- Email: [email protected]
Hypaconitine-induced QT prolongation mediated through inhibition of KCNH2 (hERG) potassium channels in conscious dogs
Journal of Ethnopharmacology 166 (2015) 375–379
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Hypaconitine-induced QT prolongation mediated through inhibition of KCNH2 (hERG) potassium channels in conscious dogs Shuilin Xie a, Ying Jia a, Aiming Liu b, Renke Dai a, Lizhen Huang a,n a b
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China Medical School of Ningbo University, Ningbo 315211, China
art ic l e i nf o
a b s t r a c t
Article history: Received 12 August 2014 Received in revised form 15 January 2015 Accepted 9 March 2015 Available online 20 March 2015
Ethnopharmacological relevance: Hypaconitine is one of the main aconitum alkaloids in traditional Chinese medicines prepared with herbs from the genus Acotinum. These herbs are widely used for the treatment of cardiac insufficiency and arrhythmias. However, Acotinum alkaloids are known for their toxicity as well as their pharmacological activity, especially cardiotoxicity including QT prolongation, and the mechanism of this toxicity is not clear. Material and methods: In this study, hypaconitine was administered orally to conscious Beagle dogs, and electrocardiograms were recorded by telemetry. Pharmacokinetic studies (6 h) were conducted to evaluate the relationship between QT prolongation and exposure level. HEK293 cells stably transfected with KCNH2 (hERG) cDNA were used to examine the effects of hypaconitine on the KCNH2 channel by using the manual patch clamp technique. Results: In the conscious dogs, all doses of hypaconitine induced QTcV (QT interval corrected according to the Van de Water formula) prolongation by more than 23% (67 ms) of control in a dose-dependent manner. The maximum QTcV prolongation was observed at 2 h after dosing. Maximum prolongation percentages were plotted against plasma concentrations of hypaconitine and showed a strong correlation (R2 ¼0.789). In the in vitro study in HEK293 cells, hypaconitine inhibited the KCNH2 currents in a concentration-dependent manner with an IC50 of 8.1 nM. Conclusion: These data suggest that hypaconitine inhibits KCNH2 potassium channels and this effect might be the molecular mechanism underlying QT prolongation in conscious dogs. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Hypaconitine QT prolongation hERG channels ECG telemetry Safety assessment
1. Introduction Prolongation of the QT in the electrocardiogram can be associated with polymorphic ventricular tachycardia or Torsades de pointes. QT prolongation has increasingly drawn attention from regulatory agencies and the pharmaceutical industry as a potentially serious adverse event (Yap and Camm, 2003). The recent withdrawal of several drugs from the market due to possibly drug-related cardiac arrhythmias has greatly increased concern about drug-induced prolongation of the QT interval (Bouchaud et al., 2009). To avoid QT prolongation and increase the chances for success of new drug candidates, a more rigorous methodology to evaluate the potential for QT prolongation is critical. The International Conference on Harmonization S7B guideline describes a nonclinical testing strategy for assessing the potential of a test substance to delay ventricular repolarization and thereby produce QT prolongation (Food and Drug
n
Corresponding author. Tel.: þ 86 13631406992; fax: þ 86 20 39380601. E-mail address: [email protected] (L. Huang).
http://dx.doi.org/10.1016/j.jep.2015.03.023 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
Administration, 2005). According to the S7B guideline, an in vivo QT assay using conscious, unrestrained animals is one of the most important elements of the testing strategy. Aconites are the common name of the genus Aconitum L., subordinated to Ranunculaceae (Xiao et al., 2006). Herbal preparations from aconites are well known worldwide for their wide use in traditional medicines of China, Japan, and Korea. These preparations are well known for their toxicity as well, including cardiotoxic and neurotoxic effects. There are quite a few clinical cases of aconite intoxication reported from China Hong Kong, Japan, Germany, and other countries (Chan, 2002; Chen et al., 2012; Poon et al., 2006). Plants of this genus commonly contain diester diterpenoid alkaloids. Alkaloids with an acetyl group at C8 and a benzoylester group at C14 are the potentially toxicalkaloids, including aconitine, mesaconitine, and hypaconitine (Fig. 1) (Hu et al., 2009). Aconitine has been shown to have substantial arrhythmogenic effects (Chen et al., 2013) Early studies indicated that aconitine blocked KCNH2 and kv1.5 potassium channels, which may contributed to action potential duration (APD) prolongation and cardiac arrhythmias (Li et al., 2010).
376
S. Xie et al. / Journal of Ethnopharmacology 166 (2015) 375–379
Fig. 1. The chemical structure of hypaconitine.
Hypaconitine has also been reported to induce arrhythmias and even death (Chan, 2012). However, few studies have been conducted to investigate the mechanism of arrhythmia induction by hypaconitine alone. Therefore, the QT prolongation potential of hypaconitine was assayed in conscious dogs. The relationship of plasma concentration to QT prolongation was also investigated. The mechanism underlying QT prolongation was studied through patch clamp tests on KCNH2 channels expressed in mammalian HEK293 cells.
