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Electropharmacological profile of an atrial-selective sodium channel blocker acehytisine assessed in the isoflurane-anesthetized guinea-pig model
Journal of Pharmacological Sciences xxx (xxxx) xxx
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Electropharmacological profile of an atrial-selective sodium channel blocker acehytisine assessed in the isoflurane-anesthetized guinea-pig model Xin Cao a, b, *, 1, Yoshinobu Nagasawa b, 1, Chengshun Zhang a, Hanxiao Zhang a, Megumi Aimoto b, Akira Takahara b, ** a Acupuncture and Tuina School/Third Teaching Hospital, Chengdu University of Traditional Chinese Medicine, 37 Shierqiao Road, Jinniu District, Chengdu, 610075, Sichuan Province, China b Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Toho University 2-2-1 Miyama, Funabashi, Chiba, 274-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 July 2019 Received in revised form 16 October 2019 Accepted 21 October 2019 Available online xxx
Experimental evidence regarding the risk of proarrhythmic potential of acehytisine is limited. We assessed its electropharmacological effect together with proarrhythmic potential at intravenous doses of 4 and 10 mg/kg (n ¼ 6) using isoflurane-anesthetized guinea pigs in comparison with that of bepridil at 1 and 3 mg/kg, intravenously (n ¼ 6). Acehytisine at therapeutic dose (4 mg/kg) decreased the heart rate, prolonged P wave duration, QRS width, QT interval, QTc, MAP90(sinus), MAP90(CL300) and MAP90(CL250). At supratherapeutic dose (10 mg/kg), it prolonged the PR interval besides enhancing the changes induced by the therapeutic dose. Quantitative assessment showed that peak changes in P wave duration by acehytisine at 10 mg/kg were 1.7 times longer than bepridil, and in MAP90(sinus), MAP90(CL300) and MAP90(CL250) by acehytisine were 1.9, 1.5 and 1.5 times shorter than bepridil, respectively. Importantly, qualitative assessment indicated that bepridil increased beat-to-beat variability and J-Tpeakc in a doserelated manner, confirming a higher proarrhythmic risk, whereas such dose-related responses were not observed in acehytisine, suggesting a lower proarrhythmic risk. These results suggest that acehytisine exhibits favorable pharmacological characters, i.e. potent atrial inhibition and lower proarrhythmic toxicity compared with bepridil, being a promising candidate for the treatment of paroxysmal supraventricular tachycardia. © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Acehytisine QT prolongation Proarrhythmic potential Early repolarization Short-term variability
1. Introduction Acehytisine (previously named Guanfu base A), a diterpene alkaloid isolated from the root of aconitum coreanum (Levl.) raipaics, has been approved for the treatment of paroxysmal supraventricular tachycardia via its atrial-selective Naþ channel-blocking
* Corresponding author. Acupuncture and Tuina School/Third Teaching Hospital, Chengdu University of Traditional Chinese Medicine, 37 Shierqiao Road, Jinniu District, Chengdu, 610075, Sichuan Province, China. Fax: þ86 28 61800000. ** Corresponding author. Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba, 2748510, Japan. Fax: þ81 47 472 3225. E-mail addresses: [email protected] (X. Cao), [email protected] (A. Takahara). Peer review under responsibility of Japanese Pharmacological Society. 1 Equally contributed.
action in China since 2005.1,2 Currently, it is undergoing phase IV clinical study. In both in vitro and in vivo studies, acehytisine has been verified as a multi-ion channel blocker, inhibiting Naþ, Ca2þ, human ether-a-go-go-related gene (hERG) Kþ channel and HCN channel.2e8 A clinical trial showed that the efficacy and safety of acehytisine for paroxysmal supraventricular tachycardia were comparable to a class IC anti-arrhythmic medication, propafenone, which has been reported to cause torsade de points.1,9 Drugs with hERG Kþ channel blocking action may prolong the QT interval, leading to a life-threatening ventricular arrhythmia termed “torsade de points”, however, in vivo experimental evidence regarding the risk of proarrhythmic potential of acehytisine is still limited. In this study, to characterize the electropharmacological profile of acehytisine, we compared it with bepridil using isofluraneanesthetized guinea pigs, firstly. Bepridil was selected because its
https://doi.org/10.1016/j.jphs.2019.10.006 1347-8613/© 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: Cao X et al., Electropharmacological profile of an atrial-selective sodium channel blocker acehytisine assessed in the isoflurane-anesthetized guinea-pig model, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.10.006
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multi-ion channel inhibition profile that inhibits Naþ, Ca2þ and HERG Kþ currents10e12 was similar to acehytisine and it induces benign QT-interval prolongation.13 To quantify the risk of druginduced lethal ventricular arrhythmias, we adopted two surrogate markers, namely, early repolarization (J-Tpeakc) and short term beat-to-beat variability (STV) of monophasic action potential, lastly.14,15 The duration of early repolarization (J-Tpeak) has been considered to be changed with the net balance between inward Naþ/L-type Ca2þ and outward hERG Kþ currents, prolongation of which may indicate high risk of lethal ventricular arrhythmias.14 STV reflects the temporal dispersion of ventricular repolarization, increase of which may trigger the early afterdepolarization.15 2. Materials and methods Experiments were performed by using 8-week-old male Hartley guinea pigs (n ¼ 12), weighing 300e400 g (Sankyo Labo Service, Tokyo, Japan). Animals were kept at 23 ± 1 C under a 12-h lightdark cycle with free access to food and water (ad libitum). All experiments were approved by the Animal Research Committee for Animal Experimentation at Faculty of Pharmaceutical Sciences of Toho University (No. 17-51-359) and performed in accordance with the Guidelines for the Care and Use of Laboratory Animal of Toho University. 2.1. Electrophysiological testing Guinea pigs were initially anesthetized with thiopental sodium (40 mg/kg, i.p.). After intubation with a tracheal cannula, 1% isoflurane vaporized with 100% oxygen was inhaled with a volumelimited ventilator (SN-480-7; Shinano Manufacturing Co., Ltd., Tokyo, Japan). Tidal volume and respiratory rate were set at 6 mL/kg and 60 breaths/min, respectively, and body temperature was maintained with 37 C by using a heating pad. The left jugular vein was cannulated for drug administration, and the left carotid artery was cannulated for measurement of the blood pressure. The surface lead II ECG was obtained from the limb electrodes. The corrected QT interval (QTc) was calculated by using Van de Water's formula: QTc ¼ QTe0.087 (RRe1000)16 and Sakaguchi's formula [QTc(S) ¼ QT/(RR/300)1/3].17 J-Tpeak was measured separately and corrected for heart rate by using a coefficient (J-Tpeakc ¼ J-Tpeak/(RR/ 300)1/3 with RR in ms).17 A MAP recording/pacing combination catheter (3 F, interelectrode distance of 4 mm, SMC-304; PhysioTech, Tokyo) was positioned at the right ventricle via the right jugular vein. The signals were amplified with a differential amplifier (DAM 50; World Precision Instruments, Sarasota, FL, USA). The duration of the MAP signals was measured as an interval, along a line horizontal to the diastolic baseline, from the monophasic action potential upstroke to the desired repolarization level. The interval (ms) at the 90% repolarization level was defined as MAP90. The heart was electrically driven with a stimulator (SEN-3301; Nihon Kohden, Tokyo) and an isolator (SS-104 J, Nihon Kohden). The stimulation pulses were rectangular in shape of 2.5 V (about twice the threshold voltage), and 3 ms in duration. The MAP90 was measured during sinus rhythm (MAP90(sinus)) and at a pacing cycle length of 300 ms (MAP90(CL300)) or 250 ms (MAP90(CL250)). Cardiovascular parameters were monitored continuously with a polygraph system (RM-6000, Nihon Kohden) and analyzed with a realtime full automatic analysis system (MP/VAS 3 for Windows ver. 1.0, Physio-Tech). For assessment of instability of the ventricular repolarization, the MAP duration (MAP90) of 51 consecutive beats under sinus rhythm was measured before and after drug administration. For analyzing beat-to-beat variability of repolarization, Poincare plots of MAP90(n) versus MAP90 (nþ1) were prepared for each analytical time point. The mean orthogonal distance from the
