Ventricular arrhythmia incidence in the rat is reduced by naloxone

Pharmacological Research 97 (2015) 64–69

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Ventricular arrhythmia incidence in the rat is reduced by naloxone M.K. Pugsley ∗,1 , E.S. Hayes 2 , W.Q. Wang, M.J.A. Walker Department of Anaesthesia, Pharmacology & Therapeutics, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada V6T 1Z3

a r t i c l e

i n f o

Article history: Received 5 March 2015 Received in revised form 16 April 2015 Accepted 16 April 2015 Available online 25 April 2015 Keywords: Ischemic arrhythmias Naloxone Electrical arrhythmia Opioid Quinidine Ventricular fibrillation Antiarrhythmic

a b s t r a c t This study characterized the antiarrhythmic effects of the opioid receptor antagonist naloxone in rats subject to electrically induced and ischemic arrhythmias. Naloxone (2, 8 and 32 ␮mol/kg/min) was examined on heart rate, blood pressure, and the electrocardiogram (EKG) as well as for effectiveness against arrhythmias produced by occlusion of the left anterior descending coronary artery or electrical stimulation of the left ventricle. Naloxone reduced blood pressure at the highest dose tested while heart rate was dose-dependently reduced. Naloxone dose-dependently prolonged the P–R and QRS intervals and increased the RSh amplitude indicative of effects on cardiac sodium (Na) channels. Naloxone prolonged the Q–T interval suggesting a delay in repolarization. Naloxone effects were comparable to the comparator quinidine. Naloxone (32 ␮mol/kg/min) reduced ventricular fibrillation (VF) incidence to 38% (from 100% in controls). This same dose significantly increased the threshold for induction of ventricular fibrillation (VFt), prolonged the effective refractory period (ERP) and reduced the maximal following frequency (MFF). The patterns of ECG changes, reduction in ischemic arrhythmia (VF) incidence and changes in electrically induced arrhythmia parameters at high doses of naloxone suggest that it directly blocks cardiac Na and potassium (K) ion channels. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Coronary heart disease commonly manifests as myocardial ischemia in the heart and results in arrhythmias. ‘Sudden death’ due to lethal ventricular fibrillation (VF) arrhythmias remains the main cause of death in the US. Death rates from VF have not changed dramatically since the inception of antiarrhythmic drug development programs [1]. There is a need for effective drugs to prevent arrhythmias despite the introduction of useful interventional cardiac techniques. It is apparent that there is no endogenous or exogenous chemical which sine qua non will solve the problem of prevention by drugs of arrhythmia-induced sudden death. Moreover, currently approved drugs and research efforts have been stopped due to serious drug safety issues encountered during drug development [2]. Thus there is a need to continue to explore new, and even old synthetic, chemical avenues in a search for useful drugs.

∗ Corresponding author at: Department of Toxicology, Pathology & Global Safety Pharmacology, Janssen Pharmaceuticals LLC, Raritan, NJ 08869, United States. Tel.: +1 908 399 8179. E-mail address: [email protected] (M.K. Pugsley). 1 Present address: Department of Global Safety Pharmacology & Toxicology, Janssen Pharmaceuticals LLC, Raritan, NJ 08869, United States. 2 Present address: Porsolt SAS, Z.A de Glatigne, 53940 Le-Genest-Saint-Isle, France. http://dx.doi.org/10.1016/j.phrs.2015.04.011 1043-6618/© 2015 Elsevier Ltd. All rights reserved.

Naloxone is a clinically used opioid receptor antagonist [3] that is a synthetic congener of oxymorphone, an opioid analgesic [4]. Naloxone reduces ventricular arrhythmias produced in multiple experimental models [5–10]. It is postulated that naloxone exhibits antiarrhythmic activity by two mechanisms. Parratt and Sitsapesan [11] and Lee [9] suggest that during ischemia endogenous opioid peptides (EOP) are released from the myocardium and mediate arrhythmias by binding to their respective opioid receptors [12]. Thus, EOP may be intrinsically arrhythmogenic [13–15] and since naloxone blocks EOP receptors this is the mechanistic basis for antiarrhythmic activity. Other studies [8,16,17] suggest that these effects of naloxone are not mediated by interactions with opioid receptors. Instead, naloxone has direct effects on the heart [18] due to blockade of cardiac ion channels [19,20] similar to the effects of naloxone observed on Na and K currents in guinea pig atria [21] and morphine in the squid giant axon [22,23]. Naloxone reduced the upstroke and prolonged the duration of the cardiac action potential (AP) [24]. Pugsley et al. [19,25] showed that cardiac Na and K channel block by arylacetamide ␬ opioid receptor agonists reduce cardiac arrhythmias [10,19,25]. The present study examined the antiarrhythmic actions of naloxone using ischemia and electrically induced arrhythmia rat models. These studies were integral to a wide ranging series of studies conducted as part of a drug discovery program aimed at finding a better antiarrhythmic drug.

