below. It was interesting that consistent, concentrationdependent inhibition developed only after mexiletine and propafenon administration, and only propafenon was able to exert a more significant effect than bisaramil. It could be also stated that, though some of the test compounds like mexiletine, propafenon and sotalol increased the time lag considerably, the effect was more or less independent of the concentration applied, therefore consistent correlation between the concentration and the length of time lag cannot be detected. Disopyramid induced only slight inhibition in the free radical production of leukocytes and only at high concentrations as 75 and 100 µg ml−1 did it increase the time necessary for initiation of radical production. Interestingly, two membranes stabilizing compounds lidocaine and procainamide showed different actions. While lidocaine slightly increased the free radical generation and showed a tendency to decrease the time lag, procainamide was found to be practically ineffective on both parameters. Sotalol induced a very slight inhibition, even at the concentration of 100 µg ml−1, however the same concentration increased significantly the time lag if compared to the initial value. Surprisingly, amiodarone stimulated the free radical production of isolated leukocytes in each concentration, but clear concentration-dependent effect could

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Fig. 3. Change on % of free radical generating capacity of PMNs in presence of various drugs. B=bisaramil, Ds=disopyramide, L=lidocaine, Pa=procainamide, M=mexiletine, Pf=propafenone, S=sotalol, A=amiodarone, V=verepamil, N=nifedipine, Dl= diltiazem.

not be observed. Amiodarone was not able to increase the time lag at any concentrations. The calcium antagonist verapamil concentrationdependently decreased the stimulated free radical production of isolated leukocytes, however its effect was less significant than that of bisaramil. The time necessary for the initiation of radical production was slightly and more or less concentration-dependently lengthened by the drug. Nifedipine at concentrations higher than 20 µg ml−1 stimulated the free radical production of leukocytes, and it increased slightly the time lag. Diltiazem exerted a strong inhibitory effect on free radical generation, its activity showed no concentration-dependency. Similarly to bisaramil, diltiazem also lengthened the time lag, but the maximal prolongation was far less than that caused by bisaramil.

DISCUSSION Experimental observation has provided evidence that early reperfusion of the ischemic myocardium can salvage a tissue before it becomes irreversibly injured [28, 29]. In the clinical practice current interventions like thrombolytic therapy with streptokinase [30] or tissue-type plasminogen activator [31] percutaneous transluminal coronary angioplasty [32], surgical

revascularization [33] are used as therapeutic strategies to assure early reperfusion. Recent observations, however, have indicated, that the reperfusion of the ischemic myocardium is associated with a number of potentially deleterious effects as well [34, 35]. Namely, that both intracellular and neutrophil derived oxygen metabolites contribute significantly to the damaging effects of reperfusion [36, 37]. Beneficial effect of non-steroid antiinflammatory agents [18] suggested that inflammatory reactions are also involved in the damage of reperfused myocardium. Ibuprofen was found to exert its beneficial effect on ischemic myocardium by inhibiting the accumulation of neutrophils leukocytes in the border zone, and by this way resulted in a 40% decrease of infarcted area. Lucchesi et al. [38] obtained the 43% reduction of infarction size by the administration of anti-neutrophil antiserum. Mullane et al. [19] produced neutropenia in dogs by the administration of hydroxyurea and detected a 67% reduction of infarction size at the end of a 5 hour reperfusion period following a 60 min LAD-ligature. Mitsos et al. [39] observed, that the administration of N-2-mercaptopropionyl-glycine (MPG)—a free radical scavenger [40]—just before the onset of reperfusion enhanced the protective effect of neutrophil depletion as the reduction in infarct size was significantly greater in the group of neutrophil depletion + MPG treatment than in the group with

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600 –1

0 µg ml –1 10 µg ml 20 µg ml–1 –1 50 µg ml–1 75 µg ml 100 µg ml–1

550 500 450 400

Min

350 300 250 200 150 100 50 0

B

Ds

L

Pa

M

Pf

S

A

V

N

Dl

Fig. 4. Time lag necessary for starting free radical generation in presence of various drugs. B=bisaramil, Ds=disopyramide, L= lidocaine, Pa=procainamide, M=mexiletine, Pf=propafenone, S=sotalol, A=amiodarone, V=verepamil, N=nifedipine, Dl= diltiazem.

neutrophil antiserum alone. At the same time, the areas at risk did not differ among the groups. The authors emphasized that this myocardial protection could not be explained on the basis of hemohynamic differences. These experiments clearly showed, that oxygen free radicals derived from extracellular and intracellular sources contribute to the deleterious effects of reperfusion and that myocardial salvage can be enhanced by free radical scavengers. Antiarrhythmic drugs classified to the different groups according to their electrophysiological properties have been already investigated on myocardial ischemia-reperfusion model and some of them were shown to have potential free-radical scavenger activity [41, 42]. Bisaramil was found to favourably influence the intensity of free radical reactions (decreased lipidperoxidation, better preservation of reduced glutathion) and in the acute reperfusion period it decreased the elevated superoxide radical production of leukocytes observed in untreated animals [25]. In the present in vitro study, bisaramil showed a clear concentration-dependent inhibitory effect on the superoxide anion generating capacity of PMN-s and on the time lag necessary for the initiation of free radical production (IC50 value=43·1 µg ml−1=0·12 mM). According to earlier studies, local-anaesthetic activity of bisaramil measured on frog nervous ischiadicus

preparation could be characterized by an IC50= 1·05 mM [20], which is very similar value to that of lidocaine (1·2 mM). Comparing these values it is apparent that the inhibitory effect of bisaramil on free radical generation can be measured in lower concentration than its membrane stabilizing activity, while—on the basis of present study—it is not true for lidocaine. On the other hand our experiment showed that significant differences exist between so called membrane stabilizing Class I antiarrhythmics in the respect of their effect on free radical generation by PMNs. Among the investigated drugs, mexiletine and propafenon were the only drugs which showed similar activity to bisaramil. Propafenon was able to cause complete inhibition at high concentrations, but in some cases, it stimulated the superoxide radical production of PMNs at the lowest concentration. Lidocaine or procainamide were found to have practically no effect on measured parameters. These results make it probable, that the inhibitory effect of a drug on free radical generation by PMNs cannot be only connected to its membrane stabilizing effect. The three investigated drugs having Ca-antagonistic activity, namely verapamil, nifedipine and diltiazem were found to differ from each other in their activity. Verapamil and nifedipine even increased the free radical generation while, diltiazem beneficially influenced the measured parameters. It means, prob-

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ably, that the Ca-antagonistic effect, in itself, cannot be responsible for the free radical generation inhibitory effect. The most surprising result was produced by amiodarone, because it increased free radical generation in the whole concentration-range investigated. Unfavourable side effects of amiodarone are wellknown [43, 44], and it may be, that present results will further restrict the clinical indication field of amiodarone. In summary, bisaramil concentration dependently inhibited the free radical generation and lengthened the time necessary to start the production of superoxide anion by PMNs. These results correlate well with our earlier observations, namely that bisaramil is able to inhibit the increase in the number and the free radical production capacity of peripheral leukocytes after coronary occlusion-reperfusion. In vitro and in vivo measured effect of bisaramil calls the attention to its possible cardioprotective activity existing beside its antiarrhythmic effect. This observation may be of significance in the acute and chronic phases of myocardial infarction as well as in the prevention of reperfusional damage.

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9. 10.

11.

12. 13. 14. 15. 16. 17.

ACKNOWLEDGEMENTS 18.

We are thankful to Ms Andrea Fekete for her assistance in preparation of the manuscript. 19.

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