Cardiac Electropharmacological Effects of Antidiarrheal Drug Loperamide and Its Antidote Naloxone in Anesthetized Guinea Pigs

Abstract

Cardiac electropharmacological effects of an antidiarrheal drug loperamide and its antidote naloxone were assessed in isoflurane-anesthetized guinea pigs. Intravenous administration of loperamide at 0.01–0.1 mg/kg did not affect parameters of electrocardiogram (ECG) or monophasic action potential (MAP) of the right ventricle. Additional administration of loperamide at 1 mg/kg prolonged the QT interval and MAP duration of the ventricle accompanied with increments of the PQ interval and QRS width. The potency of loperamide for QT-interval prolongation was about 100-times lower than that of dofetilide, in spite that similar inhibitory effects on the human Ether-a-go-go Related Gene (hERG) K⁺ channels have been reported between loperamide and dofetilide, implying lower accessibility of loperamide to the K⁺ channels. Intravenous administration of naloxone at 0.003–0.3 mg/kg, which effectively inhibits µ-opioid receptors, did not affect ECG parameters including QT interval or MAP duration. Furthermore, the loperamide-induced cardiac electrophysiological changes were not modified in the presence of naloxone at 0.3 mg/kg. These results suggest that loperamide has a potential to delay cardiac conduction and repolarization in the in vivo condition. Since naloxone did not modify ECG parameters and loperamide-induced ECG changes, naloxone is confirmed to possess acceptable cardiac safety when used as an antidote.

INTRODUCTION

Loperamide is as an antidiarrheal drug acting as a µ-opioid agonist, which can be obtained through an OTC medication. In 2016, loperamide-induced torsade de pointes arrhythmias were reported, and several case reports, usually associated with loperamide abuse, have been subsequently reported.^1–3) Previous experimental studies have demonstrated that loperamide inhibited Na_v1.5, K_vLQT1/minK and human Ether-a-go-go Related Gene (hERG) channel currents,⁴⁾ which may support clinical observations in abusers of loperamide, such as wide QRS complex and prolonged QT interval, leading to ventricular tachyarrhythmias, including torsade de pointes or Brugada syndrome.⁵⁾ However, information is still lacking regarding in vivo electropharmacological effects of loperamide to discuss causality based on bridge between cellular actions and clinical observations.

To treat cases of loperamide intoxication such as respiratory failure and neurological symptoms, a µ-opioid receptor antagonist naloxone is widely used for the addicts. However, information regarding cardiac electrophysiological effects of naloxone is limited and controversial; for example, shortening of the action potential duration of the isolated rabbit heart⁶⁾ and prolongation of the QT interval in rats.⁷⁾ Therefore, we cannot understand whether naloxone interacts loperamide-induced QT interval prolongation when used as an antidote.

In this study, we initially assessed electropharmacological effects of loperamide in anesthetized guinea pigs, which were compared with those of a typical hERG K⁺ channel blocker dofetilide, to clarify its potential of QT-interval prolongation. Next, we investigated interaction of loperamide and naloxone on the parameters of electrocardiogram to estimate cardiac safety of naloxone when used as an antidote.

MATERIALS AND METHODS

All animal experimental procedures were approved by the Toho University Animal Care and User Committee. Experiments were done according to the Guiding Principles for the Care and Use of Laboratory Animals approved by The Japanese Pharmacological Society. Twenty-seven male Hartley guinea pigs were obtained from Japan SLC (Hamamatsu, Japan).

The measurement procedures were based on our previous studies.^8,9) Guinea pigs were anesthetized with 1.0% isoflurane vaporized with room air. The blood pressure was measured from the left carotid artery. The surface lead II electrocardiogram (ECG) was obtained from the limb electrodes. A monophasic action potential (MAP) recording/pacing electrodes combination catheter (3 F, SMC-304; Physio-Tech, Tokyo, Japan) was positioned at the right ventricle, whose signals were amplified with a differential amplifier (DAM 50; World Precision Instruments, Sarasota, FL, U.S.A.). The heart was electrically driven with a cardiac stimulator (BC-03, Fukuda Denshi, Tokyo, Japan). The interval (ms) at the 90% repolarization level was defined as MAP₉₀, which was measured during sinus rhythm (MAP_90(sinus)) and at a pacing cycle length of 300 ms (MAP_90(CL300)) or 250 ms (MAP_90(CL250)).