plot was determined as shortdiagonal to the points of the Poincare term variability (¼S|MAP90 (nþ1)MAP90(n)|/[50 √2]). The ECG parameters and MAP90 were measured under sinus rhythm as the mean of 3 consecutive recordings. MAP90 during ventricular pacing was obtained at a cycle length of 300 or 250 ms as the mean of values obtained from the 3 consecutive MAP recordings. After the basal control assessment (C), a dose of 4 mg/kg of acehytisine was infused over 10 min. The ECG parameters and MAP90 were measured at 5, 10, 15, 20, 25 and 30 min after the start of drug infusion. Next, 10 mg/kg of acehytisine was additionally infused over 10 min, and each parameter was measured in the same manner. Also, the effects of bepridil at doses of 1 and 3 mg/kg were assessed in another series of animals in the same manner. The doses of acehytisine and bepridil were determined by previous reports.1,18 2.2. Drugs Acehytisine (Angene International, Ltd., Hong Kong, China) was dissolved in saline in concentrations of 2 and 5 mg/mL. Bepridil hydrochloride (Sigma-Aldrich Co., LLC., St. Louis, MO, USA) was also dissolved in saline in concentrations of 0.5 and 1.5 mg/mL. They were administered intravenously at an infusion rate of 2 mL/kg per 10 min. The other drugs, thiopental sodium (Ravonal® 0.5 g for Injection, Mitsubishi Tanabe Pharma Co., Osaka, Japan) and isoflurane (Mylan Seiyaku, Osaka, Japan) were purchased. 2.3. Statistical analysis Data are presented as the mean ± S.E.M. Differences within a parameter were evaluated with one-way repeated-measures analysis of variance (ANOVA) followed by Contrasts as a post-hoc test for mean values comparison, whereas those between the groups were analyzed by unpaired t-test. A P-value less than 0.05 was considered statistically significant. 3. Results There was no significant difference in any of the pre-drug control values of these variables between acehytisine- and bepridiladministered groups. No animals exerted any lethal ventricular arrhythmias or hemodynamic collapse, leading to the animals’ death during the experiment. 3.1. Electrophysiological effect of acehytisine and bepridil Representative traces of the electrocardiogram and MAP before and after administration of acehytisine and bepridil are depicted in Fig. 1. Time courses of changes in heart rate and mean blood pressure are summarized in Fig. 2. In the acehytisine group, the predrug control values (C) of the heart rate and mean blood pressure were 196 ± 2 bpm and 31 ± 3 mmHg, respectively. Acehytisine at 4 mg/kg significantly decreased the heart rate for 5e20 min and at 30 min, and further decreased the heart rate for 5e30 min at 10 mg/ kg. In the bepridil group, the pre-drug control values (C) of the heart rate and mean blood pressure were 205 ± 4 bpm and 36 ± 2 mmHg, respectively. Bepridil at 1 mg/kg significantly decreased the heart rate for 10e15 min, and further decreased the heart rate for 10e30 min at 3 mg/kg. The supratherapeutic doses of 10 mg/kg and 3 mg/kg at acehytisine and bepridil group were 2.5 and 3 times more than therapeutic doses, but the peak change of heart rate at supratherapeutic dose by acehytisine was 2.5 times greater than bepridil. Maximum percent decrease of heart rate in acehytisine group at therapeutic dose was 3.2 times more than bepridil group.
Please cite this article as: Cao X et al., Electropharmacological profile of an atrial-selective sodium channel blocker acehytisine assessed in the isoflurane-anesthetized guinea-pig model, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.10.006
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Acehytisine
3
Bepridil Control
Control
HR: 200 bpm
HR: 200 bpm
1 mV
ECG
MAP
10 mV P wave duration: 36 ms
MAP: 178 ms
10 min after 10 mg/kg i.v.
P wave duration: 35 ms MAP: 203 ms
15 min after 3 mg/kg i.v. HR: 171 bpm
HR: 133 bpm
1 mV
ECG
10 mV
MAP P wave duration: 46 ms
MAP: 200 ms
P wave duration: 42 ms
MAP: 248 ms 100 ms
Fig. 1. Typical traces showing the surface lead II electrocardiogram (ECG) and monophasic action potential (MAP) at pre-drug control (Control) (upper panels) and after the intravenous administration of acehytisine and bepridil (lower panels). In acehytisine group (left panel), heart rate, P wave duration and MAP were 200 bpm, 36 ms and 178 ms at pre-drug control, respectively, and then decreased to 133 bpm, prolonged to 46 ms and 200 ms at 10 min after 10 mg/kg of acehytisine, respectively. In bepridil group, heart rate, P wave duration and MAP were 200 bpm, 35 ms and 200 ms at pre-drug control, respectively, and decreased to 171 bpm, increased to 42 ms and 248 ms at 15 min after 3 mg/kg of bepridil, respectively.
Fig. 2. Time courses of changes in the heart rate (HR) and mean blood pressure (MBP) after the intravenously administration of acehytisine (left panels) at doses of 4 and 10 mg/kg and bepridil (right panels) at 1 and 3 mg/kg. Data are presented as mean ± S.E.M. (n ¼ 6 for each group). Closed symbols represent significant differences from each pre-drug control value (C) by P < 0.05.
Fig. 3 shows the time courses of changes before and after administration of acehytisine and bepridil in electrocardiogram parameters. In the acehytisine group, the pre-drug control values (C) of P wave duration, PR interval, QRS width, QT interval, QTc(V) and QTc(S) were 36 ± 2 ms, 61 ± 1 ms, 27 ± 2 ms, 218 ± 9 ms, 278 ± 9 and 217 ± 9, respectively. Acehytisine at 4 mg/kg prolonged the P wave duration for 10e30 min, QRS width at 20 min, QT
interval and QTc(V) for 5e30 min and QTc(S) for 10e30 min. Peak change of P wave duration was 7 ms (Table 1). Acehytisine at 10 mg/ kg significantly prolonged the PR interval for 10e30 min in addition to the effects observed by 4 mg/kg. Peak change of P wave duration was 10 ms (Table 1). In the bepridil group, the pre-drug control values (C) of the P wave duration, PR interval, QRS width, QT interval, QTc(V) and QTc(S) were 37 ± 1 ms, 64 ± 3 ms, 25 ± 2 ms,
Please cite this article as: Cao X et al., Electropharmacological profile of an atrial-selective sodium channel blocker acehytisine assessed in the isoflurane-anesthetized guinea-pig model, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.10.006
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Fig. 3. Time courses of changes in P wave duration, PR interval, QRS width (QRS), QT interval (QT) and corrected QT interval (QTcV and S) before and after the intravenously administration of acehytisine (4 and 10 mg/kg; left panel) and bepridil (1 and 3 mg/kg; right panel). Data are presented as mean ± S.E.M. (n ¼ 6 for each group). Closed symbols represent significant differences from each pre-drug control value (C) by P < 0.05.
Table 1 Quantitative assessment of the effect of acehytisine and bepridil on the P wave duration and MAP. Drugs
Acehytisine
Dose (mg/kg, i.v.) DP wave duration (ms) DMAP90(sinus) (ms) DMAP90(CL300) (ms) DMAP90(CL250) (ms)
4 7 22 13 17
Bepridil 10 10 29 20 19
1 4 22 14 12
3 6 55 29 28
Data are presented as the mean ± S.E.M (n ¼ 6 for each drug). DP and DMAP represent the maximum changes from their corresponding pre-drug control values. MAP90: Monophasic action potential duration (in ms) at 90% repolarization.
