Antiischemic effects of pirsidomine, a new nitric oxide donor

ELSEVIER

ejp European Journal of Pharmacology 257 (1994) 267-273

Antiischemic effects of pirsidomine, a new nitric oxide donor Piero A. Martorana *, Birgit Kettenbach, Helmut Bohn, Karl Sch6nafinger, Rainer Henning SBU Cardiovascular Therapeutics, Cassella AG, Pharmaforschung, Hanauer Landstrasse 526, 60386 Frankfurt/Main, Germany (Received 23 February 1994; accepted 8 March 1994)

Abstract

The antiischemic effect of pirsidomine (CAS 936 (3-(cis-2,6-dimethylpiperidino)-N-(4-methoxybenzoyl))-sydnonimine), a new nitric oxide donor, was investigated in a model of myocardial infarction in the dog. Dogs were anaesthetised, thoracotomized, and the left descending coronary artery was occluded for 6 h. Pirsidomine was given intraduodenally (i.d.) at the dose of 1.0 mg/kg to 11 dogs 30 min prior to coronary occlusion. Eleven dogs received the solvent i.d. and served as controls. Pirsidomine administration completely prevented the increase in left ventricular end-diastolic pressure and pulmonary artery pressure induced by the coronary occlusion and resulted in a marked decrease in systolic and diastolic blood pressure, cardiac output, left ventricular contractility, left ventricular work and left ventricular oxygen consumption. Additionally, pirsidomine completely prevented the occlusion-induced increase in flow in the non-occluded circumflex coronary artery. Regional blood flow measurements (with radioactive microspheres) revealed that pirsidomine induced a significant reduction in blood flow in the non-ischemic areas (both epi- and endocardial) but in the course of the ischemia, significantly increased flow in the ischemic epicardial areas. Infarct-size (triphenyltetrazolium chloride technique) in control dogs was 45% of the area at risk, but only 26% (P < 0.05) in pirsidomine-treated dogs. Thus, pirsidomine had a marked antiischemic effect in this model. This was probably due to the hemodynamic unloading of the heart as well as to redistribution of blood from the non-ischemic to the ischemic areas of the myocardium.

Key words: Pirsidomine; Infarct size; Hemodynamics; (Dog)

I. Introduction

Pirsidomine (CAS 936 (3-(cis-2,6-dimethylpiperidino)-N-(4-methoxybenzoyl))-sydnonimine), is a new vasodilator presently under investigation as an antianginal drug. Pirsidomine is a pro-drug (Bohn et al., 1991). It is rapidly converted in the liver into its active metabolites CAS 754 (3-cis-2,6-dimethylpiperidino) sydnonimine, and C 3786 (N-(c/s-2,6-dimethylpiperidino)-N-nitroso-2-aminoacetonitrile). The molecular mechanism(s) underlying the vasodilator activity of these agents rests on the release of nitric oxide (NO) with subsequent activation of the guanylyl cyclase (Miilsch et al., 1993). Pirsidomine decreases both preload and afterload of

* Corresponding author. Tel. (49) ( + 69) 41 09-21 17, fax (49) ( + 69) 41 09-24 13. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 1 4 - 2 9 9 9 ( 9 4 ) 0 0 1 4 7 - Y

the heart in anesthetized and conscious dogs (Bohn et al., 1992). No tolerance to the hemodynamic effects of pirsidomine a n d / o r its metabolites can be detected after repeated dosing (Bassenge et al., 1992; H u b b a r d et al., 1992; Bohn et al., 1992). Pirsidomine or its metabolites also have antiplatelet activity in vitro (Just et al., 1992; Ivanova et al., 1993) and exert antithrombotic effects in a model of carotid artery thrombosis in the anesthetized rabbit (Just et al., 1992) and in a model of coronary thrombosis in the anesthetized pig (Just et al., 1991). This effect can be reversed by the local administration of oxyhemoglobin, thus suggesting that it is also NO-related (Just et al., 1991). On the basis of these pharmacological data and in view of its clinical indication as an antianginal agent, it was of interest to investigate the antiischemic activity of pirsidomine. This was done in a model of coronary artery occlusion in the anesthetized dog. This report presents the results obtained in the course of this study.