M.K. Pugsley et al. / Pharmacological Research 97 (2015) 64–69

2. Materials Male Sprague-Dawley rats from the U.B.C. Animal Care Centre (275–325 g) were used in these studies according to the IACUC guidelines established by the University and EU Directive 2010/63/EU for animal experiments. 3. Animal preparation Adult rats were anesthetized (pentobarbital, 60 mg/kg, i.p.) and the trachea cannulated. Animals were artificially ventilated (100% oxygen) at a 10 ml/kg stroke volume and rate of 60 strokes/min. Body temperature was maintained (36–37 ◦ C) using a heating lamp. The right jugular vein was cannulated for drug administration and blood pressure (BP) recorded from the left carotid artery. A lead II configuration was used to record the ECGs [25,26].

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ECG changes occur that are associated with ischemia including a rapid increase in amplitude of the ECG signal defined by an increase in the R-wave. In rats it is difficult to assess the morphology of the T-wave of the ECG thus difficult to assess changes in the elevation of the S–T wave as a percentage of the R-wave amplitude [31]. The S–T segment changes in rats are biphasic – fall after ligation to baseline [28] and increase to a maintained maximum [32]. Since S–T segment elevation changes with ischemia and varies with each animal the maximum and time to maximum S–T segment elevation were determined in all study groups. After the post-occlusion observation period, a second blood sample was taken from surviving animals. Hearts were subjected to Langendorff perfusion with cardiogreen dye (1.0 mg/ml) to expose the occluded zone (or cardiac zone-at-risk). All aspects of these studies were performed according to the Lambeth Conventions [33,34].

4. Naloxone dose–response curves 4.2. Exclusion criteria used in occlusion studies The effects of naloxone (solubilized in isotonic saline) on heart rate, blood pressure and EKG measures were examined in anesthetized, ventilated rats (n = 8/group). Naloxone doses were infused at rates of 2.0, 8.0 or 32.0 ␮mol/kg/min. Recordings were made 5 min after infusion (at a rate of 1 ml/h) or just prior to coronary occlusion. Heart rate was calculated from the EKG using a Model 7D Grass polygraph (Quincy, MA, USA) while ECG intervals (P–R, QRS and Q–T) were directly determined from the traces. No Q–T interval correction for heart rate was applied [27]. The RSh amplitude was measured as the difference between the peak of the R-wave and the trough of the S-wave [25,26].

Exclusion criteria were applied in this study including: (1) the animal’s BP remained above 25 mmHg; (2) animals exhibited normal sinus rhythm with discernable ECG intervals prior to occlusion; (3) prior to occlusion only 15 VPB were allowed; (4) serum K levels had to be 2.9–3.9 mM before ischemia; (5) ischemia included increases in the R-wave height and elevation of the S–T segment [28]; (6) the occluded (ischemic) zone was 25–50% of the weight of the left-ventricule. Failure to meet these criteria resulted in exclusion and replacement of the animal to balance group size [33,34]. 5. Electrical stimulation

