Torsadogenic Potential of HCN Channel Blocker Ivabradine Assessed in the Rabbit Proarrhythmia Model

Abstract

Torsadogenic effects of ivabradine, an inhibitor of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, were assessed in an in vivo proarrhythmia model of acute atrioventricular block rabbit. Ivabradine at 0.01, 0.1, and 1 mg/kg was intravenously administered to isoflurane-anesthetized rabbits (n = 5) in the stable idioventricular rhythm. Ivabradine at 0.01 and 0.1 mg/kg hardly affected the atrial and ventricular automaticity, QT interval, or the monophasic action potential duration of the ventricle. Additionally administred ivabradine at 1 mg/kg decreased the atrial and ventricular rate significantly but increased the QT interval and duration of the monophasic action potential. Meanwhile, torsade de pointes arrhythmias were detected in 1 out of 5 animals and in 2 out of 5 animals after the administration of 0.1 and 1 mg/kg, respectively. Importantly, torsade de pointes arrhythmias could be observed only in 2 rabbits showing more potent suppressive effects on ventricular automaticity. These results suggest that the torsadogenic potential of ivabradine may become evident when its expected bradycardic action appears more excessively.

INTRODUCTION

Ivabradine is an inhibitor of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, approved as a heart rate-reducing drug for the treatment of coronary artery disease and chronic heart failure.¹⁾ This drug possesses antiarrhythmic properties, as shown in the isolated cardiac preparations from humans or dogs; this property may be advantageous in the treatment of patients with heart disease liable to cardiac rhythm disturbance.²⁾ The antiarrhythmic effects of ivabradine have also been experimentally shown in acute myocardial ischemia of rats.^3,4) However, clinical case reports of torsade de pontes (TdP) arrhythmias have been indicated by concomitant use of ivabradine.^5,6) To clarify torsadogenic potential of ivabradine itself, we investigated electrophysiological effects of ivabradine in this study by using a proarrhythmia model of acute atrioventricular (AV) block rabbit, which detects drug-induced TdP arrhythmias with high sensitivity and specificity.^7–9)

MATERIALS AND METHODS

All animal experimental procedures were approved by the Toho University Animal Care and User Committee (Approval No. 20-55-331). Experiments were conducted according to the Guiding Principles for the Care and Use of Laboratory Animals approved by The Japanese Pharmacological Society. New Zealand White rabbits were purchased from Japan SLC (Hamamatsu, Japan).

Production of Acute AV Block Model

Male rabbits (approximately 3 kg) were anesthetized with intramuscular administration of ketamine hydrochloride (35 mg/kg) and xylazine hydrochloride (5 mg/kg), and then inhaled 1.5% of isoflurane vaporized with pure oxygen via a tracheal tube using a ventilator (SN-480-5; Shinano, Tokyo, Japan). For measuring blood pressure (BP), heparinized catheter was inserted into the right femoral artery under the monitoring of the surface lead II electrocardiogram (ECG). Complete AV block was produced by a catheter ablation technique, as described previously.^7–9) The endpoint of the catheter ablation was the development of complete AV block with the onset of stable idioventricular escape rhythm.

Electrophysiologic Measurement

A monophasic action potential (MAP) recording/pacing combination catheter (Physio-Tech, Tokyo, Japan) was inserted through the right jugular vein to record the MAP of the right ventricle. The duration of MAP signals was measured as an interval (ms) at the 90% repolarization level, defined as MAP₉₀.

TdP was judged as a polymorphic ventricular tachycardia, of which QRS complex twisted around the baseline, lasting ≥5 consecutive beats. Premature ventricular contraction with R on T phenomenon was defined with a prematurity index of <1, which was calculated by dividing the coupling interval of premature ventricular beat (RR’) by the QT interval of the preceding normally conducted beat (=RR’/QT).¹⁰⁾

For assessing the instability of the ventricular repolarization, beat-to-beat analysis using the MAP₉₀ of 31 consecutive beats was performed before drug administration and at which a maximal change in MAP₉₀ was detected (or just before induction of the first TdP). Short-term variability of repolarization, a surrogate parameter that reliably identifies proarrhythmic risk in preclinical animal models, was obtained using Poincaré plots of MAP₉₀(n) versus MAP₉₀(n + 1), as described in our previous studies.^7–9)

Experimental Protocol

All experiments were done under idioventricular rhythm. After the basal control assessment (C), ivabradine at 0.01 mg/kg was intravenously infused over 10 min, and cardiovascular variables were continuously monitored until 30 min after the start of the infusion. Subsequently, ivabradine at 0.1 mg/kg was additionally infused, and cardiovascular variables were monitored similarly. Next, ivabradine at 1 mg/kg was additionally infused, and cardiovascular variables were monitored until 60 min.