P.A. Martorana el al. / European ,lournal of Pharmacology 257 (1994) 207-27.";

268

2.2. Protocol

2. Material and methods

2.1. General Twenty three german shepherd dogs of either sex weighing between 14.5-30.5 kg were anesthetized with pentobarbital Na (35 m g / k g i.v. as a bolus followed by an i.v. infusion at the rate of 6 m g / k g per h) and ventilated with a Bird-Mark-8 respirator delivering room air. Oxygen was supplied via the respirator as needed (1-2 I/min) in order to mantain arterial blood pO 2 values at approximately 100 mm Hg and p C O 2 values at approximately 35 mm Hg. In all animals a thoracotomy was performed through the fifth intercostal space, the lungs were retracted and the heart suspended in a pericardial cradle. The left descending coronary artery (LAD) was freed from the adjacent tissues below the first diagonal branch and a thread was placed around the artery for the later occlusion. Also, approximately 5 mm of the left circumflex coronary artery (LCX), proximal to its first descending branch, were carefully dissected free and an electromagnetic flow probe (Hellige Recomed, Freiburg) was placed around the artery for continuous coronary flow (CF) measurement. Hemodynamic data were recorded continuously during the course of the experiment on a MK 260 Brush Gould (Cleveland, Ohio) polygraph. The systemic arterial pressure ( B P s / m / d ) was measured with a catheter-tip manometer (Type PC 350 Millar Instruments, Houston, Texas) placed in a femoral artery and the left ventricular pressure (LVP) was recorded with another tip-catheter passed retrogradely from a carotid artery into the left ventricle. Heart rate (HR) was calculated from the LVP waveform and the left ventricular end-diastolic pressure (LVEDP) was measured on a high sensitivity scale. Myocardial contractility (dP/dtm~ ×) was measured as the rate of rise of LVP. Pulmonary artery pressure (PAP) was measured by means of a thermodilution catheter (Statham 5F, SP 5105) positioned in the pulmonary artery via a jugular vein. Cardiac output (CO) was determined by the thermodilution method (CO computer Statham SP 1435, Gould Statham Instruments, CA) with the thermodilution catheter. Stroke volume (SV) was calculated as the C O / H R quotient and left ventricular work (LVW) as (BPm - LVEDP) × CO × 0.0136. Myocardial oxygen consumption (MVO 2) was calculated as

Following a 60 min equilibration period and a 15 min control period, pirsidomine (dissolved in propylenc glycol) was given intraduodenally (i.d.) to a group of 12 dogs at the dose of 1.0 mg/kg. Another group of 11 dogs received the solvent i.d. and served as control. Thirty minutes after d r u g / s o l v e n t administration the LAD was ligated. Six hours after continuous coronary occlusion (without reperfusion) the animals were killed with an overdose of barbiturates and the heart dissected for assessment of infarct-size.

2.3. Infarct-size determination The isolated heart was then perfused simultaneously with two solutions at the same pressure (100 mm Hg). One solution of 0.5% Evans blue was infused retrogradely via the stump of the aorta to determine the anatomic area at risk and the non-ischemic area. The second solution of 1.5% 2,3,5-triphenyltetrazolium chloride in a 20 mM phosphate buffer (pH 7.4) was infused in the LAD just distal to the site of occlusion. With this procedure the area distal of the occlusion, the so-called area at risk was stained in a brick-red • Control ( N - 1 1 , c . v . - 1 8 . 7 ± 0.7) o P i r s i d o m i n e ( N - 1 1 , c . v . - 2 1 . 0 ± 0.8) 6"

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Fig. l. Effects of p i r s i d o m i n e on h e m o d y n a m i c p a r a m e t e r s . U p p e r panel: effect on m e a n p u l m o n a r y a r t e r y p r e s s u r e ( 3 P A P m ) . L o w e r panel: effect on left v e n t r i c u l a r e n d - d i a s t o l i c p r e s s u r e ( a L V E D P ) . T h e d a t a are m e a n s ± S.E.M.; N = n u m b e r of animals; full circles = controls; e m p t y circles = p i r s i d o m i n e ; c.v. = b a s e l i n e values.