2. Materials and methods 2.1. Chemicals and reagents Hypaconitine (499%), quinidine (499%), terfenadine (499%) and phenacetin (499%) were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China); Terfenadine, acetonitrile, ethanol (HPLC grade) and G418 were obtained from Sigma-Aldrich (St. Louis, MO 63178, USA); HEK293 cells purchased from ATCC (Manassas, VA 20110, USA) were stably transfected with hERG cDNA (GenBank accession number NM_ 000238); DMEM, FBS, pCDNA3.1 (þ) and lipofectmine 2000 were provided by Life technologies (Grand Island, NY 14072, USA). 2.2. Animals and treatment Six beagle dogs (3 males and 3 females, 7.570.3 kg, Marshall Biotechnology Co., Ltd (Beijing, China)) were randomly assigned in a Latin Square design. All animals were housed in individual stainless steel cages in a room maintained at 22–26 1C, 60–70% relative humidity and 12 h light/12 h dark cycle. They were fed approximately 300 g of standard laboratory diet daily and had access to water ad libitum. Animals were kept in the fasted state for approximately 12 h pre-dose and 6 h post-dose. All study procedures were reviewed and approved by the Laboratory Animal Care and Use Committee of Zhongshan PharmaSS Corporation, Guangdong, China. Each dog was orally administered three doses of hypaconitine (50, 150, and 450 μg/kg), quinidine (1, 3, and 9 mg/kg), a drug known to prolong the QT interval in animals (Olivier et al., 2003; Testai et al., 2004) and humans (Cubeddu, 2003), as the positive control, and vehicle in the same volume. Each animal received the treatments with a 7-day washout period between doses. The dose-finding study had been conducted according to mice LD50 of 5.8 mg/kg (Bisset, 1981). And finally, the present dosage was selected for dog study. 2.3. ECG recording EMKA non-invasive telemetry for large animals (Emka Technologies, Paris 75015, France) was used for this study. The system
consisted of an emkaPACK transmitter with a 5-wire ECG cable to collect 7-lead ECG data with iox2 data acquisition and real-time analysis software (Emka Technologies, Paris 75015, France). All dogs were habituated to the EMKA jacket that was used to carry the non-invasive electrodes and keep them in place for at least 24 h. A seven lead surface ECG configuration (limb I, II, III, aVR, aVL, aVF, and V) was continuously recorded. After ECG signals were stabilized, baseline QT interval corrected using to the Van de Water formula interval was continuously recorded from pre-dose to 6 h post-dose. Data were recorded at 0, 0.5, 1, 2, 4, and 6 h. During the recording, access to the animal room was restricted to the personnel administering the test compounds. At the end of each experiment, the surface ECG electrode patches and jackets were removed and animals were returned to their home cages. The ECG signals were analyzed using iox2 data analysis software. Baseline values were calculated from the pre-dose data. The QT interval was corrected for heart rate using Van de Water's method:QTC V ¼ QT 0:1087ðRR 1000Þ, one of the most common formulae used in conscious dogs (Soloviev et al., 2006; Spence et al., 1998). In the equation, QTcV is the corrected QT internal, QT is the raw QT internal, and RR is the R–R interval. 2.4. Plasma hypaconitine analysis To evaluate the relationship between plasma hypaconitine and QT prolongation, plasma hypaconitine concentrations were determined. Six-hour pharmacokinetic analyses were carried out to evaluate the relationship between hypaconitine blood levels and QTcV prolongation. To measure hypaconitine blood levels, 0.5 mL blood samples were drawn at 0, 0.5, 1, 2, 4, and 6 h after dosing. The blood samples were immediately centrifuged at 3000 g for 10 min, the plasma was collected and then stored at 80 1C until analysis. Before analysis, 80 μL of acetonitrile was added to the 20 μL of plasma to denature the plasma proteins and then the plasma was centrifuged at 14,000 g for 30 min. An aliquot of the supernatant (10 μL) was subjected to liquid chromatography/tandem mass spectroscopy (LC-MS/MS) analysis coupled with Analyst 1.4.2 workstation software. The separation was performed by a CAPCELL PAK C18 column (5 μm, 2.0 mm IDn50 mm) with a flow rate of 0.2 mL/min. 2.5. The effect of hypaconitine on the KCNH2 channel The hERG gene (KCNH2) was subcloned into pCDNA3.1 (þ ) vector. Human embryonic kidney 293 (HEK293) cells were cultured at 37 1C (5% CO2) in DMEM supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). Cells were transfected with linearized KCNH2 plasmid DNA using lipofectmine 2000 (Invitrogen, Carlsbad, CA). Human KCNH2 stable cell line (HEK293-HuKCNH2) was established using G418 screening and validated using manual patch clamp. KCNH2 expressing HEK293 cells were subcultured 2–3 times per week in order to maintain optimal cell health. The cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and 200 μg/mL of G418. HEK293 cells are plated in 35 mm dishes at least 24 h prior to the day of experiment and maintained at 37 1C/5% CO2. The extracellular solution for the whole-cell patch clamp recordings was composed of (mM): NaCl 145; MgCl2 1.0; KCl 4.0; Glucose 10; HEPES 10; and CaCl2 2.0. The pH was adjusted to 7.4 with NaOH. The osmolarity was adjusted to 300 mOsm with sucrose. The intracellular solution for whole-cell patch clamp recordings was composed of (mM): KCl 140; MgCl2 1; EGTA 5; HEPES 10 and Na2ATP 4. The pH was adjusted to 7.2 with KOH. The osmolarity was adjusted to 295 mOsm with sucrose.