Materials

Ivabradine hydrochloride was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and was dissolved in saline. Heparin sodium, isoflurane, ketamine hydrochloride and xylazine hydrochloride were purchased from AY pharmaceuticals (Tokyo, Japan), Mylan Seiyaku (Osaka, Japan), Daiichi Sankyo (Tokyo, Japan) and Bayer Yakuhin (Tokyo, Japan), respectively.

Statistical Analysis

Data are shown as mean ± standard error of the mean (S.E.M.). The statistical significance within a parameter was evaluated by one-way repeated-measures ANOVA, followed by Dunnett’s test for comparison of each treatment with a pre-drug control, which was analyzed using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, U.S.A.). A p-value <0.05 was considered significant.

RESULTS

Torsadogenic Action of Ivabradine

Figure 1A shows the typical tracing of TdP arrhythmia during the administration of 0.1 mg/kg of ivabradine to an acute AV block rabbit. The arrhythmia followed a premature ventricular contraction that occurred during the preceding T wave, known as R on T phenomenon (PVC with R on T phenomenon). Figure 1B summarizes the time course showing the onset of arrhythmias after the administration of ivabradine. After the administration of 0.01 mg/kg, there were no proarrhythmic signs including PVCs with R on T phenomenon. After the administration of 0.1 mg/kg, episodes of PVCs with R on T phenomenon and TdP arrhythmias were detected in 1 out of 5 animals, which were also observed in 2 out of 5 animals after the administration of 1 mg/kg. In rabbits #2 and #5 who received 1 mg/kg of ivabradine, ventricular standstill was continuously observed after the termination of latest TdP, and it was found to lead to cardiac arrest. Meanwhile, significant prolongation of MAP₉₀ (p < 0.05) and increment of short-term variability (p = 0.06) were detected after the administration of 1 mg/kg (Table 1).

Fig. 1. Proarrhythmic Effects of Ivabradine

(A) Typical tracings of ivabradine-induced torsade de pointes (TdP) in the rabbit with acute atrioventricular block (#5). The arrhythmia appeared at 7 min 20 s after the administration of 0.1 mg/kg, which terminated spontaneously. (B) Summary of proarrhythmic effect of ivabradine. Each column indicates the responses of each rabbit to drug, and the columns are marked in accordance with the severity of arrhythmias developed in each minute. Orange, red and black columns represent the occurrence of premature ventricular contraction with R on T phenomenon (R on T PVC), TdP and cardiac arrest (CA), respectively. In rabbit #2, 2 episodes of short type of TdP (<10 s) and 3 episodes of longer type of the arrhythmias (≥10 s) were discontinuously observed at 15–16, 17–19 and 20–22 min after the high-dose administration. In rabbit #5, 2 episodes of the short type and 2 episodes of the longer type were discontinuously observed at 7–10 and 15–19 min after the administration of 0.1 mg/kg, whereas 1 episode of the short type and 1 episode of longer type were discontinuously observed at 2–5 min after the high-dose administration. In rabbits #2 and #5 receiving the high dose, ventricular standstill was continuously observed after the termination of latest TdP, leading to cardiac arrest. ECG: electrocardiogram, MAP: monophasic action potential, BP: blood pressure. (Color figure can be accessed in the online version.)