4.1. Coronary artery occlusion Surgical procedures are described by Pugsley et al. [10]. A left thoracotomy was produced and a polyethylene occluder placed around the left coronary artery in anesthetized, ventilated, cannulated rats. The chest was closed and the animal was allowed to recover (45 min) prior to drug administration. The serum K level (Ionetics Potassium Analyzer) was determined from a blood sample taken prior to occlusion since this ensures adequate ventilation of the animal and that physiologically relevant K levels exist since this electrolyte can influence arrhythmia incidence [10,28]. Animals (n = 8/group) were given an infusion of either isotonic saline (control) or naloxone (at either 2.0, 8.0, or 32.0 ␮mol/kg/min) using a random block design study protocol. The BP and ECG were recorded 5 min after infusion and a blood sample (0.25 mL) taken before occlusion. Quinidine (n = 8) was the positive control in this study in order to compare naloxone findings since it blocks Na and K channels in the rat and is antiarrhythmic [29]. Quinidine (2.0 ␮mol/kg) was given as a slow bolus dose (given over 5 min). ECG, arrhythmias, BP, heart rate and mortality were monitored for 30 min after occlusion. Arrhythmias designated as ventricular premature beats (VPB), ventricular tachycardia (VT) and ventricular fibrillation (VF) were summed using an Arrhythmia Score (AS) [30] for each animal. Values were assigned based on the time of occurrence, incidence and duration of arrhythmia type: 0 (0–49 VPBs), 1 (50–499 VPBs), 2 (>499 VPBs ± 1 episode of spontaneously reverting VT or VF), 3 (>1 VT/VF episode or both with a duration <60 s), 4 (VT/VF alone or both with 60–120 s duration), 5 (VT/VF or both with duration >120 s), 6 (lethal VF starting <15 min post-occlusion), 7 (lethal VF starting 4–15 min), 8 (lethal VF starting 1–4 min postocclusion) and 9 (lethal VF starting <1 min post-occlusion) [10,30]. The ECG had a positive S–T segment during the pre-dose interval so signs of drug-mediated changes could be discerned prior to occlusion and allowed for assessment of post-occlusion changes. Specific

Arrhythmias can be produced by electrical stimulation of the left ventricle [35]. Thus, the influence of drugs on ventricular vulnerability (i.e., arrhythmia induction) can be assessed as well as a probe of the drug effects on Na and K channels. In anesthetized rats, left-ventricle electrical stimulation using two TeflonTM -coated silver wire electrodes inserted through the chest wall was conducted [36]. A constant current square wave Grass stimulator (Grass, model SD9 or S88) was used to stimulate the left ventricle. The threshold current for capture (iT – ␮A), threshold pulse-width (tT – ms) for induction of extrasystoles, threshold current for induction of VF (VFt – ␮A), maximum following frequency (MFF – Hz) and effective refractory period (ERP-ms) were determined [19]. The variables iT and tt assess excitability [37] and measure Na channel availability. Ventricular fibrillation threshold (VFt) is the current required to develop VF [35]. ERP and MFF are measures of AP refractoriness [25]. ERP measured drug changes in the absolute refractory period while MFF probes the relative refractory period. The electrical stimulation variables were determined 5 min after each dose of infused saline or naloxone (n = 8/group). Naloxone was given cumulatively at 2.0, 8.0 and 32.0 ␮mol/kg/min. Quinidine was given as a single 2.0 ␮mol/kg slow bolus dose (n = 8). 6. Drugs Naloxone hydrochloride dihydrate ((5␣)-4,5-epoxy-3,14dihydroxy-17-(2-propen-1-yl)morphinan-6-one) (PubChem CID: 5464092) and quinidine hydrochloride monohydrate (6 methoxycinchonan-9-ol hydrochloride monohydrate) (PubChem CID: 16219921) were purchased from Sigma–Adrich Chemical Co. (St. Louis, MO). Naloxone was dissolved in 0.9% sodium chloride while quinidine was dissolved in 22% ethanol/78% distilled water.

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Table 1 The effects of naloxone and quinidine on hemodynamic and EKG measures prior to coronary artery ligation in anesthetized rats. Dose

BP (mmHg)

Saline 118 Naloxone (␮mol/kg/min) 2.0 112 8.0 110 78 32.0 Quinidine (␮mol/kg) 103 2.0

HR (beats/min)

P–R (ms)

QRS (ms)

RSh (mV)

Q–T (ms)

±5

359 ± 8

55 ± 2

29 ± 0.5

0.42 ± 0.04

38 ± 1

±7 ±6 ± 8*

341 ± 12 329 ± 10* 311 ± 9*

58 ± 1 60 ± 1 64 ± 2*

31 ± 1 31 ± 0.4 34 ± 2*

0.46 ± 0.03 0.51 ± 0.04* 0.58 ± 0.06*

39 ± 1 41 ± 1 46 ± 2*

± 9*

332 ± 16*

65 ± 5*

35 ± 0.3*

0.61 ± 0.05*

49 ± 1*

Values are the mean ± s.e.m. for the effects of naloxone, infused at the doses indicated and quinidine at the dose given, on blood pressure (BP), heart rate (HR), and the P–R, QRS, RSh, and Q–T intervals of the EKG. Values were obtained 5 min after beginning infusion of the naloxone dose or completion of the slow bolus quinidine dose, prior to occlusion (n = 8 for all treatment groups). * P < 0.05 for difference from saline control. Table 2 The effects of naloxone and quinidine on arrhythmia incidence in coronary artery ligated anesthetized rats. Drug