S. Xie et al. / Journal of Ethnopharmacology 166 (2015) 375–379
A micropipette was pulled from borosilicate glass with the pipette tip resistance of 3–5 MΩ. For each experiment, a single dish of cells was removed from the incubator, washed twice with room temperature extracellular solution, and placed on the microscope stage. A commercial patch clamp amplifier (Molecular Devices, Sunnyvale, CA, USA) was used for the whole cell recordings. The tail currents were evoked in room temperature once every 30 s by a 3 s 50 mV repolarizing pulse following a 2 s þ 50 mV depolarizing pulse with a hold voltage of 80 mV. A 50 ms depolarizing pulse to 50 mV at the beginning of the voltage protocol served as a baseline for calculating the amplitude of the peak tail current. Only stable cells with recording parameters above threshold were used for the drug application procedure. Terfenadine, a known compound inducing the blockade of HERG channels, was used for the positive control. Hypaconitine or terfenadine was added into the test solution when the KCNH2 currents were stable over a 3 min period in the presence of vehicle alone. The cells were kept in the test solution until the peak tail current was stable (o 5% change) for approximately five sweeps or for a maximum of 6 min, whichever came first. Data were recorded by Clampex (version 10.1, Sunnyvale, CA 94089 USA) and stored for off-line analysis. Data acquisition and analyses were performed using a pCLAMP (version 10.1, Sunnyvale, CA 94089 USA). Peak tail amplitudes were then plotted as a function of the sweep number. Five peak tail current measurements at steady state before hypaconitine or terfenadine application were averaged and used as the control current amplitude. Four or five peak tail current measurements at steady state after hypaconitine or terfenadine application were averaged and used as the current amplitude after inhibition by the test article. The inhibition percentage (%) of the hypaconitine and terfenadine was calculated from the following equation: Inhibition percentage (%)¼ {1 (remaining current amplitude)/ (control current amplitude)} 100 2.6. Statistical analysis All data were expressed as the mean 7the standard deviation (SD). Statistical analysis among groups was performed by was performed with oneway ANOVA and LSD-t test by SPSS16.0. Differences were considered statistically significant if the probability (P value) was less than 0.05 (Po 0.05).
377
Fig. 2. Electrocardiogram recordings before (A) and 2 h after oral administration of 450 μg/kg hypaconitine (B) in a conscious dog. The QTcV intervals were 271 ms for (A) and 438 ms for (B).
Table 1 The maximum value of QT interval corrected according to the Van de Water formula (QTcV) prolongation induced by quinidine and hypaconitine in conscious dogs and the corresponding pharmacokinetic parameters. Group
Maximum value of QTcV iterval Pharmacokinetic prolongation and time points parameters (ms)
Quinidine (mg/kg) Hypaconitine (μg/kg)
1 287 6n 3 607 13n 9 977 14nn 50 677 9n 150 1087 25nn 450 1747 32nn
(%)
(h)
Tmax (h)
Cmax (ng/mL)
107 3 217 5 357 11 237 6 377 7 617 13
0.5 0.5 0.5 2.0 2.0 2.0
0.5 0.5 0.5 1.0 1.0 1.0
958 2107 5868 1.53 5.74 10.11
The maximum value of QTcV interval prolongation between the vehicle and each dose of quinidine and hypaconitine were calculated individually, and the difference values from six dogs were averaged at each time point. The Cmax and Tmax represent means of values from six dogs. All Data is represented as mean 7SD (n¼ 6). n
P o 0.05. Po 0.01 vs. control.
nn
3. Results 3.1. QTcV interval prolongation in conscious dog Typical ECG waveforms before and 2 h after oral administrations of 450 μg/kg hypaconitine are shown in Fig. 2. The QTcV interval after hypaconitine administration was substantially longer than vehicle. Changes in QTcV interval in response to the hypaconitine and positive control, quinidine are summarized in Table 1. All doses prolonged the QTcV interval compared to the vehicle group in a dose-dependent manner. The maximum QTcV prolongation from time-matched vehicle was observed at 0.5 h after administration of quinidine and 2 h after administration of hypaconitine respectively. The maximum prolongation value for hypaconitine was 23% or 67 ms for the 50 μg/kg of group, 37% or 108 ms for the 150 μg/ kg of group and 61% or 174 ms for the 450 μg/kg of group. 3.2. Hypaconitine exposure and toxicity-concentration correlation A validated LC-MS/MS method was used for the determination of hypaconitine in plasma. The mass spectrometer was operated in
positive ion mode with m/z 616.40 for hypaconitine (Fig. 3A). A typical chromatograph of hypaconitine and phenacetin, a frequently-used internal standard owing to its good stability and high response, is shown in Fig. 3B. The concentrations of hypaconitine corresponding to the time points of QTcV measurement were analyzed and the average concentrations were calculated (Fig. 4A and B). The maximum percent prolongation of QTcV from six dogs was plotted in relation to plasma concentrations of hypaconitine. These results showed a strong plasma concentration-response correlation (R²¼0.789) (Fig. 4C). The maximum value of QT interval corrected according to the Van de Water formula (QTcV) prolongation after oral administration of quinidine (0.5 h) and hypaconitine (2 h) in conscious dogs (A). Serum concentration of hypaconitine at each time-point for electrocardiogram recording (B).The relationship between plasma concentration and the percentage of QTcV prolongation in Conscious Dog (C). All Data is represented as mean 7SD (n ¼ 6); n Po 0.05, nnP o0.01 vs. control.