Table 1. Effects of Ivabradine on the Beat-to-Beat Variability of Ventricular Repolarization in the Rabbit with Acute Atrioventricular Block

Ivabradine (mg/kg)	Control	0.01	0.1	1
STV (ms)	2.3 ± 0.9	3.0 ± 0.8	2.9 ± 0.6	5.4 ± 1.5
LTV (ms)	1.9 ± 0.7	2.7 ± 0.8	2.3 ± 0.5	4.3 ± 1.1
MAP90 (ms)	177 ± 33	190 ± 35	202 ± 38	228 ± 44*

Poincaré plots of MAP₉₀(n) versus MAP₉₀(n + 1) were prepared for each analysis time point, and short-term variability (=Σ|MAP₉₀(n + 1) − MAP₉₀(n)|/[30 × ]) and long-term variability (=Σ|MAP₉₀(n + 1) + MAP₉₀(n) − 2MAP₉₀(mean) |/[30 × ]) were calculated. Data are presented as mean ± S.E.M. of 5 experiments except for the dose of 1 mg/kg (n = 4) because the MAP₉₀ of 31 consecutive beats was not obtained due to arrhythmias after the drug administration in the #5 animal. STV: short-term variability, LTV: long-term variability, MAP₉₀: monophasic action potential duration at 90% repolarization level. * p < 0.05, significantly different from corresponding pre-drug control.

Effects on the Hemodynamic and Electrophysiological Parameters

Hemodynamic and electrophysiological effects of ivabradine (n = 5) are summarized in Fig. 2. No significant change was detected in the any of the parameters after the administration of 0.01 or 0.1 mg/kg. The high dose of 1 mg/kg significantly increased the QT interval, corrected QT interval (QTc), QRS width and MAP₉₀ but decreased the atrial and ventricular rates. Meanwhile, there was no significant change in the mean BP. As summarized in Table 2, bradycardic effects of ivabradine on the ventricle were greater in TdP-positive rabbits than those in TdP-negative ones.

Fig. 2. Time Courses of Effects of Ivabradine on the Hemodynamic and Electrophysiological Parameters in Atrioventricular Block Rabbits (n = 5)

The control values (C) of the atrial rate, ventricular rate, mean blood pressure (MBP), QT interval, QTc, QRS width and the monophasic action potential duration at 90% repolarization level (MAP₉₀) were 305 ± 9 beats/min, 96 ± 8 beats/min, 41 ± 3 mmHg, 217 ± 32 ms, 249 ± 29 ms, 51 ± 5 ms and 179 ± 32 ms, respectively. After the high-dose administration, maximal changes in the atrial rate, ventricular rate, QT interval, QTc and MAP₉₀ were −48 ± 3 beats/min, −45 ± 17 beats/min, +112 ± 65 ms, +60 ± 30 ms and +77 ± 42 ms, respectively. The corrected QT interval (QTc) was calculated using Fridericia’s formula. The closed symbols represent the significant differences from each pre-drug control value (C) by p < 0.05.

Table 2. Changes in the Atrial Rate, Ventricular Rate and Ventricular Repolarization Period by Ivabradine in the AV Block Rabbits

Animal No.	Atrial rate (bpm)	Ventricular rate (bpm)	MAP90 (ms)	TdP
Ivabradine 0.1 mg/kg
#5	−20	−32	+57	+
#1 to #4 (n = 4)	−3 ± 15	−5 ± 6	+19 ± 3	−
Ivabradine 1 mg/kg
#2	−88	−86	+216	+
#5	−34	−42	−7	+
#1, #3, #4 (n = 3)	−49 ± 4	−29 ± 8	+35 ± 8	−

Data are shown as changes from corresponding pre-drug control values just before proceeding to onset of TdP, which can be compared with average of maximal changes in rabbits without induction of TdP (n = 3–4) at doses of 0.1 and 1 mg/kg of ivabradine.

DISCUSSION

Proarrhythmic effects of ivabradine were investigated using the acute AV block rabbit model.⁸⁾ Ivabradine did not affect cardiovascular variables at the low dose, whereas its middle and high doses exerted proarrhythmic actions. Although ivabradine has been demonstrated to act as an aggravating factor for proarrhythmic risk in the isolated rabbit heart,¹¹⁾ this is the first report demonstrating that ivabradine itself has torsadogenic actions in the in vivo experimental condition.