P.A. Martorana et al. / European Journal of Pharmacology 257 (1994) 267-273

color if still viable, or left unstained (pale yellowish color) if infarcted. The right ventricle was then removed and the left ventricle plus septum (LV + S) was cut in slices, approximately 1.0 cm thick, from the base to the apex. Each slice was placed in a petri dish filled with saline and fotographed. The area of the (LV + S), the area at risk, and the infarcted area were measured on color prints of each slice by using a digitizing pen and the computer software Elas (Leitz, Wetzlar). The area at risk was calculated as percentage of the (LV + S) and the infarcted area as percentage of the area at risk.

2.4. Regional myocardial blood flow Regional myocardial blood flow was determined by the radioactive microsphere technique (Winkler, 1984) in six dogs of both the pirsidomine and control groups. Microspheres were administered 5 rain prior to drug solvent administration, 5 min prior to coronary occlusion and 5 and 360 rain after coronary occlusion. Carbonized plastic microspheres (15.5 /~m diameter, New England Nuclear, Boston, Mass labeled with 5tCr, 85Sr, 14iCe, 95Nb were suspended in isotonic saline with 0.01% Tween 80. To prevent lumping the suspension was sonified for 5 min followed by stirring immediately

269

before injection. Approximately 1 ml of the microsphere suspension (ca. 2 × 106 microspheres) were injected into the left atrium followed by several saline flushes. A reference blood sample was withdrawn from the femoral artery at a constant rate of 20 m l / m i n for 70 s. The heart slices (which after photography for the assessment of infarct-size were kept in a solution of 4% formaldehyde for 3 - 4 days) were cut into wedges. These segments were further subdivided into subepicardial, midmyocardial and subendocardial samples. The weight of each sample was approximately 120-180 mg. Subepicardial and subendocardial samples and the reference blood sample were counted in a gamma counter to determine the activity of each isotope. Myocardial blood flow was calculated by using the equation Qr × Cm Qm

Cr

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Fig. 2. Effects of pirsidomine on hemodynamic parameters. U p p e r left panel: effect on systolic blood pressure (ABPs). Lower left panel: effect on diastolic blood pressure (zlBPd). Upper right panel: effect on heart rate (zlHR). Lower right panel: effect on contractility (AdP/dtm~x). The data are means _+ S.E.M; N = number of animals; full circles = controls; empty circles = pirsidomine; c.v. = baseline values.

P.A. Martorana et al. / European Journal of Pharmacolo~' 257 (1994) 267-273

270

2.5. Data analysis The significance of the differences between the treated and the control animals was calculated using the one-sided unpaired Student's t-test. A P-value less than 0.05 was considered significant.

3. Results

3.1. Hemodynamics Twenty three dogs were entered into the study. One dog treated with pirsidomine died 9 rain after coronary occlusion. Death was due to ventricular fibrillation. The hemodynamic parameters of the remaining 22 dogs (11 per group) are presented in Figs. 1-3. Pirsidomine administered 30 min before occlusion, resulted in a marked and significant decrease in PAPm (Fig. i, upper panel) and LVEDP (Fig. 1, lower panel). Thus, the increase in both these parameters brought about by the coronary occlusion, restored in the pirsidomine dogs the original baseline values. Additionally, pirsidomine lowered the systolic and diastolic BP (Fig. 2, upper and lower left panels). This effect was

• Control

maximal by the time of the occlusion and lasted for approximately 240 min after the occlusion. The HR was not affected (Fig. 2, upper right). The lack of a reflectory increase in H R is not unusual in pentobarbital-anesthetized animals with an already high basal HR. The lowering of the LVEDP with the consequent decrease in filling volume was probably the cause of the decrease in dP/dtm~ x (Fig. 2, lower right) as well as of the decrease in CO (Fig. 3, upper left) since as mentioned above, H R was not increased. Coronary flow, assessed in the non-occluded artery, was significantly decreased by the administration of pirsidomine. Thus, in pirsidomine-treated animals the increase in coronary flow induced by the occlusion of the LAD restored the original blood flow in the nonoccluded vessel (Fig. 3, lower left). Left ventricular work and oxygen consumption were markedly lower in the pirsidomine dogs. These effects lasted approximately for 240 min of the occlusion period (Fig. 3, right panels).