378
S. Xie et al. / Journal of Ethnopharmacology 166 (2015) 375–379
3.3. Inhibition of hypaconitine on hERG currents Hypaconitine (1, 3, 10, and 30 nM) inhibited KCNH2 currents by 5.597 2.56%, 32.30 73.34%, and 58.95 73.87%, and 73.64 73.51%, respectively. Table 2 shows the percent inhibition in the individual cells and the statistics of the percent inhibition at different hypaconitine concentrations. The representative KCNH2 voltageclamp current recordings acquired at the steady state before and after application of hypaconitine are superimposed in Fig. 5A. Hypaconitine inhibited KCNH2 currents with an IC50 of 8.1 nM (Fig. 5B). Table 2 summarizes the effect of application of hypaconitine and terfenadine on KCNH2 currents.
4. Discussion Traditional Chinese medicines (TCM) are widely used around the world. However, with the increasing cases of adverse drug reactions (ADRs), the ADRs induced by TCM are becoming more widely recognized (Zeng and Jiang, 2010). A number of the ADRs are cardiovascular including arrhythmias, myocardial damage, heart failure, increased or decreased blood pressure, and cardiac arrest. Up to date, most studies are limited to the isolation and identification Fig. 3. The mass spectrometry of hypaconitine (A) and the typical chromatograph of hypaconitine (RT: 2.98) and phenacetin (RT: 2.47) as the internal standard(B).
Table 2 Percentage inhibition of KCNH2 currents by hypaconitine and terfenadine. Concentration (nM)
Hypaconitine
Terfenadine
Fig. 4. Correlation of hypaconitine serum concentration and QT prolongation.
1 3 10 30 3 10 30 100
Inhibition (%)
Mean (%)
Cell 1
Cell 2
Cell 3
4.14 28.78 54.50 71.11 22.78 30.23 72.10 94.90
4.09 35.43 60.79 72.16 14.28 37.16 61.98 83.55
8.55 32.69 61.56 77.64 16.57 32.69 63.46 80.58
5.59 32.30 58.95 73.64 17.88 33.36 65.85 86.34
SD
2.56 3.34 3.87 3.51 4.40 3.51 5.47 7.58
Fig. 5. The representative KCNH2 current traces before and after hypaconitine application (A) and the concentration-response relationship of hypaconitine on KCNH2 currents (B). Percentage inhibition of KCNH2 currents at each concentration of hypaconitine (n¼ 3) is shown. The IC50 value was 8.1 nM.
S. Xie et al. / Journal of Ethnopharmacology 166 (2015) 375–379
of active ingredients and their pharmacological aspects, toxicological research is clearly insufficient (Lv et al., 2012). Our study on the toxicity of hypaconitine will help us understand its toxic mechanism, which is also the basis for the control and treatment of corresponding indication. In this study, the non-invasive EMKA jacket system was used to detect QT prolongation in conscious dogs. The data indicated that the ECG signal from the EMKA jacket system was robust and sensitive. The analysis revealed significant prolongation of QTcV interval (23% greater than the vehicle control) by a single dose of hypaconitine in a dose dependent manner. There was also a strong correlation between plasma concentration of hypaconitine and prolongation QTcV interval (Fig. 4C, R²¼0.789). The maximum plasma concentration (Cmax) of single-dose hypaconitine was 10.11 ng/mL, and time to maximum plasma concentration (Tmax) was 1.0 h. The maximum QTcV prolongation was observed with different doses of hypaconitine at 2 h. This delayed peak effect on QT prolongation (compared to peak plasma concentration) was consistent with other studies (Toyoshima et al., 2005). One reason may be the delayed distribution of hypaconitine from the blood to the myocardial cells, the other reasons for the delayed maximal effect may be the formation of active metabolites of hypaconitine and mixed ion channel blocking properties. It is clear that potassium efflux through KCNH2 is a major determinant of repolarization and APD in ventricular cardiomyocytes. Blockade of IKCNH2 could lengthen APD and thus lead to QT prolongation. In this study, hypaconitine directly inhibited IKCNH2 in a concentration-dependent manner, with a maximum blockade of 73.6473.51% at the highest concentration tested (30 nM). This suggested that hypaconitine produced QT interval prolongation mainly through KCNH2 channel inhibition. The ratio of Cmax (10.11 ng/mL ) to the IC50 (8.1 nM) was far lower than 30, which usually means the compound can be concerned with toxicity (Redfern et al., 2003). Thus, the data in the present study may indicate a safety margin for hypaconitine-induced arrhythmogenes is that could be useful in guiding future clinical applications. Compared with two other toxic Acotinum alkaloids, aconitine and mesaconitine, the content of hypaconitine in aconites was much higher (Lu et al., 2010). Therefore, the therapeutic index of hypaconitine was lowest among the three toxic alkaloids (Zhang, 2007). In the present study, the pharmacological effects of hypaconitine on the QT interval were examined along with the inhibitory effect on the cloned KCNH2 potassium channel. These results may make the mechanism of cardiovascular risk of hypaconitine clearer. QT prolongation studies in vivo showed that the non-invasive system used was quantitatively comparable to invasive telemetry. The noninvasive system could be used successfully to acquire continuous ECG data from conscious freely moving dogs for at least 6 h (Prior et al., 2009). For the studies on the KCNH2 channel, the percent inhibition by hypaconitine of KCNH2 currents was evaluated using manual patch clamp studies. In previous studies, inhibition of KCNH2 inwardly rectifying potassium channels closely predicted the cardiac action potential prolongation by non-cardiac drugs (Yap and Camm, 2000). Such cardiac action potential prolongation may lead to a potentially lethal form of cardiac arrhythmia called Torsades de pointes. KCNH2 potassium channels were expressed in a human embryonic kidney (HEK293) cell line that does not have any endogenous inwardly rectifying potassium channels. In summary, this study shows that oral administration of hypaconitine can induce QT prolongation in a dose-dependent manner. Hypaconitine also blocked KCNH2 channels expressed in HEK293 cells and this may be the molecular mechanism for the QT interval prolongation in conscious dogs. However, further studies are required to determine the effect of hypaconitine on other cardiac ion channels to fully elucidate the mechanism of hypaconitineinduced QT prolongation.