222 ± 5 ms, 283 ± 4 and 224 ± 4, respectively. Bepridil at 1 mg/kg significantly prolonged the P wave duration, PR interval, QT interval, QTc(V) and QTc(S) at 15 min. Peak change of P wave duration at 1 mg/kg was 4 ms (Table 1). Bepridil at 3 mg/kg prolonged QRS width for 5e30 min at 3 mg/kg in addition to the effects observed by 1 mg/kg. Peak change of P wave duration at 3 mg/kg was 6 ms (Table 1). Mobitiz II 2nd degree atrioventricular conduction block was observed in one out of six guinea pigs at 3 mg/kg. During the sinus rhythm, acehytisine prolonged P wave duration and QRS width by 18% and 8% at therapeutic dose and 28% and 20% at supratherapeutic dose, respectively. Meanwhile, bepridil prolonged P wave duration and QRS width by 11% and 7% at therapeutic dose and by 15% and 17% at supratherapeutic dose, respectively. More importantly, the peak changes in P wave duration by acehytisine were 1.8 and 1.7 times longer than those of bepridil at each corresponding dose. Fig. 4 summarized the time courses of changes before and after administration of acehytisine and bepridil in monophasic action potential at 90% repolarization level. In the acehytisine group, the pre-drug control values (C) of MAP90(sinus), MAP90(CL300) and MAP90(CL250) were 193 ± 4, 190 ± 5 and 174 ± 6 ms, respectively. Acehytisine at 4 mg/kg significantly prolonged the MAP90(sinus) for 5e20 min and at 30 min, MAP90(CL300) and MAP90(CL250) for
5e30 min, the peak changes of which were 22, 13 and 17 ms at 10 min after the start of administration, respectively (Table 1). Acehytisine at 10 mg/kg further prolonged MAP90(sinus), MAP90(CL300) and MAP90(CL250) for 5e30 min, the peak changes of which were 29, 20 and 19 ms at 10 min after the start of administration, respectively. In bepridil group, the pre-drug control values (C) of MAP90(sinus), MAP90(CL300) and MAP90(CL250) were 193 ± 4, 199 ± 5 and 184±4 ms, respectively. Bepridil at 1 mg/kg significantly prolonged the MAP90(sinus) for 10e30 min, MAP90(CL300) for 10e25 min and MAP90(CL250) for 5e30 min, the peak changes of which were 22, 14 and 12 ms at 10 min after the start of administration, respectively, and further prolonged all variables for 5e30 min at 3 mg/kg. Peak changes of MAP90(sinus), MAP90(CL300) and MAP90(CL250) were 55, 29 and 28 ms at 10 min after the start of administration, respectively. The peak changes in MAP90(sinus), MAP90(CL300) and MAP90(CL250) by acehytisine were 1.9, 1.5 and 1.5 times shorter than those of bepridil at supratherapeutic dose. 3.2. Proarrhythmic effect of acehytisine and bepridil plot of the MAP90 is depicted in Fig. 5 (upper Typical Poincare panel), and the effects of acehytisine and bepridil on the J-Tpeak and STV after the start of administration of each dose are summarized in Fig. 5 (lower panel). In the acehytisine group, pre-drug control of J-Tpeak and STV were 164 ± 8 and 1.17 ± 0.26 ms, respectively. Acehytisine hardly altered the J-Tpeakc. On the other hand, it increased the STV at 4 mg/kg, but hardly affected it at 10 mg/kg. In the bepridil group, pre-drug control values (C) of J-Tpeak and STV are 163 ± 2 and 1.26 ± 0.28 ms, respectively. Bepridil increased both JTpeakc and STV in a dose-related manner. 4. Discussion We assessed the electropharmacological effects together with its proarrhythmic potential of acehytisine using well-established
Please cite this article as: Cao X et al., Electropharmacological profile of an atrial-selective sodium channel blocker acehytisine assessed in the isoflurane-anesthetized guinea-pig model, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.10.006
X. Cao et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx
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Fig. 4. Time courses of changes in monophasic action potential duration at 90% repolarization level at sinus rhythm (MAP90 (sinus)), a cycle length of 300 ms (MAP90 (CL300)) and 250 ms (MAP90 (CL250)) before and after the intravenously administration of acehytisine (4 and 10 mg/kg; left panel) and bepridil (1 and 3 mg/kg; right panel). Data are presented as mean ± S.E.M. (n ¼ 6 for each group). Closed symbols represent significant differences from each pre-drug control value (C) by P < 0.05.
plot of the monophasic action potential (MAP90) (upper panel). Fifty-one beats were plotted for each of the 3 analysis time points before and after Fig. 5. Typical traces of Poincare the administration of acehytisine (4 and 10 mg/kg) or bepridil (1 and 3 mg/kg). Summary of the effects of acehytisine and bepridil on the short-term variability of ventricular repolarization (STV) and J-Tpeakc (lower panel). Data are presented as mean ± S.E.M. Closed symbols represent significant differences from the corresponding pre-drug basal control value (C) by P < 0.05.
isoflurane-anesthetized in vivo guinea-pig model in comparison with bepridil. Quantitative assessment with P wave duration and MAP90 showed that acehytisine exerted potent atrial inhibition and lower proarrhythmic toxicity. Qualitative assessment with J-Tpeakc and STV further indicated that the proarrhythmic risk of acehytisine was lower than bepridil. Thus, acehytisine may be a promising candidate for the treatment of paroxysmal supraventricular tachycardia.
4.1. The rationale for the drug doses Two escalating i.v. doses (4 and 10 mg/kg) of acehytisine were selected. The clinically recommended intravenously daily dose is described to be 4 mg/kg in China in drug information from the manufacturer. The concentrations in healthy subjects were 4,866 and 5,739 ng/mL at 15 min and 90 min, respectively, after intravenous dose of 4 mg/kg in 5 min and then additional intravenous dose of 40
Please cite this article as: Cao X et al., Electropharmacological profile of an atrial-selective sodium channel blocker acehytisine assessed in the isoflurane-anesthetized guinea-pig model, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.10.006
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mg/kg/min for 90 min19. Based on our previous experience with halothane-anesthetized guinea pig model, Cmax of acehytisine was estimated to 4,000 and 10,000 ng/mL,20,21 and thus the doses of acehytisine assessed in this study could be considered to provide therapeutic to supratherapeutic levels of the plasma drug concentrations. In the clinical practice, the therapeutic plasma concentrations of bepridil during repeated oral administration have been reported to be 367e1,210 ng/mL.22 Meanwhile, according to our previous experience with halothane-anesthetized guinea pig model,20,21 Cmax after the administration of 1 and 3 mg/kg of bepridil could be estimated to be approximately 1,000 and 3,000 ng/mL, respectively. Thus, the plasma concentration in the present study was estimated to be therapeutic to supratherapeutic levels.
overload and have less potential to induce lethal arrhythmia than bepridil.26 Bepridil increased the temporal instability of ventricular repolarization in a dose-related manner, whereas acehytisine lack such dose-related response, suggesting high dose of bepridil may increase the chance to trigger early after depolarization,15 whereas acehytisine lack such proarrhythmic potential. The mechanism might be its potent Naþ and Ca2þ channel inhibition than those of bepridil. Thus, acehytisine poses lower proarrhythmic risk than bepridil for patients with elevated plasma drug concentration and/ or compromised cardiac repolarization.
4.2. Electrophysiological effect of acehytisine and bepridil
The electropharmacological effects of acehytisine and bepridil were similar, but their potency for each cardiovascular variable varied significantly, as summarized in Table 1. These results indicate that acehytisine in comparison with bepridil exhibits favorable pharmacological characters, i.e. potent atrial inhibition and lower proarrhythmic toxicity, being a promising candidate for the treatment of paroxysmal supraventricular tachycardia.