Arrhythmias Log10 PVC

Saline 2.0 ± Naloxone (␮mol/kg/min) 1.8 ± 2.0 8.0 1.5 ± 1.6 ± 32.0 Quinidine (␮mol/kg) 1.8 ± 2.0

AS VT (%)

VF (%)

0.20

100

100

0.20 0.40 0.30

88 88 88

0.10

88

OZ (%)

Serum K+ (mM) Pre

Post (n)

5.8 ± 0.4

35 ± 2.0

3.4 ± 0.3

3.8 ± 0.3 (4)

88 50* 38*

5.1 ± 0.5 3.8 ± 0.3* 3.0 ± 0.5*

37 ± 2.5 32 ± 1.4 31 ± 1.5

3.6 ± 0.5 3.5 ± 0.1 3.7 ± 0.1

4.0 ± 0.2 (4) 3.9 ± 0.2 (4) 4.2 ± 0.2 (5)

0*

1.6 ± 0.7*

32 ± 2.3

3.4 ± 0.1

3.2 ± 0.1 (7)

Values are the mean ± s.e.m. during coronary artery occlusion. Drug effects are expressed as group incidence (n = 8) of one or more episodes of ventricular tachycardia (VT) or fibrillation (VF). Log10 PVC denotes transformation of the number of ventricular premature beats (VPB). AS is arrhythmia score and OZ is occluded zone. Serum K was determined prior to drug infusion (pre) and 30 min post-occlusion (post), provided the animal survived (value in parentheses). * P < 0.05 for difference from saline control.

7. Statistical analyses Values are mean ± standard error of the mean (s.e.m). Statistical significance (P < 0.05) was determined with the NCSS statistical package [38]. Repeated measures ANOVA was used to determine significance and multiple comparisons were made using Duncan’s test. Significant arrhythmia incidence was assessed by Mainland’s contingency tables [39]. VPB count was log10 transformed for parametric analysis. 8. Results 8.1. Hemodynamic and EKG effects of naloxone Naloxone, at the highest dose tested, 32 ␮mol/kg/min, reduced blood pressure when compared to the saline control group (Table 1). Heart rate was dose-dependently decreased from a control of 359 ± 8 beats/min to 331 ± 9 at this same dose. Quinidine produced the anticipated reduction in both blood pressure and heart rate for the dose administered [29]. Some ECG measures were changed by naloxone at the mid and high doses, the lowest dose tested (2.0 ␮mol/kg/min) did not produce any changes in the ECG (Table 1). Naloxone prolonged the P–R interval from a control of 55 ± 2 ms to 64 ± 2 ms at the high dose. This same naloxone dose increased RSh amplitude from a control of 0.42 ± 0.04 mV to 0.58 ± 0.06 mV concurrent to a prolongation of the QRS interval (∼17%). The Q–T interval was also increased from a control of 38 ± 1 ms to 46 ± 2 ms at the highest dose tested (Table 1). Saline was without effect on the ECG measures. Quinidine produced changes that result from both Na channel block (i.e., prolongation of the QRS interval) as well as K channel block (i.e., prolongation of the Q–T interval) (Table 1). 8.2. Antiarrhythmic actions of naloxone The effects of naloxone on the in vivo ECG block of both Na and K channels and previous studies show that a single dose of

naloxone has antiarrhythmic actions against ischemic arrhythmias [10]. Antiarrhythmic activity, when given as an infusion, was examined in coronary artery ligated animals. The antiarrhythmic actions are summaried in Table 2 and compared to those of quinidine (2.0 ␮mol/kg). Naloxone decreased arrhythmia incidence at the mid and high dose but not at the low dose. The mid and high doses reduced the incidence of VF (from 8/8 to 4/8 and 3/8 rats, respectively) and arrhythmia score (AS) but, like quinidine, did not reduce VPBs (Table 2). Quinidine did abolish VF at the dose examined. Only quinidine reduced ischemic mortality (7/8 animals survived). The antiarrhythmic activity of naloxone and quinidine did not result from altered occluded zone size or serum K concentrations. Arrhythmic insult was similar in all groups since the ischemic zone size was not different between groups and serum K concentrations were similar in all groups (Table 2).