379
Acknowledgments This study was supported by the Fundamental Research Funds for the Central Universities (2014ZM0067) and National Natural Science Foundation of China (81202585).
References Bisset, N.G., 1981. Arrow poisons in China. Part II. Aconitum—botany, chemistry, and pharmacology. J. Ethnopharmacol. 4, 247–336. Bouchaud, O., Imbert, P., Touze, J.E., Dodoo, A.N., Danis, M., Legros, F., 2009. Fatal cardiotoxicity related to halofantrine: a review based on a worldwide safety data base. Malar. J. 8, 281–289. Chan, T.Y., 2002. Incidence of herb-induced aconitine poisoning in Hong Kong: impact of publicity measures to promote awareness among the herbalists and the public. Drug Saf. 25, 823–828. Chan, T.Y., 2012. Contributory factors in herb-induced fatal aconite poisoning. Forensic Sci. Int. 223, 40–43. Chen, R.C., Sun, G.B., Zhang, Q., Ye, Z.G., Sun, X.B., 2013. Advances in studies on toxicity of aconite. Zhongguo Zhong Yao Za Zhi 38, 1126–1129. Chen, S.P., Ng, S.W., Poon, W.T., Lai, C.K., Ngan, T.M., Tse, M.L., Mak, T.W., 2012. Aconite poisoning over 5 years: a case series in Hong Kong and lessons towards herbal safety. Drug Saf. 35, 575–587. Cubeddu, L.X., 2003. QT prolongation and fatal arrhythmias: a review of clinical implications and effects of drugs. Am. J. Ther. 10, 452–457. Food and Drug Administration, HHS, 2005. The nonclinical evaluation of the potential for delayed ventricular repolarization (QT interval prolongation) by human pharmaceuticals. Fed. Regist. 70 (202), 61133–61134. Hu, R., Zhao, J., Qi, L.W., Li, P., Jing, S.L., Li, H.J., 2009. Structural characterization and identification of C(19)- and C(20)-diterpenoid alkaloids in roots of Aconitum carmichaeli by rapid-resolution liquid chromatography coupled with time-offlight mass spectrometry. Rapid Commun. Mass Spectrom. 23, 1619–1635. Li, Y., Tu, D., Xiao, H., Du, Y., Zou, A., Liao, Y., Dong, S., 2010. Aconitine blocks HERG and Kv1.5 potassium channels. J. Ethnopharmacol. 131, 187–195. Lu, G., Dong, Z., Wang, Q., Qian, G., Huang, W., Jiang, Z., Zhao, Z., 2010. Toxicity assessment of nine types of decoction pieces from the daughter root of Aconitum carmichaeli (Fuzi) based on the chemical analysis of their diester diterpenoid alkaloids. Planta Med. 76, 825–830. Lv, W., Piao, J.H., Jiang, J.G., 2012. Typical toxic components in traditional Chinese medicine. Expert Opin. Drug Saf. 11, 985–1002. Olivier, N.B., Eyster, G.E., Sanders, R., Cheng, J., Bohart, G., Girand, M., Bailie, M., 2003. Atrioventricular nodal ablation and His-bundle pacing: an acute canine model for proarrhythmic risk assesment. J. Cardiovasc. Electrophysiol. 14, 1356–1360. Prior, H., McMahon, N., Schofield, J., Valentin, J.P., 2009. Non-invasive telemetric electrocardiogram assessment in conscious beagle dogs. J. Pharmacol. Toxicol. Methods 60, 167–173. Redfern, W.S., Carlsson, L., Davis, A.S., Lynch, W.G., MacKenzie, I., Palethorpe, S., Siegl, P.K., Strang, I., Sullivan, A.T., Wallis, R., Camm, A.J., Hammond, T.G., 2003. Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: evidence for a provisional safety margin in drug development. Cardiovasc. Res. 58, 32–45. Soloviev, M.V., Hamlin, R.L., Barrett, R.M., Chengelis, C.P., Schaefer, G.J., 2006. Different species require different correction factors for the QT interval. Cardiovasc. Toxicol. 6, 145–157. Spence, S., Soper, K., Hoe, C.M., Coleman, J., 1998. The heart rate-corrected QT interval of conscious beagle dogs: a formula based on analysis of covariance. Toxicol. Sci. 45, 247–258. Testai, L., Calderone, V., Salvadori, A., Breschi, M.C., Nieri, P., Martinotti, E., 2004. QT prolongation in anaesthetized guinea-pigs: an experimental approach for preliminary screening of torsadogenicity of drugs and drug candidates. J. Appl. Toxicol. 24, 217–222. Toyoshima, S., Kanno, A., Kitayama, T., Sekiya, K., Nakai, K., Haruna, M., Mino, T., Miyazaki, H., Yano, K., Yamamoto, K., 2005. QT PRODACT: in vivo QT assay in the conscious dog for assessing the potential for QT interval prolongation by human pharmaceuticals. J. Pharmacol. Sci. 99, 451–471. Poon, W.T., Lai, C.K., Ching, C.K., et al., 2006. Aconite poisoning in camouflage. Hong Kong Med. J. 12, 456–459. Xiao, P.G., Wang, F.P., Gao, F., Yan, L.P., Chen, D.L., Liu, Y., 2006. A pharmacophylogenetic study of Aconitum L. (Ranunculaceae) from China. Acta Phytotaxon. 