Acehytisine as well as bepridil at therapeutic to supratherapeutic doses decreased the heart rate, which was in good accordance with previous studies.18,21,23,24 This chronotropic effect of acehytisine may be explained by the blocking effect on pacemaker currents (IC50 ¼ 9.9 mM) in sinus node in addition to cardiac L-type Ca2þ channel which was inhibited by 23.5% at 250 mM,4,8 while the bradycardic effect of bepridil may be associated with cardiac L-type (IC50 ¼ 50 mM) as well as T-type Ca2þ channelblocking actions (IC50 ¼ 0.4 mM).10,25 This stronger inhibition on pacemaker currents by acehytisine may explain its greater changes in heart rate than bepridil. Acehytisine prolonged the PR interval in a dose-related manner, confirming its blockade actions on Ca2þ channels as previously reported in the in vitro,4 which is essentially consistent with previous in vivo observation.7 Similar negative dromotropic effect was also observed in bepridil at 1 and 3 mg/kg. The stronger inhibition of bepridil on T-type calcium (IC50 ¼ 0.4 mM) may explain the onset of Mobitiz II 2nd degree atrioventricular conduction block in one of six guinea pigs. The prolongation of P wave duration and QRS width by acehytisine are in good accordance with the human study1 and in vitro study,2 and the greater changes in P wave duration than QRS width may be ascribed to the stronger blocking effects on sodium channels (IC50 ¼ 1.57 mM) in atria than in ventricles.2,3 The prolongation of P wave duration and QRS width by bepridil confirmed its wellknown inhibitory actions on the Naþ (IC50 ¼ 30 mM) and Ca2þ channels in both in vitro10 and in vivo observations.18,21 Importantly, the greater peak change in P wave duration by acehytisine indicated its greater atrial inhibition than bepridil. Both acehytisine and bepridil prolonged the QT interval, MAP90(sinus), MAP90(CL300) and MAP90(CL250) at therapeutic doses, which were consistent with previous in vivo studies.6,13 In addition, a reverse use-dependence was observed in the repolarization delay in both drugs, reflecting the inhibition on hERG Kþ channel.5,12 Bepridil has been reported to inhibit both hERG Kþ channel (IC50 ¼ 13.2 mM)12 and slow component of delayed rectifier Kþ current (IKs, IC50 ¼ 6.2 mM),12 whereas acehytisine was reported to inhibit hERG Kþ channel with an IC50 of 466 mM.5 These results may explain the much greater QT interval-prolonging effect of bepridil than acehytisine. 4.3. Proarrhythmic potential As discussed above, both acehytisine and bepridil may decrease outward hERG Kþ as well as inward Naþ and Ca2þ currents. However, acehytisine hardly altered the early repolarization (J-Tpeakc), showing that the impact of Naþ and Ca2þ channel inhibition might counterbalance the hERG Kþ channel suppression in this period, which suggests that acehytisine may less likely induce Ca2þ
5. Conclusions
Declaration of Competing Interest The authors declare no conflicts of interest. Acknowledgements This study was supported in part by Grant-in-aid for research activity start-up (17H07135), the National Natural Science Foundation of China (81704187), Sichuan Science and Technology Program (2019YJ0587), Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital Research Fund (2018ZX05). References 1. Gao X, Zhu J, Yang YM, Li JD, Yang ZM, Liu JH. Efficacy of intravenous acehytisine hydrochloride versus propafenone on terminating paroxysmal supraventricular tachycardia: a double-blinded, randomized multi-center study. Zhonghua Xinxueguanbing Zazhi. 2007;35:151e154 [In Chinese]. 2. Fan X, Wang C, Wang N, et al. Atrial-selective block of sodium channels by acehytisine in rabbit myocardium. J Pharmacol Sci. 2016;132:235e243. 3. Jin SS, Guo Q, Xu J, Yu P, Liu JH, Tang YQ. Antiarrhythmic ionic mechanism of guanfu base ASelective inhibition of late sodium current in isolated ventricular myocytes from Guinea pigs. Chin J Nat Med. 2015;13:361e367. 4. Gao X, Pu JL, Yang YM, Wu H, Huang A, Shi SY. Effects of guanfu base a hydrochloride on heart L-type calcium channel of rat ventricular myocytes. Chin J N Drugs. 2006;15:1926e1929. 5. Huang X, Yang Y, Zhu J, Dai Y, Pu J. The effects of a novel anti-arrhythmic drug, acehytisine hydrochloride, on the human ether-a-go-go relat ed gene K channel and its trafficking. Basic Clin Pharmacol Toxicol. 2009;104:145e154. 6. Liang Y. Hemodynamic and electrophysiological effect of acehytisine hydrochloride on the acute ischemia and chronic cardiac dysfunction animal models. Zhong Guo Xie He Yi Ke Da Xue. 2005:71e94 [In Chinese]. 7. Liang Y, Zhu J, Yang YM, et al. Electrophysiological effects of acehytisine hydrochloride in a porcine model of acute coronary occlusion. Zhonghua Xinxueguanbing Zazhi. 2006;34:1035e1039 [In Chinese]. 8. Fan X, Chen Y, Xing J, et al. Blocking effects of acehytisine on pacemaker currents (i(f)) in sinoatrial node cells and human HCN4 channels expressed in xenopus laevis oocytes. J Ethnopharmacol. 2012;139:42e51. 9. Hii JT, Wyse DG, Gillis AM, Cohen JM, Mitchell LB. Propafenone-induced torsade de pointes: cross-reactivity with quinidine. Pacing Clin Electrophysiol. 1991;14: 1568e1570. 10. Yatani A, Brown AM, Schwartz A. Bepridil block of cardiac calcium and sodium channels. J Pharmacol Exp Ther. 1986;237:9e17. 11. Anno T, Furuta T, Itoh M, Kodama I, Toyama J, Yamada K. Electromechanical effects of bepridil on rabbit isolated hearts. Br J Pharmacol. 1984;81:41e47. 12. Wang JC, Kiyosue T, Kiriyama K, Arita M. Bepridil differentially inhibits two delayed rectifier Kþ currents, IKr and IKs, in guinea-pig ventricular myocytes. Br J Pharmacol. 1999;128:1733e1738. 13. Takahara A, Nakamura Y, Sugiyama A. Beat-to-beat variability of repolarization differentiates the extent of torsadogenic potential of multi ion channelblockers bepridil and amiodarone. Eur J Pharmacol. 2008;596:127e131.
Please cite this article as: Cao X et al., Electropharmacological profile of an atrial-selective sodium channel blocker acehytisine assessed in the isoflurane-anesthetized guinea-pig model, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.10.006
X. Cao et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx 14. Cao X, Nakamura Y, Wada T, Izumi-Nakaseko H, Ando K, Sugiyama A. Electropharmacological effects of amantadine on cardiovascular system assessed with J-Tpeak and Tpeak-Tend analysis in the halothane-anesthetizes beagle dogs. J Toxicol Sci. 2016;41:439e447. 15. Thomsen MB, Verduyn SC, Stengl M, et al. Increased short-term variability of repolarization predicts d-sotalol-induced torsades de pointes in dogs. Circulation. 2004;110:2453e2459. 16. Van de Water A, Verheyen J, Xhonneux R, Reneman RS. An improved method to correct the QT interval of the electrocardiogram for changes in heart rate. J Pharmacol Methods. 1989;22:207e217. 17. Sakaguchi Y, Takahara A, Nakamura Y, Akie Y, Sugiyama A. Halothane-anesthetized, closed-chest, guinea-pig model for assessment of drug-induced QTinterval prolongation. Basic Clin Pharmacol Toxicol. 2009;104:43e48. 18. Ishizaka T, Takahara A, Iwasaki H, et al. Comparison of electropharmacological effects of bepridil and sotalol in halothane-anesthetized dogs. Circ J. 2008;72: 1003e1011. 19. Zhu J, Li P, Yang YM, et al. Electrophysiological effect of acehytisine hydrochloride. Chin J Cardiol. 2001;29:124 [In Chinese]. 20. Matsuo K, Fujiwara K, Omuro N, et al. Effects of the fluoroquinolone antibacterial drug ciprofloxacin on ventricular repolarization in the halothaneanesthetized guinea pig. J Pharmacol Sci. 2013;122:205e212.