Ivabradine is clinically supplied as oral tablets containing 2.5, 5 and 7.5 mg in Japan, whereas intravenous administration of 0.1 to 0.25 mg/kg of ivabradine has been used for previous clinical investigations.^12,13) Thus, the present study used 0.01 to 1 mg/kg, i.v. of ivabradine that can be considered to be carried out under subtherapeutic to supratherapeutic levels. Based on pharmacokinetic parameters obtained from human and animals, maximum plasma concentrations of ivabradine at 1 mg/kg can be estimated to be 88–410 nM in this study,^14–16) which is relatively higher than Cmax values of 13.5–45 nM in healthy human after the administration of oral tablets containing 2.5–7.5 mg.¹⁴⁾ In this study, ivabradine induced TdP in 1 out of 5 rabbits at 0.1 mg/kg and in 2 out of 5 rabbits at 1 mg/kg (Fig. 1), which is roughly corresponded to a therapeutic to supratherapeutic dose range. In addition, the short-term variability of MAP₉₀, a predicting marker for the onset of TdP,¹⁷⁾ tended to increase in rabbits, as shown in Table 1, which is essentially similar to the property of other torsadogenic drugs as demonstrated in the AV block rabbit model.^7–9)

As shown in Fig. 2, the high dose of ivabradine significantly prolonged the QT interval and MAP₉₀ with decrement of atrial and ventricular spontaneous rate of AV block rabbit heart, whereas no significant change in these parameters was detected at low and middle doses. In previous in vitro electrophysiological studies, IC₅₀s of ivabradine for HCN4 and human ether-a-go-go related gene (hERG) K⁺ currents have been demonstrated to be 2.2 and 3.5 µM, respectively,^18,19) which may reflect our current results that ivabradine affected automaticity and ventricular repolarization from the same dose of 1 mg/kg. Importantly, as shown in Table 2, TdP arrhythmias could be only observed in the 2 rabbits that showed more potent suppressive effects on ventricular automaticity (#2, #5), which might depend on a physiological condition of HCN channels that are functionally regulated by cAMP.²⁰⁾ This may provide significant information on distinctive torsadogenicity of ivabradine because there is a possibility that reverse use-dependent QT-interval prolongation by hERG K⁺-channel blockade²¹⁾ could be enhanced by pharmacological inhibition of HCN channels.

Based on Table 2, TdP arrhythmias appeared when advanced bradycardia was present as an individual-specific reaction, thus the TdP risk may be low in the heart rate-reducing therapy with an appropriate dose of ivabradine under the observation of patients’ heart rate. Therefore, ivabradine is positioned as a Conditional Risk, as suggested by an online resource that promotes the safe use of medicines, CredibleMeds^®.