44, 1–46. Yap, Y.G., Camm, A.J., 2003. Drug induced QT prolongation and torsades de pointes. Heart 89, 1363–1372. Yap, Y.G., Camm, J., 2000. Risk of torsades de pointes with non-cardiac drugs. Doctors need to be aware that many drugs can cause qt prolongation. BMJ 320, 1158–1159. Zeng, Z.P., Jiang, J.G., 2010. Analysis of the adverse reactions induced by natural product-derived drugs. Br. J. Pharmacol. 159, 1374–1391. Zhang, S.F., 2007. The relationship between toxicity and curable effect induced by diester diterpenoid alkaloids and intervention of liquiritin. Dissertation of Beijing University of Chinese Medicine.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Hypaconitine-induced QT prolongation mediated through inhibition of KCNH2 (hERG) potassium channels in conscious dogs Shuilin Xie a, Ying Jia a, Aiming Liu b, Renke Dai a, Lizhen Huang a,n a b
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China Medical School of Ningbo University, Ningbo 315211, China
art ic l e i nf o
a b s t r a c t
Article history: Received 12 August 2014 Received in revised form 15 January 2015 Accepted 9 March 2015 Available online 20 March 2015
Ethnopharmacological relevance: Hypaconitine is one of the main aconitum alkaloids in traditional Chinese medicines prepared with herbs from the genus Acotinum. These herbs are widely used for the treatment of cardiac insufficiency and arrhythmias. However, Acotinum alkaloids are known for their toxicity as well as their pharmacological activity, especially cardiotoxicity including QT prolongation, and the mechanism of this toxicity is not clear. Material and methods: In this study, hypaconitine was administered orally to conscious Beagle dogs, and electrocardiograms were recorded by telemetry. Pharmacokinetic studies (6 h) were conducted to evaluate the relationship between QT prolongation and exposure level. HEK293 cells stably transfected with KCNH2 (hERG) cDNA were used to examine the effects of hypaconitine on the KCNH2 channel by using the manual patch clamp technique. Results: In the conscious dogs, all doses of hypaconitine induced QTcV (QT interval corrected according to the Van de Water formula) prolongation by more than 23% (67 ms) of control in a dose-dependent manner. The maximum QTcV prolongation was observed at 2 h after dosing. Maximum prolongation percentages were plotted against plasma concentrations of hypaconitine and showed a strong correlation (R2 ¼0.789). In the in vitro study in HEK293 cells, hypaconitine inhibited the KCNH2 currents in a concentration-dependent manner with an IC50 of 8.1 nM. Conclusion: These data suggest that hypaconitine inhibits KCNH2 potassium channels and this effect might be the molecular mechanism underlying QT prolongation in conscious dogs. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Hypaconitine QT prolongation hERG channels ECG telemetry Safety assessment
1. Introduction Prolongation of the QT in the electrocardiogram can be associated with polymorphic ventricular tachycardia or Torsades de pointes. QT prolongation has increasingly drawn attention from regulatory agencies and the pharmaceutical industry as a potentially serious adverse event (Yap and Camm, 2003). The recent withdrawal of several drugs from the market due to possibly drug-related cardiac arrhythmias has greatly increased concern about drug-induced prolongation of the QT interval (Bouchaud et al., 2009). To avoid QT prolongation and increase the chances for success of new drug candidates, a more rigorous methodology to evaluate the potential for QT prolongation is critical. The International Conference on Harmonization S7B guideline describes a nonclinical testing strategy for assessing the potential of a test substance to delay ventricular repolarization and thereby produce QT prolongation (Food and Drug
n
Corresponding author. Tel.: þ 86 13631406992; fax: þ 86 20 39380601. E-mail address: [email protected] (L. Huang).