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21. Katagi J, Nakamura Y, Cao X, et al. Why can dl-Sotalol prolong the QT interval in vivo despite its weak inhibitory effect on hERG Kþ Channels in vitro? Electrophysiological and pharmacokinetic analysis with the halothaneanesthetized guinea pig model. Cardiovasc Toxicol. 2016;16:138e146. 22. Omata T, Kasai C, Hashimoto M, Hombo T, Yamamoto K. QT PRODACT: comparison of non-clinical studies for drug-induced delay in ventricular repolarization and their role in safety evaluation in humans. J Pharmacol Sci. 2005;99: 531e541. 23. Dong YL, Chen WZ. Effects of guan-fu base A on experimental cardiac arrhythmias and myocardial contractility. Yao Xue Xue Bao. 1995;30:577e582 [In Chinese]. 24. Wang YQ, Su L, Gong GQ, Liu JH. Guanfu base A inhibits the post infarction ventricular arrhythmias in dogs. Yao Xue Xue Bao. 1994;29:141e144 [In Chinese]. 25. Uchino T, Lee TS, Kaku T, Yamashita N, Noguchi T, Ono K. Voltage-dependent and frequency-independent inhibition of recombinant Cav3.2 T-type Ca2þ channel by bepridil. Pharmacology. 2005;74:174e181. 26. Johannesen L, Vicente J, Mason JW, et al. Differentiating drug-induced multichannel block on the electrocardiogram: randomized study of dofetilide, quinidine, ranolazine, and verapamil. Clin Pharmacol Ther. 2014;96: 549e558.
Please cite this article as: Cao X et al., Electropharmacological profile of an atrial-selective sodium channel blocker acehytisine assessed in the isoflurane-anesthetized guinea-pig model, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.10.006
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Electropharmacological profile of an atrial-selective sodium channel blocker acehytisine assessed in the isoflurane-anesthetized guinea-pig model Xin Cao a, b, *, 1, Yoshinobu Nagasawa b, 1, Chengshun Zhang a, Hanxiao Zhang a, Megumi Aimoto b, Akira Takahara b, ** a Acupuncture and Tuina School/Third Teaching Hospital, Chengdu University of Traditional Chinese Medicine, 37 Shierqiao Road, Jinniu District, Chengdu, 610075, Sichuan Province, China b Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Toho University 2-2-1 Miyama, Funabashi, Chiba, 274-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 July 2019 Received in revised form 16 October 2019 Accepted 21 October 2019 Available online xxx
Experimental evidence regarding the risk of proarrhythmic potential of acehytisine is limited. We assessed its electropharmacological effect together with proarrhythmic potential at intravenous doses of 4 and 10 mg/kg (n ¼ 6) using isoflurane-anesthetized guinea pigs in comparison with that of bepridil at 1 and 3 mg/kg, intravenously (n ¼ 6). Acehytisine at therapeutic dose (4 mg/kg) decreased the heart rate, prolonged P wave duration, QRS width, QT interval, QTc, MAP90(sinus), MAP90(CL300) and MAP90(CL250). At supratherapeutic dose (10 mg/kg), it prolonged the PR interval besides enhancing the changes induced by the therapeutic dose. Quantitative assessment showed that peak changes in P wave duration by acehytisine at 10 mg/kg were 1.7 times longer than bepridil, and in MAP90(sinus), MAP90(CL300) and MAP90(CL250) by acehytisine were 1.9, 1.5 and 1.5 times shorter than bepridil, respectively. Importantly, qualitative assessment indicated that bepridil increased beat-to-beat variability and J-Tpeakc in a doserelated manner, confirming a higher proarrhythmic risk, whereas such dose-related responses were not observed in acehytisine, suggesting a lower proarrhythmic risk. These results suggest that acehytisine exhibits favorable pharmacological characters, i.e. potent atrial inhibition and lower proarrhythmic toxicity compared with bepridil, being a promising candidate for the treatment of paroxysmal supraventricular tachycardia. © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Acehytisine QT prolongation Proarrhythmic potential Early repolarization Short-term variability
1. Introduction Acehytisine (previously named Guanfu base A), a diterpene alkaloid isolated from the root of aconitum coreanum (Levl.) raipaics, has been approved for the treatment of paroxysmal supraventricular tachycardia via its atrial-selective Naþ channel-blocking
* Corresponding author. Acupuncture and Tuina School/Third Teaching Hospital, Chengdu University of Traditional Chinese Medicine, 37 Shierqiao Road, Jinniu District, Chengdu, 610075, Sichuan Province, China. Fax: þ86 28 61800000. ** Corresponding author. Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba, 2748510, Japan. Fax: þ81 47 472 3225. E-mail addresses: [email protected] (X. Cao), [email protected] (A. Takahara). Peer review under responsibility of Japanese Pharmacological Society. 1 Equally contributed.
action in China since 2005.1,2 Currently, it is undergoing phase IV clinical study. In both in vitro and in vivo studies, acehytisine has been verified as a multi-ion channel blocker, inhibiting Naþ, Ca2þ, human ether-a-go-go-related gene (hERG) Kþ channel and HCN channel.2e8 A clinical trial showed that the efficacy and safety of acehytisine for paroxysmal supraventricular tachycardia were comparable to a class IC anti-arrhythmic medication, propafenone, which has been reported to cause torsade de points.1,9 Drugs with hERG Kþ channel blocking action may prolong the QT interval, leading to a life-threatening ventricular arrhythmia termed “torsade de points”, however, in vivo experimental evidence regarding the risk of proarrhythmic potential of acehytisine is still limited. In this study, to characterize the electropharmacological profile of acehytisine, we compared it with bepridil using isofluraneanesthetized guinea pigs, firstly. Bepridil was selected because its
https://doi.org/10.1016/j.jphs.2019.10.006 1347-8613/© 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: Cao X et al., Electropharmacological profile of an atrial-selective sodium channel blocker acehytisine assessed in the isoflurane-anesthetized guinea-pig model, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.10.006
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multi-ion channel inhibition profile that inhibits Naþ, Ca2þ and HERG Kþ currents10e12 was similar to acehytisine and it induces benign QT-interval prolongation.13 To quantify the risk of druginduced lethal ventricular arrhythmias, we adopted two surrogate markers, namely, early repolarization (J-Tpeakc) and short term beat-to-beat variability (STV) of monophasic action potential, lastly.14,15 The duration of early repolarization (J-Tpeak) has been considered to be changed with the net balance between inward Naþ/L-type Ca2þ and outward hERG Kþ currents, prolongation of which may indicate high risk of lethal ventricular arrhythmias.14 STV reflects the temporal dispersion of ventricular repolarization, increase of which may trigger the early afterdepolarization.15 2. Materials and methods Experiments were performed by using 8-week-old male Hartley guinea pigs (n ¼ 12), weighing 300e400 g (Sankyo Labo Service, Tokyo, Japan). Animals were kept at 23 ± 1 C under a 12-h lightdark cycle with free access to food and water (ad libitum). All experiments were approved by the Animal Research Committee for Animal Experimentation at Faculty of Pharmaceutical Sciences of Toho University (No. 17-51-359) and performed in accordance with the Guidelines for the Care and Use of Laboratory Animal of Toho University. 