In conclusion, the present study suggests that the torsadogenic potential of ivabradine may become evident when its expected bradycardic action appears more excessively.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Ide T, Ohtani K, Higo T, Tanaka M, Kawasaki Y, Tsutsui H. Ivabradine for the treatment of cardiovascular diseases. Circ. J., 83, 252–260 (2019).
• 2) Koncz I, Szél T, Bitay M, Cerbai E, Jaeger K, Fülöp F, Jost N, Virág L, Orvos P, Tálosi L, Kristóf A, Baczkó I, Papp JG, Varró A. Electrophysiological effects of ivabradine in dog and human cardiac preparations: potential antiarrhythmic actions. Eur. J. Pharmacol., 668, 419–426 (2011).
• 3) Mackiewicz U, Gerges JY, Chu S, Duda M, Dobrzynski H, Lewartowski B, Mączewski M. Ivabradine protects against ventricular arrhythmias in acute myocardial infarction in the rat. J. Cell. Physiol., 229, 813–823 (2014).
• 4) Vaillant F, Timour Q, Descotes J, Manati W, Belhani D, Bui-Xuan B, Tabib A, Bricca G, Chevalier P. Ivabradine induces an increase in ventricular fibrillation threshold during acute myocardial ischemia: an experimental study. J. Cardiovasc. Pharmacol., 52, 548–554 (2008).
• 5) Jang JH, Kwon SW, Lee MJ, Ko KY, Park JH, Yoon GS, Choi SH, Beak YS, Park SD, Shin SH, Woo SI, Kim DH, Kwan J. Ivabradine-induced torsade de pointes in patients with heart failure reduced ejection fraction. Int. Heart J., 61, 1044–1048 (2020).
• 6) Cocco G, Jerie P. Torsades de pointes induced by the concomitant use of ivabradine and azithromycin: an unexpected dangerous interaction. Cardiovasc. Toxicol., 15, 104–106 (2015).
• 7) Kawakami S, Nagasawa Y, Hagiwara-Nagasawa M, Omura K, Aimoto M, Takahara A. Torsadogenic potential of a novel remyelinating drug clemastine for multiple sclerosis assessed in the rabbit proarrhythmia model. J. Pharmacol. Sci., 144, 123–128 (2020).
• 8) Hagiwara M, Shibuta S, Takada K, Kambayashi R, Nakajo M, Aimoto M, Nagasawa Y, Takahara A. The anaesthetized rabbit with acute atrioventricular block provides a new model for detecting drug-induced Torsade de Pointes. Br. J. Pharmacol., 174, 2591–2605 (2017).
• 9) Hagiwara M, Kambayashi R, Aimoto M, Nagasawa Y, Takahara A. In vivo analysis of torsadogenic potential of an antipsychotic drug paliperidone using the acute atrioventricular block rabbit as a proarrhythmia model. J. Pharmacol. Sci., 132, 48–54 (2016).
• 10) Adgey AA. Warning arrhythmias in ischemic heart disease. Int. J. Cardiol., 4, 224–228 (1983).
• 11) Frommeyer G, Weller J, Ellermann C, Leitz P, Kochhäuser S, Lange PS, Dechering DG, Eckardt L. Ivabradine aggravates the proarrhythmic risk in experimental models of long QT syndrome. Cardiovasc. Toxicol., 19, 129–135 (2019).
• 12) Manz M, Reuter M, Lauck G, Omran H, Jung W. A single intravenous dose of ivabradine, a novel I_f inhibitor, lowers heart rate but does not depress left ventricular function in patients with left ventricular dysfunction. Cardiology, 100, 149–155 (2003).
• 13) De Ferrari GM, Mazzuero A, Agnesina L, Bertoletti A, Lettino M, Campana C, Schwartz PJ, Tavazzi L. Favourable effects of heart rate reduction with intravenous administration of ivabradine in patients with advanced heart failure. Eur. J. Heart Fail., 10, 550–555 (2008).
• 14) Ono Pharmaceutical Co. Ltd. “Interview Form: CORALAN^® Tablets., 5th ed.”: ‹https://www.ononavi1717.jp/system/files/2021-07/CRL_IF.pdf›, accessed 11 August, 2021.
• 15) Riesen SC, Ni W, Carnes CA, Lindsey KJ, Phelps MA, Schober KE. Pharmacokinetics of oral ivabradine in healthy cats. J. Vet. Pharmacol. Ther., 34, 469–475 (2011).
• 16) Chen XP, Zheng HT, Cai WW, Li MK, Zhang JW, Hu J. The effect of silibinin on the pharmacokinetics of ivabradine and N-desmethylivabradine in rats. Pharmacology, 96, 107–111 (2015).
• 17) Varkevisser R, Wijers SC, van der Heyden MAG, Beekman JDM, Meine M, Vos MA. Beat-to-beat variability of repolarization as a new biomarker for proarrhythmia in vivo. Heart Rhythm, 9, 1718–1726 (2012).
• 18) Bois P, Bescond J, Renaudon B, Lenfant J. Mode of action of bradycardic agent, S 16257, on ionic currents of rabbit sinoatrial node cells. Br. J. Pharmacol., 118, 1051–1057 (1996).
• 19) Melgari D, Brack KE, Zhang C, Zhang Y, El Harchi A, Mitcheson JS, Dempsey CE, Ng GA, Hancox JC. hERG potassium channel blockade by the HCN channel inhibitor bradycardic agent ivabradine. J. Am. Heart Assoc., 4, e001813 (2015).
• 20) Marger L, Mesirca P, Alig J, Torrente A, Dubel S, Engeland B, Kanani S, Fontanaud P, Striessnig J, Shin HS, Isbrandt D, Ehmke H, Nargeot J, Mangoni ME. Functional roles of Ca_v1.3, Ca_v3.1 and HCN channels in automaticity of mouse atrioventricular cells: insights into the atrioventricular pacemaker mechanism. Channels (Austin), 5, 251–261 (2011).
• 21) Hondeghem LM, Snyders DJ. Class III antiarrhythmic agents have a lot of potential but a long way to go. Reduced effectiveness and dangers of reverse use dependence. Circulation, 81, 686–690 (1990).