http://dx.doi.org/10.1016/j.jep.2015.03.023 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
Administration, 2005). According to the S7B guideline, an in vivo QT assay using conscious, unrestrained animals is one of the most important elements of the testing strategy. Aconites are the common name of the genus Aconitum L., subordinated to Ranunculaceae (Xiao et al., 2006). Herbal preparations from aconites are well known worldwide for their wide use in traditional medicines of China, Japan, and Korea. These preparations are well known for their toxicity as well, including cardiotoxic and neurotoxic effects. There are quite a few clinical cases of aconite intoxication reported from China Hong Kong, Japan, Germany, and other countries (Chan, 2002; Chen et al., 2012; Poon et al., 2006). Plants of this genus commonly contain diester diterpenoid alkaloids. Alkaloids with an acetyl group at C8 and a benzoylester group at C14 are the potentially toxicalkaloids, including aconitine, mesaconitine, and hypaconitine (Fig. 1) (Hu et al., 2009). Aconitine has been shown to have substantial arrhythmogenic effects (Chen et al., 2013) Early studies indicated that aconitine blocked KCNH2 and kv1.5 potassium channels, which may contributed to action potential duration (APD) prolongation and cardiac arrhythmias (Li et al., 2010).
376
S. Xie et al. / Journal of Ethnopharmacology 166 (2015) 375–379
Fig. 1. The chemical structure of hypaconitine.
Hypaconitine has also been reported to induce arrhythmias and even death (Chan, 2012). However, few studies have been conducted to investigate the mechanism of arrhythmia induction by hypaconitine alone. Therefore, the QT prolongation potential of hypaconitine was assayed in conscious dogs. The relationship of plasma concentration to QT prolongation was also investigated. The mechanism underlying QT prolongation was studied through patch clamp tests on KCNH2 channels expressed in mammalian HEK293 cells.
2. Materials and methods 2.1. Chemicals and reagents Hypaconitine (499%), quinidine (499%), terfenadine (499%) and phenacetin (499%) were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China); Terfenadine, acetonitrile, ethanol (HPLC grade) and G418 were obtained from Sigma-Aldrich (St. Louis, MO 63178, USA); HEK293 cells purchased from ATCC (Manassas, VA 20110, USA) were stably transfected with hERG cDNA (GenBank accession number NM_ 000238); DMEM, FBS, pCDNA3.1 (þ) and lipofectmine 2000 were provided by Life technologies (Grand Island, NY 14072, USA). 2.2. Animals and treatment Six beagle dogs (3 males and 3 females, 7.570.3 kg, Marshall Biotechnology Co., Ltd (Beijing, China)) were randomly assigned in a Latin Square design. All animals were housed in individual stainless steel cages in a room maintained at 22–26 1C, 60–70% relative humidity and 12 h light/12 h dark cycle. They were fed approximately 300 g of standard laboratory diet daily and had access to water ad libitum. Animals were kept in the fasted state for approximately 12 h pre-dose and 6 h post-dose. All study procedures were reviewed and approved by the Laboratory Animal Care and Use Committee of Zhongshan PharmaSS Corporation, Guangdong, China. Each dog was orally administered three doses of hypaconitine (50, 150, and 450 μg/kg), quinidine (1, 3, and 9 mg/kg), a drug known to prolong the QT interval in animals (Olivier et al., 2003; Testai et al., 2004) and humans (Cubeddu, 2003), as the positive control, and vehicle in the same volume. Each animal received the treatments with a 7-day washout period between doses. The dose-finding study had been conducted according to mice LD50 of 5.8 mg/kg (Bisset, 1981). And finally, the present dosage was selected for dog study. 2.3. ECG recording EMKA non-invasive telemetry for large animals (Emka Technologies, Paris 75015, France) was used for this study. The system
consisted of an emkaPACK transmitter with a 5-wire ECG cable to collect 7-lead ECG data with iox2 data acquisition and real-time analysis software (Emka Technologies, Paris 75015, France). All dogs were habituated to the EMKA jacket that was used to carry the non-invasive electrodes and keep them in place for at least 24 h. A seven lead surface ECG configuration (limb I, II, III, aVR, aVL, aVF, and V) was continuously recorded. After ECG signals were stabilized, baseline QT interval corrected using to the Van de Water formula interval was continuously recorded from pre-dose to 6 h post-dose. Data were recorded at 0, 0.5, 1, 2, 4, and 6 h. During the recording, access to the animal room was restricted to the personnel administering the test compounds. At the end of each experiment, the surface ECG electrode patches and jackets were removed and animals were returned to their home cages. The ECG signals were analyzed using iox2 data analysis software. Baseline values were calculated from the pre-dose data. The QT interval was corrected for heart rate using Van de Water's method:QTC V ¼ QT 0:1087ðRR 1000Þ, one of the most common formulae used in conscious dogs (Soloviev et al., 2006; Spence et al., 1998). In the equation, QTcV is the corrected QT internal, QT is the raw QT internal, and RR is the R–R interval. 