2.1. Electrophysiological testing Guinea pigs were initially anesthetized with thiopental sodium (40 mg/kg, i.p.). After intubation with a tracheal cannula, 1% isoflurane vaporized with 100% oxygen was inhaled with a volumelimited ventilator (SN-480-7; Shinano Manufacturing Co., Ltd., Tokyo, Japan). Tidal volume and respiratory rate were set at 6 mL/kg and 60 breaths/min, respectively, and body temperature was maintained with 37 C by using a heating pad. The left jugular vein was cannulated for drug administration, and the left carotid artery was cannulated for measurement of the blood pressure. The surface lead II ECG was obtained from the limb electrodes. The corrected QT interval (QTc) was calculated by using Van de Water's formula: QTc ¼ QTe0.087 (RRe1000)16 and Sakaguchi's formula [QTc(S) ¼ QT/(RR/300)1/3].17 J-Tpeak was measured separately and corrected for heart rate by using a coefficient (J-Tpeakc ¼ J-Tpeak/(RR/ 300)1/3 with RR in ms).17 A MAP recording/pacing combination catheter (3 F, interelectrode distance of 4 mm, SMC-304; PhysioTech, Tokyo) was positioned at the right ventricle via the right jugular vein. The signals were amplified with a differential amplifier (DAM 50; World Precision Instruments, Sarasota, FL, USA). The duration of the MAP signals was measured as an interval, along a line horizontal to the diastolic baseline, from the monophasic action potential upstroke to the desired repolarization level. The interval (ms) at the 90% repolarization level was defined as MAP90. The heart was electrically driven with a stimulator (SEN-3301; Nihon Kohden, Tokyo) and an isolator (SS-104 J, Nihon Kohden). The stimulation pulses were rectangular in shape of 2.5 V (about twice the threshold voltage), and 3 ms in duration. The MAP90 was measured during sinus rhythm (MAP90(sinus)) and at a pacing cycle length of 300 ms (MAP90(CL300)) or 250 ms (MAP90(CL250)). Cardiovascular parameters were monitored continuously with a polygraph system (RM-6000, Nihon Kohden) and analyzed with a realtime full automatic analysis system (MP/VAS 3 for Windows ver. 1.0, Physio-Tech). For assessment of instability of the ventricular repolarization, the MAP duration (MAP90) of 51 consecutive beats under sinus rhythm was measured before and after drug administration. For analyzing beat-to-beat variability of repolarization, Poincare plots of MAP90(n) versus MAP90 (nþ1) were prepared for each analytical time point. The mean orthogonal distance from the
plot was determined as shortdiagonal to the points of the Poincare term variability (¼S|MAP90 (nþ1)MAP90(n)|/[50 √2]). The ECG parameters and MAP90 were measured under sinus rhythm as the mean of 3 consecutive recordings. MAP90 during ventricular pacing was obtained at a cycle length of 300 or 250 ms as the mean of values obtained from the 3 consecutive MAP recordings. After the basal control assessment (C), a dose of 4 mg/kg of acehytisine was infused over 10 min. The ECG parameters and MAP90 were measured at 5, 10, 15, 20, 25 and 30 min after the start of drug infusion. Next, 10 mg/kg of acehytisine was additionally infused over 10 min, and each parameter was measured in the same manner. Also, the effects of bepridil at doses of 1 and 3 mg/kg were assessed in another series of animals in the same manner. The doses of acehytisine and bepridil were determined by previous reports.1,18 2.2. Drugs Acehytisine (Angene International, Ltd., Hong Kong, China) was dissolved in saline in concentrations of 2 and 5 mg/mL. Bepridil hydrochloride (Sigma-Aldrich Co., LLC., St. Louis, MO, USA) was also dissolved in saline in concentrations of 0.5 and 1.5 mg/mL. They were administered intravenously at an infusion rate of 2 mL/kg per 10 min. The other drugs, thiopental sodium (Ravonal® 0.5 g for Injection, Mitsubishi Tanabe Pharma Co., Osaka, Japan) and isoflurane (Mylan Seiyaku, Osaka, Japan) were purchased. 2.3. Statistical analysis Data are presented as the mean ± S.E.M. Differences within a parameter were evaluated with one-way repeated-measures analysis of variance (ANOVA) followed by Contrasts as a post-hoc test for mean values comparison, whereas those between the groups were analyzed by unpaired t-test. A P-value less than 0.05 was considered statistically significant. 3. Results There was no significant difference in any of the pre-drug control values of these variables between acehytisine- and bepridiladministered groups. No animals exerted any lethal ventricular arrhythmias or hemodynamic collapse, leading to the animals’ death during the experiment. 3.1. Electrophysiological effect of acehytisine and bepridil Representative traces of the electrocardiogram and MAP before and after administration of acehytisine and bepridil are depicted in Fig. 1. Time courses of changes in heart rate and mean blood pressure are summarized in Fig. 2. In the acehytisine group, the predrug control values (C) of the heart rate and mean blood pressure were 196 ± 2 bpm and 31 ± 3 mmHg, respectively. Acehytisine at 4 mg/kg significantly decreased the heart rate for 5e20 min and at 30 min, and further decreased the heart rate for 5e30 min at 10 mg/ kg. In the bepridil group, the pre-drug control values (C) of the heart rate and mean blood pressure were 205 ± 4 bpm and 36 ± 2 mmHg, respectively. Bepridil at 1 mg/kg significantly decreased the heart rate for 10e15 min, and further decreased the heart rate for 10e30 min at 3 mg/kg. The supratherapeutic doses of 10 mg/kg and 3 mg/kg at acehytisine and bepridil group were 2.5 and 3 times more than therapeutic doses, but the peak change of heart rate at supratherapeutic dose by acehytisine was 2.5 times greater than bepridil. Maximum percent decrease of heart rate in acehytisine group at therapeutic dose was 3.2 times more than bepridil group.
Please cite this article as: Cao X et al., Electropharmacological profile of an atrial-selective sodium channel blocker acehytisine assessed in the isoflurane-anesthetized guinea-pig model, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.10.006
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Acehytisine
3
Bepridil Control
Control
HR: 200 bpm
HR: 200 bpm
1 mV
ECG
MAP
10 mV P wave duration: 36 ms
MAP: 178 ms
10 min after 10 mg/kg i.v.
P wave duration: 35 ms MAP: 203 ms
15 min after 3 mg/kg i.v. HR: 171 bpm
HR: 133 bpm
1 mV
ECG
10 mV
MAP P wave duration: 46 ms
MAP: 200 ms
P wave duration: 42 ms
MAP: 248 ms 100 ms
Fig. 1. Typical traces showing the surface lead II electrocardiogram (ECG) and monophasic action potential (MAP) at pre-drug control (Control) (upper panels) and after the intravenous administration of acehytisine and bepridil (lower panels). In acehytisine group (left panel), heart rate, P wave duration and MAP were 200 bpm, 36 ms and 178 ms at pre-drug control, respectively, and then decreased to 133 bpm, prolonged to 46 ms and 200 ms at 10 min after 10 mg/kg of acehytisine, respectively. In bepridil group, heart rate, P wave duration and MAP were 200 bpm, 35 ms and 200 ms at pre-drug control, respectively, and decreased to 171 bpm, increased to 42 ms and 248 ms at 15 min after 3 mg/kg of bepridil, respectively.
Fig. 2. Time courses of changes in the heart rate (HR) and mean blood pressure (MBP) after the intravenously administration of acehytisine (left panels) at doses of 4 and 10 mg/kg and bepridil (right panels) at 1 and 3 mg/kg. Data are presented as mean ± S.E.M. (n ¼ 6 for each group). Closed symbols represent significant differences from each pre-drug control value (C) by P < 0.05.
Fig. 3 shows the time courses of changes before and after administration of acehytisine and bepridil in electrocardiogram parameters. In the acehytisine group, the pre-drug control values (C) of P wave duration, PR interval, QRS width, QT interval, QTc(V) and QTc(S) were 36 ± 2 ms, 61 ± 1 ms, 27 ± 2 ms, 218 ± 9 ms, 278 ± 9 and 217 ± 9, respectively. Acehytisine at 4 mg/kg prolonged the P wave duration for 10e30 min, QRS width at 20 min, QT
interval and QTc(V) for 5e30 min and QTc(S) for 10e30 min. Peak change of P wave duration was 7 ms (Table 1). Acehytisine at 10 mg/ kg significantly prolonged the PR interval for 10e30 min in addition to the effects observed by 4 mg/kg. Peak change of P wave duration was 10 ms (Table 1). In the bepridil group, the pre-drug control values (C) of the P wave duration, PR interval, QRS width, QT interval, QTc(V) and QTc(S) were 37 ± 1 ms, 64 ± 3 ms, 25 ± 2 ms,
Please cite this article as: Cao X et al., Electropharmacological profile of an atrial-selective sodium channel blocker acehytisine assessed in the isoflurane-anesthetized guinea-pig model, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.10.006
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Fig. 3. Time courses of changes in P wave duration, PR interval, QRS width (QRS), QT interval (QT) and corrected QT interval (QTcV and S) before and after the intravenously administration of acehytisine (4 and 10 mg/kg; left panel) and bepridil (1 and 3 mg/kg; right panel). Data are presented as mean ± S.E.M. (n ¼ 6 for each group). Closed symbols represent significant differences from each pre-drug control value (C) by P < 0.05.