8.3. Effects of naloxone on ischemia-induced changes in the EKG EKG changes show that neither naloxone nor quinidine reduced the maximum size of the R-wave that developed post-occlusion. However both drugs reduced the S–T segment change and significantly prolonged the time to development of the maximum S–T segment elevation (Table 3). Table 3 also shows that only the high dose of naloxone prolonged the time to maximal R-wave development, similar to quinidine, potentially indicative of a limited anti-ischemic effect.

8.4. Effects of naloxone on electrically induced arrhythmias Naloxone produced similar changes in BP, heart rate and the ECG in electrically induced arrhythmia animals at the same infused doses as in coronary ligated animals (data not shown). Naloxone did not alter thresholds for capture for ventricular extrasystoles (iT ) or time to threshold (tT – data not shown) but significantly increased VFt at the highest dose tested. VFt increased from 130 ± 15 to

M.K. Pugsley et al. / Pharmacological Research 97 (2015) 64–69 Table 3 The effect of naloxone and quinidine on EKG changes during coronary artery occlusion. Group

Max. S–T (% of R)

Saline 79 ± 11 Naloxone (␮mol/kg/min) 2.0 67 ± 4 8.0 62 ± 7 32.0 63 ± 8* Quinidine (␮mol/kg) 2.0 60 ± 5*

Time to maximum S–T (min)

Maximum R-wave (mV)

Time to max. R-wave (s)

5.2 ± 1.8

1.8 ± 0.4

30 ± 2

5.9 ± 1.1 6.8 ± 2.0 8.0 ± 0.5*

1.5 ± 0.3 1.6 ± 0.2 1.3 ± 0.4

33 ± 5 35 ± 6 38 ± 3*

11 ± 2.2*

1.2 ± 0.3

45 ± 5*

Values are the mean ± s.e.m. during coronary artery occlusion (n = 8). The maximal S–T segment elevation is expressed as a % of the height of the R-wave. * P < 0.05 for difference from saline control. Table 4 The effect of naloxone and quinidine on electrical stimulation measures in anesthetized rats. Drug

iT (␮A)

Naloxone (␮mol/kg/min) Saline 63 ± 4.2 2.0 65 ± 3.1 8.0 67 ± 2.8 32.0 71 ± 4.8 Quinidine (␮mol/kg) 2.0 78 ± 6.0*

VFT (␮A) 130 137 153 161

± ± ± ±

15 12 11* 13*

168 ± 12*

ERP (ms) 47 50 57 71

± ± ± ±

4.0 5.1 4.4* 5.0*

85 ± 6.5*

MFF (ms) 17 16 14 12

± ± ± ±

0.3 0.5 0.4 0.2*

10 ± 0.3*

Values are the mean ± s.e.m for drug effects on left-ventricle electrical stimulation measures. Effects on threshold current for capture (iT ) or fibrillation (VFT), effective refractory period (ERP) and maximum following frequency (MFF) were obtained 5 min after infusion at the doses indicated or after completion of the slow bolus quinidine dose (n = 8). * P < 0.05 for difference from saline control.

161 ± 13 ␮A with naloxone. Quinidine produced effects indicative of Na channel blockade. Naloxone (32.0 ␮mol/kg/min) dose-dependently reduced MFF from 17 ± 0.3 Hz in saline control to 12 ± 0.8 Hz. Measured ERP was dose-dependently prolonged by naloxone (Table 4). ERP was increased from 47 ± 4.0 ms in saline control to 57 ± 4.4 ms and 71 ± 5.0 ms at the mid and high doses (8.0 and 32.0 ␮mol/kg/min), respectively. Quinidine prolonged ERP by ∼80% and reduced MFF by ∼41%. The vehicle control values for were constant over the treatment period. 9. Discussion In this study we examined the dose-dependent effects of naloxone on heart rate, blood pressure, EKG changes and incidence of arrhythmias in anesthetized rats subject to either left-anterior descending (LAD) coronary artery occlusion or electrical myocardial stimulation. At high doses, naloxone produced consistent effects on EKG measures examined unrelated to opioid receptor antagonism. Naloxone blockade of opioid receptors occurs at nonclinical doses of 0.15 ␮mol/kg [40]; thus, blood concentrations resulting in opioid receptor blockade would be much lower than those achieved in this study. Thus, while opioid receptors would be expected to be fully blocked at the lowest dose examined in this study, it did not protect the heart from variant forms of arrhythmia induction. This suggests that naloxone antagonism of opioid receptors is not the probable mechanism for cardiac effects. These findings dispute the early literature with regard to the nature of the antiarrhyhmic mechanism proposed. Many initial studies suggested that naloxone blockade of EOP release in the myocardium was responsible for the antiarrhythmic activity observed in a multitude of models. From our studies, significant antiarrhythmic effects of naloxone were only observed at the highest dose tested