3.2. Regional myocardial blood flow As shown in Fig. 4 (upper left panel), there was no difference in epicardial flow between the two groups

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Fig. 3. Effects of pirsidomine on hemodynamic parameters. U ppe r left panel: effects on cardiac output (ACO). Lower left panel: effect on coronary flow (ACF) in the non-occluded LCX. U p p e r right panel: effect on left ventricular work (ALVW). Lower right panel: effect on myocardial oxygen consumption (AMVO2). The data are means +_ S.E.M; N = number of animals; full circles = controls; empty circles = pirsidomine; c.v. = baseline values.

P.A. Martorana et al. / European Journal of Pharmacology 257 (1994) 267-273

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Fig. 4. Myocardial regional blood flow in non-ischemic and ischemic areas of control (white columms) and pirsidomine-treated (black columms) dogs with coronary occlusion. Upper and lower left panels: epicardial blood flow. Upper and lower right panels: endocardial blood flow. Data are means + S.E.M.

prior to drug treatment. Administration of pirsidomine resulted in a 25% ( P < 0.01) decrease in epicardial flow. Occlusion of the LAD resulted within 5 rain in a similar increase in epicardial flow of the non-ischemic myocardium both in the control and in the treated groups. At this time, there was no difference between the groups in epicardial flow of the ischemic areas (Fig. 4, lower left panel). This indicates that the extent of preformed functional collaterals was similar in both groups. Threehundred and sixty minutes after continuous occlusion there was no more difference in epicardial flow in the non-ischemic areas (Fig. 4, upper left panel). On the other hand, blood flow was significantly greater in the ischemic areas of the treated dogs

( + 47%; P < 0.01) (Fig. 4, lower left panel). A similar pattern of regional blood flow was also seen in the endocardial layers (Fig. 4, upper and lower right panels) with the difference that the blood flow in the ischemic areas of the pirsidomine dogs was significantly increased at 5 but not at 360 min following coronary occlusion.

Table 1 Effect of pirsidomine on infarct-size in anesthetized dogs

4. Discussion

Group

N

AR/(LV

Control Pirsidomine

11 11

3 8 . 6 + 1.8 37.9 + 1.8

+ S)

I/AR

A

44.6+3.8 26.2 + 2.9 a

- 41

N = n u m b e r o f a n i m a l s ; A R = a r e a at risk; ( L V + S) = left v e n t r i c l e p l u s s e p t u m ; I = i n f a r c t ; V a l u e s a r e m e a n s _ + S . E . M . ( % ) . a p < 0.05.

3.3. Infarct-size The myocardial area at risk involved = 40% of the left ventricle in both groups. Infarct-size, as percentage of the area at risk, was significantly smaller ( - 4 1 % , Table 1) in the pirsidomine-treated dogs.

The present study demonstrates that the new NO donor pirsidomine has a marked antiischemic activity resulting in a reduction of infarct-size by approximately 40%. Infarct-size in the anesthetized dog has been related