Table 1 Quantitative assessment of the effect of acehytisine and bepridil on the P wave duration and MAP. Drugs
Acehytisine
Dose (mg/kg, i.v.) DP wave duration (ms) DMAP90(sinus) (ms) DMAP90(CL300) (ms) DMAP90(CL250) (ms)
4 7 22 13 17
Bepridil 10 10 29 20 19
1 4 22 14 12
3 6 55 29 28
Data are presented as the mean ± S.E.M (n ¼ 6 for each drug). DP and DMAP represent the maximum changes from their corresponding pre-drug control values. MAP90: Monophasic action potential duration (in ms) at 90% repolarization.
222 ± 5 ms, 283 ± 4 and 224 ± 4, respectively. Bepridil at 1 mg/kg significantly prolonged the P wave duration, PR interval, QT interval, QTc(V) and QTc(S) at 15 min. Peak change of P wave duration at 1 mg/kg was 4 ms (Table 1). Bepridil at 3 mg/kg prolonged QRS width for 5e30 min at 3 mg/kg in addition to the effects observed by 1 mg/kg. Peak change of P wave duration at 3 mg/kg was 6 ms (Table 1). Mobitiz II 2nd degree atrioventricular conduction block was observed in one out of six guinea pigs at 3 mg/kg. During the sinus rhythm, acehytisine prolonged P wave duration and QRS width by 18% and 8% at therapeutic dose and 28% and 20% at supratherapeutic dose, respectively. Meanwhile, bepridil prolonged P wave duration and QRS width by 11% and 7% at therapeutic dose and by 15% and 17% at supratherapeutic dose, respectively. More importantly, the peak changes in P wave duration by acehytisine were 1.8 and 1.7 times longer than those of bepridil at each corresponding dose. Fig. 4 summarized the time courses of changes before and after administration of acehytisine and bepridil in monophasic action potential at 90% repolarization level. In the acehytisine group, the pre-drug control values (C) of MAP90(sinus), MAP90(CL300) and MAP90(CL250) were 193 ± 4, 190 ± 5 and 174 ± 6 ms, respectively. Acehytisine at 4 mg/kg significantly prolonged the MAP90(sinus) for 5e20 min and at 30 min, MAP90(CL300) and MAP90(CL250) for
5e30 min, the peak changes of which were 22, 13 and 17 ms at 10 min after the start of administration, respectively (Table 1). Acehytisine at 10 mg/kg further prolonged MAP90(sinus), MAP90(CL300) and MAP90(CL250) for 5e30 min, the peak changes of which were 29, 20 and 19 ms at 10 min after the start of administration, respectively. In bepridil group, the pre-drug control values (C) of MAP90(sinus), MAP90(CL300) and MAP90(CL250) were 193 ± 4, 199 ± 5 and 184±4 ms, respectively. Bepridil at 1 mg/kg significantly prolonged the MAP90(sinus) for 10e30 min, MAP90(CL300) for 10e25 min and MAP90(CL250) for 5e30 min, the peak changes of which were 22, 14 and 12 ms at 10 min after the start of administration, respectively, and further prolonged all variables for 5e30 min at 3 mg/kg. Peak changes of MAP90(sinus), MAP90(CL300) and MAP90(CL250) were 55, 29 and 28 ms at 10 min after the start of administration, respectively. The peak changes in MAP90(sinus), MAP90(CL300) and MAP90(CL250) by acehytisine were 1.9, 1.5 and 1.5 times shorter than those of bepridil at supratherapeutic dose. 3.2. Proarrhythmic effect of acehytisine and bepridil plot of the MAP90 is depicted in Fig. 5 (upper Typical Poincare panel), and the effects of acehytisine and bepridil on the J-Tpeak and STV after the start of administration of each dose are summarized in Fig. 5 (lower panel). In the acehytisine group, pre-drug control of J-Tpeak and STV were 164 ± 8 and 1.17 ± 0.26 ms, respectively. Acehytisine hardly altered the J-Tpeakc. On the other hand, it increased the STV at 4 mg/kg, but hardly affected it at 10 mg/kg. In the bepridil group, pre-drug control values (C) of J-Tpeak and STV are 163 ± 2 and 1.26 ± 0.28 ms, respectively. Bepridil increased both JTpeakc and STV in a dose-related manner. 4. Discussion We assessed the electropharmacological effects together with its proarrhythmic potential of acehytisine using well-established
Please cite this article as: Cao X et al., Electropharmacological profile of an atrial-selective sodium channel blocker acehytisine assessed in the isoflurane-anesthetized guinea-pig model, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.10.006
X. Cao et al. / Journal of Pharmacological Sciences xxx (xxxx) xxx
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Fig. 4. Time courses of changes in monophasic action potential duration at 90% repolarization level at sinus rhythm (MAP90 (sinus)), a cycle length of 300 ms (MAP90 (CL300)) and 250 ms (MAP90 (CL250)) before and after the intravenously administration of acehytisine (4 and 10 mg/kg; left panel) and bepridil (1 and 3 mg/kg; right panel). Data are presented as mean ± S.E.M. (n ¼ 6 for each group). Closed symbols represent significant differences from each pre-drug control value (C) by P < 0.05.
plot of the monophasic action potential (MAP90) (upper panel). Fifty-one beats were plotted for each of the 3 analysis time points before and after Fig. 5. Typical traces of Poincare the administration of acehytisine (4 and 10 mg/kg) or bepridil (1 and 3 mg/kg). Summary of the effects of acehytisine and bepridil on the short-term variability of ventricular repolarization (STV) and J-Tpeakc (lower panel). Data are presented as mean ± S.E.M. Closed symbols represent significant differences from the corresponding pre-drug basal control value (C) by P < 0.05.
isoflurane-anesthetized in vivo guinea-pig model in comparison with bepridil. Quantitative assessment with P wave duration and MAP90 showed that acehytisine exerted potent atrial inhibition and lower proarrhythmic toxicity. Qualitative assessment with J-Tpeakc and STV further indicated that the proarrhythmic risk of acehytisine was lower than bepridil. Thus, acehytisine may be a promising candidate for the treatment of paroxysmal supraventricular tachycardia.
4.1. The rationale for the drug doses Two escalating i.v. doses (4 and 10 mg/kg) of acehytisine were selected. The clinically recommended intravenously daily dose is described to be 4 mg/kg in China in drug information from the manufacturer. The concentrations in healthy subjects were 4,866 and 5,739 ng/mL at 15 min and 90 min, respectively, after intravenous dose of 4 mg/kg in 5 min and then additional intravenous dose of 40
Please cite this article as: Cao X et al., Electropharmacological profile of an atrial-selective sodium channel blocker acehytisine assessed in the isoflurane-anesthetized guinea-pig model, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.10.006
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mg/kg/min for 90 min19. Based on our previous experience with halothane-anesthetized guinea pig model, Cmax of acehytisine was estimated to 4,000 and 10,000 ng/mL,20,21 and thus the doses of acehytisine assessed in this study could be considered to provide therapeutic to supratherapeutic levels of the plasma drug concentrations. In the clinical practice, the therapeutic plasma concentrations of bepridil during repeated oral administration have been reported to be 367e1,210 ng/mL.22 Meanwhile, according to our previous experience with halothane-anesthetized guinea pig model,20,21 Cmax after the administration of 1 and 3 mg/kg of bepridil could be estimated to be approximately 1,000 and 3,000 ng/mL, respectively. Thus, the plasma concentration in the present study was estimated to be therapeutic to supratherapeutic levels.
overload and have less potential to induce lethal arrhythmia than bepridil.26 Bepridil increased the temporal instability of ventricular repolarization in a dose-related manner, whereas acehytisine lack such dose-related response, suggesting high dose of bepridil may increase the chance to trigger early after depolarization,15 whereas acehytisine lack such proarrhythmic potential. The mechanism might be its potent Naþ and Ca2þ channel inhibition than those of bepridil. Thus, acehytisine poses lower proarrhythmic risk than bepridil for patients with elevated plasma drug concentration and/ or compromised cardiac repolarization.