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(32.0 ␮mol/kg/min) and the reduction in arrhythmias observed was primarily confined to those of a high frequency, i.e., VF. Naloxone limited did not eliminate the incidence of either VT or VPBs. This pattern was similar to that observed for quinidine used in these studies [29]. Naloxone produced a decrease in blood pressure and heart rate and a prolongation in the P–R and QRS intervals as well as an increase in the RSh amplitude of the EKG suggesting Na channel blockade. Naloxone also prolonged the Q–T interval suggesting a delay in repolarization from K channel blockade. These changes are similar in profile to those seen with the administration of many antiarrhythmic drugs, most typically those defined as class Ia agents. Studies by Pugsley et al. [19,25,41] show that at high doses many structurally different highly potent opioid compounds are antiarrhythmic due to Na and/or K channel blockade in the heart [25,42]. Naloxone was used in many of these studies as a pharmacological tool to block opioid receptors and reveal these non-opioid ion channel properties; however, naloxone itself, when given as a single bolus dose (8 ␮mol/kg), exhibited antiarrhythmic activity [10]. As observed in this study, naloxone only reduced the incidence of VF, nor did it have an effect on the QRS or Q–T intervals of the EKG. Rather naloxone only produced a prolongation of the P–R interval, as was observed in this study. These effects corroborate studies highlighting the antiarrhythmic properties not related to opioid receptor antagonism [18,43]. The determination of opioid receptor binding affinity for the active enantiomer, (−)-naloxone, range between 0.56 and 4.9 nM for ␮ and ␬ receptors [44]. When the stereoisomer, (+)-naloxone (with an opioid potency ∼1000–10,000 times less than that of (−)-naloxone) was used, it produced comparable antiarrhythmic activity [17,45]. The authors conclude that non-opioid pharmacological activity reduced arrhythmia incidence. Sarne et al. [8] then examined the antiarrhythmic activity of a series of opioid agonists, antagonists and their inactive enantiomers in an ischemic rat model. Similarly, the inactive (+)-naloxone stereoisomer was equipotent with naloxone at reducing ischemic arrhythmias [8]. These findings complement previous studies suggesting a direct effect on cardiac ionic currents as the mechanism responsible for antiarrhythmic activity. These findings corroborate studies by Frazier et al. [22] who showed that when morphine was perfused into the squid giant axon it had local anesthetic (Na channel blocking) activity and when naloxone was co-perfused with morphine the effects were additive [23]. Under voltage clamp conditions in the frog node of Ranvier both Na and K currents were decreased with naloxone (68 ␮M) perfusion [46]. Thus naloxone studies in multiple preparations in different species show Na and K channel blocking properties. Studies with k opioid receptor agonists U-50,488H [10,19], (−)PD129,290 [25] and spiradoline (U-62,066E) [47] consistently showed antiarrhythmic activities due to Na and K channel blockade. In several studies these arylacetamide compounds produce concentration-dependent tonic and frequency-dependent block of cardiac Na channels [25,41,47] and a hyperpolarizing shift in the steady-state voltage-dependence of inactivation of the Na channel. We confirmed that naloxone produced direct Na channel block by examining cardiac (rH1) currents expressed in Xenopus oocytes [20] which lack endogenous opioid receptors. Thus at the doses and concentrations examined in these studies, direct blockade of Na and K currents in the heart is the most likely explanation for the antiarrhythmic actions observed. The naloxone dose that reduced VF incidence in the rat (32.0 ␮mol/kg/min) also slowed the rate at which EKG changes occur following ischemia. Naloxone reduced the maximum S–T segment change that developed but not the R-wave height. However, it delayed the time to the development of these changes suggesting a possible anti-ischemic action at the high dose. But when the magnitude of the naloxone changes are compared to