Cardiovascular parameters were analyzed with a real-time full automatic analysis system (MP/VAS 3 for Windows ver. 1.1, Physio-Tech). The ECG parameters and MAP₉₀ were measured as the mean of three consecutive recordings. After the basal control assessment (C), a dose of 0.01 mg/kg of loperamide was administered intravenously (n = 5), and the ECG parameters and MAP₉₀ were measured until 30 min after the start of drug infusion. Similarly, 0.1 and 1 mg/kg of loperamide was additionally infused over 10 min, and each parameter was measured. In another series of animals, the effects of dofetilide at doses of 0.001, 0.01 and 0.1 mg/kg and those of naloxone at doses of 0.003, 0.03 and 0.3 mg/kg were assessed in a similar manner. Influence of naloxone on electropharmacological action of loperamide was assessed using another series of animals. After the treatment with naloxone (0.3 mg/kg) or saline, 1 mg/kg of loperamide was additionally administered, and each parameter was measured until 30 min after the start of loperamide infusion.

Loperamide hydrochloride (Tokyo Kasei, Tokyo, Japan) and dofetilide (Sigma-Aldrich Co., St. Louis, MO, U.S.A.) were dissolved in 0.5% lactic acid and 0.1 mM hydrochloride, respectively. Naloxone hydrochloride (Abcam, Cambridge, U.K.) was dissolved in saline. Drugs were administered via the left jugular vein. Isoflurane and heparin sodium were purchased from Mylan (Tokyo, Japan) and EA Pharma (Tokyo, Japan), respectively.

All data are expressed as mean ± standard error of mean (S.E.M.). Statistical analysis was undertaken using unpaired t-test and Dunnett’s test as implemented in GraphPad Prism (ver. 8.4.3; GraphPad Software, Inc., La Jolla, CA, U.S.A.). A p-value less than 0.05 was considered statistically significant.

RESULTS

Typical traces of effects of loperamide on the ECG and MAP are depicted in Fig. 1. The time courses of effects of loperamide (n = 5) and dofetilide (n = 5) on the heart rate are summarized in Fig. 2. In the loperamide group, no significant change was detected in any parameters after the low dose. The middle dose prolonged the QT interval, whereas no significant change was detected in any parameters except for the QT interval. The high dose decreased the heart rate, but increased the PQ interval, QT interval and QTc(S) and the QRS width. In the dofetilide group, no significant change was detected in the ECG parameters after the low dose. The middle dose decreased the heart rate but increased the QT interval and QTc(S). The high dose further decreased the heart rate but increased the PQ interval and the QT interval and QTc(S).

Fig. 1. Typical Tracings of the Effects of Loperamide on the Electrocardiogram (ECG) and Monophasic Action Potential (MAP) in the Isoflurane-Anesthetized Guinea Pig

Loperamide was administered intravenously at 1 mg/kg over 10 min. ECG and MAP tracings were obtained before and 10 min after the start of drug infusion. Since the MAP signal is recorded by the contact electrode technique, its amplitude depends on extent of press against the myocardium.

Fig. 2. Time Courses of the Effects of Loperamide (n = 5) and Dofetilide (n = 5) on the Heart Rate (HR) and ECG Parameters: PQ Interval (Triangles), QRS Width (Squares), QT Interval (Circles) and QTc(S) (Diamonds)

The corrected QT interval (QTc) was calculated using Sakaguchi’s formula [QTc(S) = QT/(RR/300)^1/3]. In the loperamide group (left panel), pre-drug control values of the heart rate, PQ interval, QRS width, QT interval and QTc(S) were 207 ± 5 bpm, 60 ± 3, 24 ± 1, 206 ± 12, and 208 ± 11 ms, whereas those in the dofetilide group (right panel) were 203 ± 8 bpm, 65 ± 3, 28 ± 3, 217 ± 13, and 217 ± 10 ms, respectively. The maximum changes in the QT interval by loperamide were 36 ± 6 ms after the high dose, and those by dofetilide were 40 ± 12 and 105 ± 14 ms after the middle and high dose, respectively. Loperamide or dofetilide was intravenously infused over 10 min. Data are presented as mean ± S.E.M. The solid symbols represent significant differences from the corresponding pre-drug control value (C) for each parameter at p < 0.05.