4.2. Electrophysiological effect of acehytisine and bepridil
The electropharmacological effects of acehytisine and bepridil were similar, but their potency for each cardiovascular variable varied significantly, as summarized in Table 1. These results indicate that acehytisine in comparison with bepridil exhibits favorable pharmacological characters, i.e. potent atrial inhibition and lower proarrhythmic toxicity, being a promising candidate for the treatment of paroxysmal supraventricular tachycardia.
Acehytisine as well as bepridil at therapeutic to supratherapeutic doses decreased the heart rate, which was in good accordance with previous studies.18,21,23,24 This chronotropic effect of acehytisine may be explained by the blocking effect on pacemaker currents (IC50 ¼ 9.9 mM) in sinus node in addition to cardiac L-type Ca2þ channel which was inhibited by 23.5% at 250 mM,4,8 while the bradycardic effect of bepridil may be associated with cardiac L-type (IC50 ¼ 50 mM) as well as T-type Ca2þ channelblocking actions (IC50 ¼ 0.4 mM).10,25 This stronger inhibition on pacemaker currents by acehytisine may explain its greater changes in heart rate than bepridil. Acehytisine prolonged the PR interval in a dose-related manner, confirming its blockade actions on Ca2þ channels as previously reported in the in vitro,4 which is essentially consistent with previous in vivo observation.7 Similar negative dromotropic effect was also observed in bepridil at 1 and 3 mg/kg. The stronger inhibition of bepridil on T-type calcium (IC50 ¼ 0.4 mM) may explain the onset of Mobitiz II 2nd degree atrioventricular conduction block in one of six guinea pigs. The prolongation of P wave duration and QRS width by acehytisine are in good accordance with the human study1 and in vitro study,2 and the greater changes in P wave duration than QRS width may be ascribed to the stronger blocking effects on sodium channels (IC50 ¼ 1.57 mM) in atria than in ventricles.2,3 The prolongation of P wave duration and QRS width by bepridil confirmed its wellknown inhibitory actions on the Naþ (IC50 ¼ 30 mM) and Ca2þ channels in both in vitro10 and in vivo observations.18,21 Importantly, the greater peak change in P wave duration by acehytisine indicated its greater atrial inhibition than bepridil. Both acehytisine and bepridil prolonged the QT interval, MAP90(sinus), MAP90(CL300) and MAP90(CL250) at therapeutic doses, which were consistent with previous in vivo studies.6,13 In addition, a reverse use-dependence was observed in the repolarization delay in both drugs, reflecting the inhibition on hERG Kþ channel.5,12 Bepridil has been reported to inhibit both hERG Kþ channel (IC50 ¼ 13.2 mM)12 and slow component of delayed rectifier Kþ current (IKs, IC50 ¼ 6.2 mM),12 whereas acehytisine was reported to inhibit hERG Kþ channel with an IC50 of 466 mM.5 These results may explain the much greater QT interval-prolonging effect of bepridil than acehytisine. 4.3. Proarrhythmic potential As discussed above, both acehytisine and bepridil may decrease outward hERG Kþ as well as inward Naþ and Ca2þ currents. However, acehytisine hardly altered the early repolarization (J-Tpeakc), showing that the impact of Naþ and Ca2þ channel inhibition might counterbalance the hERG Kþ channel suppression in this period, which suggests that acehytisine may less likely induce Ca2þ
5. Conclusions
Declaration of Competing Interest The authors declare no conflicts of interest. Acknowledgements This study was supported in part by Grant-in-aid for research activity start-up (17H07135), the National Natural Science Foundation of China (81704187), Sichuan Science and Technology Program (2019YJ0587), Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital Research Fund (2018ZX05). References 1. Gao X, Zhu J, Yang YM, Li JD, Yang ZM, Liu JH. Efficacy of intravenous acehytisine hydrochloride versus propafenone on terminating paroxysmal supraventricular tachycardia: a double-blinded, randomized multi-center study. Zhonghua Xinxueguanbing Zazhi. 2007;35:151e154 [In Chinese]. 2. Fan X, Wang C, Wang N, et al. Atrial-selective block of sodium channels by acehytisine in rabbit myocardium. J Pharmacol Sci. 2016;132:235e243. 3. Jin SS, Guo Q, Xu J, Yu P, Liu JH, Tang YQ. Antiarrhythmic ionic mechanism of guanfu base ASelective inhibition of late sodium current in isolated ventricular myocytes from Guinea pigs. Chin J Nat Med. 2015;13:361e367. 4. Gao X, Pu JL, Yang YM, Wu H, Huang A, Shi SY. Effects of guanfu base a hydrochloride on heart L-type calcium channel of rat ventricular myocytes. Chin J N Drugs. 2006;15:1926e1929. 5. Huang X, Yang Y, Zhu J, Dai Y, Pu J. The effects of a novel anti-arrhythmic drug, acehytisine hydrochloride, on the human ether-a-go-go relat ed gene K channel and its trafficking. Basic Clin Pharmacol Toxicol. 2009;104:145e154. 6. Liang Y. Hemodynamic and electrophysiological effect of acehytisine hydrochloride on the acute ischemia and chronic cardiac dysfunction animal models. Zhong Guo Xie He Yi Ke Da Xue. 2005:71e94 [In Chinese]. 7. Liang Y, Zhu J, Yang YM, et al. Electrophysiological effects of acehytisine hydrochloride in a porcine model of acute coronary occlusion. Zhonghua Xinxueguanbing Zazhi. 2006;34:1035e1039 [In Chinese]. 8. Fan X, Chen Y, Xing J, et al. Blocking effects of acehytisine on pacemaker currents (i(f)) in sinoatrial node cells and human HCN4 channels expressed in xenopus laevis oocytes. J Ethnopharmacol. 2012;139:42e51. 9. Hii JT, Wyse DG, Gillis AM, Cohen JM, Mitchell LB. Propafenone-induced torsade de pointes: cross-reactivity with quinidine. Pacing Clin Electrophysiol. 1991;14: 1568e1570. 10. Yatani A, Brown AM, Schwartz A. Bepridil block of cardiac calcium and sodium channels. J Pharmacol Exp Ther. 1986;237:9e17. 11. Anno T, Furuta T, Itoh M, Kodama I, Toyama J, Yamada K. Electromechanical effects of bepridil on rabbit isolated hearts. Br J Pharmacol. 1984;81:41e47. 12. Wang JC, Kiyosue T, Kiriyama K, Arita M. Bepridil differentially inhibits two delayed rectifier Kþ currents, IKr and IKs, in guinea-pig ventricular myocytes. Br J Pharmacol. 1999;128:1733e1738. 13. Takahara A, Nakamura Y, Sugiyama A. Beat-to-beat variability of repolarization differentiates the extent of torsadogenic potential of multi ion channelblockers bepridil and amiodarone. Eur J Pharmacol. 2008;596:127e131.
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Please cite this article as: Cao X et al., Electropharmacological profile of an atrial-selective sodium channel blocker acehytisine assessed in the isoflurane-anesthetized guinea-pig model, Journal of Pharmacological Sciences, https://doi.org/10.1016/j.jphs.2019.10.006