272

P.A. Mart orana et al. / European Journal of Pharmacology 257 (1994) 267-273

to 3 main factors: (1) the size of the area at risk; (2) the level of collateral flow to the ischemic region; (3) the myocardial oxygen demand (Reimcr et al., 1985; Schaper et al., 1987). In the present study the size of the area at risk was similar in both groups, thus this factor was not responsible for the myocardial salvage seen in the pirsidomine-treated dogs. Collateral flow was estimated by means of tracer radioactive microspheres. Collateral flow in the epicardial ischemic areas was not significantly different in both groups, 5 min after the coronary occlusion. Thus, the level of the preformed functional collaterals was similar in both groups. At this time, collateral flow in the endocardial ischemic areas was 4.82 ml. m i n - ~ • 100 g - i in the pirsidomine-treated dogs and 3.28 ml" m i n - l . 100 g - I in the control dogs. A blood flow of approximately 5 m l / m i n per 100 g is a threshold value below which it becomes difficult to discern flow from random analytical noise. When blood flow is approximately at this level, or below, then an unpractically large number of microspheres must be injected and a large volume of tissue must be analyzed to obtain reliable flow values (Schaper, 1984). Thus, the difference seen in the endocardial collateral flow does not rest on a valid analysis. During the period from 5 to 360 rain after the coronary occlusion the epicardial collateral flow in the ischemic zones increased by 13% in the control dogs. This is consistent with early observations showing that collateral flow in the ischemic areas increases with time, but that the increase is only to the advantage of the epicardial layers which eventually survive (Schaper, 1979, 1984). This effect was 4.6 × greater in the pirsidomine-treated than in control dogs and most likely, played an important role in the antiischemic activity of this compound. The mechanism(s) of action of this effect however, must remain speculative. Pirsidomine may act via its peripheral hemodynamic activity consisting in a sustained lowering of the preload (LVEDP) as well as of the afterload (BPs) of the heart. Under these circumstances a decrease in LV wall tension and heart size would be expected. This may in turn lead to better perfusion conditions for the preformed functional epicardial collaterals. Additionally, pirsidomine antiplatelet activity (Just et al., 1992; Ivanova et al., 1993) may prevent plugging of underperfused vessels. Also, its endothelial protective activity (Siegfried et al., 1992; Carey et al., 1992) coupled with the inhibition of freeradical generation from leukocytes (Wainwright and Martorana, 1993), which have been reported to be activated in acute myocardial infarction (Radomsky et al., 1990), may prevent endothelial damage and swelling in the underperfused areas. Pirsidomine markedly lowered myocardial oxygen consumption, i.e. the third factor which affects infarct-

size. This effect is probably related to lowering of the pre- and afterload of the heart with consequent reduction of the left ventricular work. The decrease in LV d P / d t m a X after pirsidomine is an effect which is common to the class of NO donors and which occurs only in the anesthetized dog (Chatterjee and Parmley, 1980; Bohn et al., 1992; Yamada et al., 1993). This effect is thought to be the result of a decreased filling volume instead of a depressed cardiac contractility (Chatterjee and Parmley, 1980). In conscious dogs with intact reflexes, pirsidomine does not lower the LV d P / d t m a x (Bohn et al., 1992; Wang et al., 1993) even after the induction of LV failure (Wang et al., 1993). In pirsidomine-treated dogs, myocardial oxygen consumption was at its lowest point at the time of coronary occlusion and remained lower than in controls for approximately 240 of the 360 min occlusion. It is thus likely that this effect played a pivotal role in the antiischemic activity of pirsidomine. Recently pirsidomine was reported to suppress ischemic arrhythmia in anesthetized pigs (Wainwright and Martorana, 1993). This effect was also thought to rest on the hemodynamic unloading of the heart. CAS 754, the main metabolite of pirsidomine, reduced infarct-size in a model of myocardial ischemiareperfusion in the anesthetized cat. After 80 rain of a 90 min ischemic period CAS 754 was given as a bolus and then infused for the entire 4.5 h reperfusion period. Thus, in the above-mentioned study the reduction of infarct-size probably represents a cardioprotective effect taking place in the reperfusion phase (against the reperfusion injury) rather than in the ischemic period (Siegfried et al., 1992). Similary to pirsidomine other NO donors such as nitroglycerin and sodium nitroprusside have been previously reported to reduce infarct-size in the dog (Jugdutt, 1983, 1985; Inou et al., 1987). In conclusion in the present study pirsidomine significantly reduced infarct-size. This effect can be explained on the basis of a reduction in myocardial oxygen consumption. An effect on regional blood flow may also play an important role in this beneficial action. Since it has been shown that the mechanism of action of pirsidomine rests on the liberation of NO (Mtilsch et al., 1993), and that in vivo the activity of pirsidomine is reversed by oxyhemoglobin (Just et al., 1991), it is reasonable to assume that these effects are NO-mediated.

Acknowledgements We are indebted to Prof. W. Schaper for his constructive critism of the manuscript, Dr. B. Winkler, Mr. H. G6bel and Dr. B. Jablonka for their help with the tracer microspheremethodologyand Ms. K. Dombrofskyfor her friendly secretarial assistance.

P.A. Martorana et al. / European Journal of Pharmacology 257 (1994) 267-273

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