The time courses of effects on the MAP₉₀ are summarized in Fig. 3. In the loperamide group, no significant change was detected in any parameters after the low dose. The middle dose prolonged the MAP_90(sinus), and the high dose increased the MAP_90(sinus), MAP_90(CL300), and MAP_90(CL250). In the dofetilide group, the middle dose increased the MAP_90(sinus), MAP_90(CL300), and MAP_90(CL250), and high dose increased the MAP_90(sinus) and MAP_90(CL300).

Fig. 3. Time Courses of the Effects of Loperamide (n = 5) and Dofetilide (n = 5) on the Duration of the Monophasic Action Potential at a Level of 90% Repolarization during Sinus Rhythm (MAP_90(sinus), Circles), and That during Ventricular Pacing at a Cycle Length of 300 ms (MAP_90(CL300), Squares) and 250 ms (MAP_90(CL250), Triangles)

In the loperamide group (left panel), pre-drug control values of the MAP_90(sinus), MAP_90(CL300) and MAP_90(CL250) were 182 ± 11, 182 ± 8, and 167 ± 6 ms, whereas those in the dofetilide group (right panel) were 183 ± 17, 175 ± 17, and 157 ± 17 ms, respectively. Data of MAP_90(CL250) during the high dose of dofetilide were n = 4 because of pacing failure due to excessive prolongation of ventricular repolarization. Data are presented as mean ± S.E.M. The solid symbols represent significant differences from the corresponding pre-drug control value (C) for each parameter at p < 0.05.

Effects of naloxone itself on the ECG and MAP parameters are summarized in Fig. 4, where there was no significant change in any of the parameters after the administration of naloxone (n = 5). As shown in Fig. 5, no significant difference was detected in the prolongation of PQ interval, QRS width, QT interval, QTc(S) or MAP_90(CL300) induced by loperamide in the presence and absence of naloxone (n = 6).

Fig. 4. Time Courses of the Effects of Naloxone (n = 5) on the Heart Rate (HR) and ECG Parameters: PQ Interval (Triangles), QRS Width (Squares), QT Interval (Circles) and QTc(S) (Diamonds)

The corrected QT interval (QTc) was calculated using Sakaguchi’s formula [QTc(S) = QT/(RR/300)^1/3]. Pre-drug control values of the heart rate, PQ interval, QRS width, QT interval and QTc(S) were 197 ± 6 bpm, 64 ± 1, 21 ± 2, 198 ± 8, and 194 ± 7 ms, whereas those of the MAP_90(sinus), MAP_90(CL300) and MAP_90(CL250) were 172 ± 5, 176 ± 9, and 165 ± 6 ms, respectively. Naloxone was administered intravenously over 30 s. Data are presented as mean ± S.E.M. The solid symbols represent significant differences from the corresponding pre-drug control value (C) for each parameter at p < 0.05.

Fig. 5. Comparison of Effects of Loperamide (1 mg/kg) on the ECG and MAP Parameters in the Presence ((+), n = 6) and Absence ((−), n = 6) of Naloxone (0.3 mg/kg, i.v.)

The corrected QT interval (QTc) was calculated using Sakaguchi’s formula [QTc(S) = QT/(RR/300)^1/3]. Naloxone or saline was administered intravenously over 30 s, and then loperamide was additionally infused over 10 min. Data were obtained from maximum changes of the ECG and MAP variables during the 30-min observation period. Data are presented as mean ± S.E.M.

DISCUSSION

In this study, loperamide prolonged the QT interval and MAP duration of the ventricle accompanied with increment of the PQ interval and QRS width at 1 mg/kg, as shown in Figs. 2 and 3, which are similar electrophysiological properties to that in the abusers of loperamide.¹⁾ The cardiac electrophysiological changes by loperamide were not affected in the presence of naloxone.