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drugs (verapamil) that truly provide marked anti-ischemic actions [31], it is only a minimally protective effect. The changes in these ischemic markers likely result from some drug effect on Ca currents [48,49] since there was some P–R interval prolongation. These weak anti-ischemic actions of high dose naloxone are the first to be reported for this opioid receptor antagonist and require further investigation. By exploring the antiarrhythmic activity of drugs in ischemia and electrical arrhythmia models and assessing the ion channel blocking properties of multiple compounds, it proved possible, by selecting enantiomers lacking in opioid receptor actions and using conventional structure activity relationship studies in functional in vivo and in vitro preparations, to develop a compound, vernakalant, currently used clinically to terminate acute atrial fibrillation in many countries. 10. Conclusion Naloxone, a benzomorphan opioid antagonist, has a similar nonopioid profile in the heart to arylacetamide ␬ opioid agonists. The profile suggests Na and K channel blocking actions which are similar to the class Ia antiarrhythmic drug, quinidine. While high dose non-opioid pharmacological properties of naloxone may have limited clinical relevance, the benzomorphan structure of naloxone may provide additional important structural information regarding the nature of drug blockade of cardiac ion channels. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgements This study was funded by the Heart & Stroke Foundation of B.C. and Yukon and the Science Council of British Columbia. References [1] M.K. Pugsley, S. Authier, M.J. Curtis, Cardiovascular disease: drug development struggles against a global epidemic, Pharmacol. Matters 3 (2010) 25–30. [2] M.K. Pugsley, Antiarrhythmic drug development: historical review and future perspective, Drug Dev. Res. 55 (2002) 3–16. [3] W.R. Martin, Pharmacology of opioids, Pharmacol. Rev. 35 (4) (1983) 283–323. [4] A.P. Feinberg, I. Creese, S.H. Snyder, The opiate receptor: a model explaining structure–activity relationships of opiate agonists and antagonists, Proc. Natl. Acad. Sci. U. S. A. 73 (1976) 4215–4219. [5] O. Fagbemi, I. Lepran, J.R. Parratt, L. Szekeres, Naloxone inhibits early arrhythmias resulting from acute coronary ligation, Br. J. Pharmacol. 76 (4) (1982) 504–506. [6] A.Y.S. Lee, Y.T. Chen, M.N. Kan, F.K. P’eng, C.Y. Chai, J.S. Kuo, Consequences of opiate agonist and antagonist in myocardial ischemia suggest a role of endogenous opioid peptides in ischemic heart disease, Cardiovasc. Res. 26 (4) (1992) 392–395. [7] X.D. Huang, A.Y.S. Lee, T.M. Wong, C.Y. Zhan, Y.Y. Zhao, Naloxone inhibits arrhythmias induced by coronary artery occlusion and reperfusion in anesthetized dogs, Br. J. Pharmacol. 87 (3) (1986) 475–477. [8] Y. Sarne, A. Flitstein, E. Oppenheimer, Anti-arrhythmic activities of opioid agonists and antagonists and their stereoisomers, Br. J. Pharmacol. 102 (3) (1991) 696–698. [9] A.Y.S. Lee, Stereospecific antiarrhythmic effects of naloxone against myocardial ischemia and reperfusion in the dog, Br. J. Pharmacol. 107 (4) (1992) 1057–1060. [10] M.K. Pugsley, W.P. Penz, M.J.A. Walker, T.M. Wong, Antiarrhythmic effects of U-50,488H in rats subject to coronary artery occlusion, Eur. J. Pharmacol. 212 (1) (1992) 15–19. [11] J.R. Parratt, R. Sitsapesan, Stereospecific antiarrhythmic effect of opioid receptor antagonists in myocardial ischaemia, Br. J. Pharmacol. 87 (4) (1986) 621–627. [12] L.N. Maslov, Iu.B. Lishmanov, J.I. Székely, The mechanism of the anti-arrhythmia action of opioid receptor agonists and antagonists, Bull. Exp. Biol. Med. 116 (1993) 169–171. [13] A.Y. Lee, C.Y. Zhan, T.M. Wong, Effects of ␤-endorphin on the contraction and electrical activity of the isolated perfused rat heart, Int. J. Pept. Protein Res. 24 (5) (1984) 525–528.

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