Drug-induced QT interval prolongation is closely associated with an inhibitory action on the hERG K⁺ channels. Since loperamide has been demonstrated to exert potent inhibitory effects on the hERG K⁺ channels (IC₅₀ value = 33–89 nM) and prolongation of the action potential duration at a concentration of 10 nM in the swine ventricular myocytes,^4,10) these cellular mechanisms could explain the current results of prolongation of the ventricular repolarization in guinea pigs. Also, the increment of the PQ interval and QRS width by loperamide might be associated with its inhibitory action on Na_v1.5 (4), which would explain clinically observed loperamide-induced Brugada ECG pattern.⁵⁾

As shown in Fig. 2, similar QT-interval prolongation (about 40 ms) was detected after administration of 1 mg/kg of loperamide and 0.01 mg/kg of dofetilide, suggesting about 100-times lower potency of loperamide than dofetilide in spite that the potency of inhibitory effects of loperamide on the hERG K⁺ channels is similar to those of dofetilide (IC₅₀; 35 nM,¹¹⁾). This may imply lower accessibility of loperamide to the hERG K⁺ channels. Orally administered loperamide, whose usual sustained doses are 1 to 2 mg for diarrhea, has been reported to be absorbed well from the gastrointestinal tract and is almost completely extracted and metabolized in the liver, thereby little loperamide reaches the systemic circulation (bioavailability is <2%).¹²⁾ On the contrary, excessive oral doses of loperamide, presumably leading to saturation of hepatic metabolism, might induce drug-induced ECG abnormalities, as observed in patients who took about 134–600 mg of loperamide daily, including history of heroin abuse,^1–3,5) which is closely associated with the current results of intravenous administration of loperamide in the isoflurane-anesthetized guinea pig. We have no information regarding chronic effects of toxic doses of loperamide on ECG as well as toxicokinetic parameters,¹²⁾ which should be checked extensively. The electropharmacological as well as pharmacokinetic profiles may suggest that a torsadogenic risk of loperamide is estimated to be low as far as the drug is used properly based on a package insert or prescriptions.

Injection form of 0.2 mg of naloxone hydrochloride is clinically available for drug-induced respiratory depression, which is roughly corresponded to 0.003 mg/kg. Antidiarrheal activity of loperamide (1 mg/kg) in rats has been reported to be completely inhibited by subcutaneous administration of naloxone (0.5 mg/kg).¹³⁾ Therefore, the dose of 0.3 mg/kg of naloxone in this study is considered enough to inhibit µ-opioid receptor-mediated actions of loperamide. In this study, naloxone at 0.003–0.3 mg/kg did not affect any of ECG parameters, as shown in Fig. 4. Furthermore, the cardiac electrophysiological changes by loperamide were not affected by naloxone at 0.3 mg/kg, estimated to be 100 times higher dose than clinical one (Fig. 5). Although abbreviation of the action potential duration has been shown by naloxone at 0.1 to 2.0 µM in a previous study using the isolated rabbit heart,⁶⁾ we are unable to comment about the discrepancy. Previous electrophysiological experiments have shown effective suppression of Na⁺ current (I_Na), transient outward K⁺ current (I_to) and Ca²⁺ current (I_Ca) by naloxone at 10–30 µM,¹⁴⁾ which may explain electrophysiological effects of naloxone at a higher dose of 32 µmol/kg/min (≈10 mg/kg/min) in rats; namely, prolongations of PQ interval, QRS width and QT interval.⁹⁾ Therefore, the clinically relevant dose (=0.003 mg/kg) of naloxone possesses acceptable cardiac safety when used as an antidote of loperamide intoxication.

In conclusion, the present results show that loperamide has a potential to delay cardiac conduction and repolarization in the in vivo condition. Since naloxone did not modify ECG parameters and loperamide-induced ECG changes, naloxone is confirmed to possess acceptable cardiac safety when used as an antidote.

Conflict of Interest

The authors declare no conflict of interest.

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