2.4. Plasma hypaconitine analysis To evaluate the relationship between plasma hypaconitine and QT prolongation, plasma hypaconitine concentrations were determined. Six-hour pharmacokinetic analyses were carried out to evaluate the relationship between hypaconitine blood levels and QTcV prolongation. To measure hypaconitine blood levels, 0.5 mL blood samples were drawn at 0, 0.5, 1, 2, 4, and 6 h after dosing. The blood samples were immediately centrifuged at 3000 g for 10 min, the plasma was collected and then stored at 80 1C until analysis. Before analysis, 80 μL of acetonitrile was added to the 20 μL of plasma to denature the plasma proteins and then the plasma was centrifuged at 14,000 g for 30 min. An aliquot of the supernatant (10 μL) was subjected to liquid chromatography/tandem mass spectroscopy (LC-MS/MS) analysis coupled with Analyst 1.4.2 workstation software. The separation was performed by a CAPCELL PAK C18 column (5 μm, 2.0 mm IDn50 mm) with a flow rate of 0.2 mL/min. 2.5. The effect of hypaconitine on the KCNH2 channel The hERG gene (KCNH2) was subcloned into pCDNA3.1 (þ ) vector. Human embryonic kidney 293 (HEK293) cells were cultured at 37 1C (5% CO2) in DMEM supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). Cells were transfected with linearized KCNH2 plasmid DNA using lipofectmine 2000 (Invitrogen, Carlsbad, CA). Human KCNH2 stable cell line (HEK293-HuKCNH2) was established using G418 screening and validated using manual patch clamp. KCNH2 expressing HEK293 cells were subcultured 2–3 times per week in order to maintain optimal cell health. The cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum and 200 μg/mL of G418. HEK293 cells are plated in 35 mm dishes at least 24 h prior to the day of experiment and maintained at 37 1C/5% CO2. The extracellular solution for the whole-cell patch clamp recordings was composed of (mM): NaCl 145; MgCl2 1.0; KCl 4.0; Glucose 10; HEPES 10; and CaCl2 2.0. The pH was adjusted to 7.4 with NaOH. The osmolarity was adjusted to 300 mOsm with sucrose. The intracellular solution for whole-cell patch clamp recordings was composed of (mM): KCl 140; MgCl2 1; EGTA 5; HEPES 10 and Na2ATP 4. The pH was adjusted to 7.2 with KOH. The osmolarity was adjusted to 295 mOsm with sucrose.
S. Xie et al. / Journal of Ethnopharmacology 166 (2015) 375–379
A micropipette was pulled from borosilicate glass with the pipette tip resistance of 3–5 MΩ. For each experiment, a single dish of cells was removed from the incubator, washed twice with room temperature extracellular solution, and placed on the microscope stage. A commercial patch clamp amplifier (Molecular Devices, Sunnyvale, CA, USA) was used for the whole cell recordings. The tail currents were evoked in room temperature once every 30 s by a 3 s 50 mV repolarizing pulse following a 2 s þ 50 mV depolarizing pulse with a hold voltage of 80 mV. A 50 ms depolarizing pulse to 50 mV at the beginning of the voltage protocol served as a baseline for calculating the amplitude of the peak tail current. Only stable cells with recording parameters above threshold were used for the drug application procedure. Terfenadine, a known compound inducing the blockade of HERG channels, was used for the positive control. Hypaconitine or terfenadine was added into the test solution when the KCNH2 currents were stable over a 3 min period in the presence of vehicle alone. The cells were kept in the test solution until the peak tail current was stable (o 5% change) for approximately five sweeps or for a maximum of 6 min, whichever came first. Data were recorded by Clampex (version 10.1, Sunnyvale, CA 94089 USA) and stored for off-line analysis. Data acquisition and analyses were performed using a pCLAMP (version 10.1, Sunnyvale, CA 94089 USA). Peak tail amplitudes were then plotted as a function of the sweep number. Five peak tail current measurements at steady state before hypaconitine or terfenadine application were averaged and used as the control current amplitude. Four or five peak tail current measurements at steady state after hypaconitine or terfenadine application were averaged and used as the current amplitude after inhibition by the test article. The inhibition percentage (%) of the hypaconitine and terfenadine was calculated from the following equation: Inhibition percentage (%)¼ {1 (remaining current amplitude)/ (control current amplitude)} 100 2.6. Statistical analysis All data were expressed as the mean 7the standard deviation (SD). Statistical analysis among groups was performed by was performed with oneway ANOVA and LSD-t test by SPSS16.0. Differences were considered statistically significant if the probability (P value) was less than 0.05 (Po 0.05).
377
Fig. 2. Electrocardiogram recordings before (A) and 2 h after oral administration of 450 μg/kg hypaconitine (B) in a conscious dog. The QTcV intervals were 271 ms for (A) and 438 ms for (B).
Table 1 The maximum value of QT interval corrected according to the Van de Water formula (QTcV) prolongation induced by quinidine and hypaconitine in conscious dogs and the corresponding pharmacokinetic parameters. Group
Maximum value of QTcV iterval Pharmacokinetic prolongation and time points parameters (ms)
Quinidine (mg/kg) Hypaconitine (μg/kg)
1 287 6n 3 607 13n 9 977 14nn 50 677 9n 150 1087 25nn 450 1747 32nn
(%)
(h)
Tmax (h)
Cmax (ng/mL)
107 3 217 5 357 11 237 6 377 7 617 13
0.5 0.5 0.5 2.0 2.0 2.0
0.5 0.5 0.5 1.0 1.0 1.0
958 2107 5868 1.53 5.74 10.11
The maximum value of QTcV interval prolongation between the vehicle and each dose of quinidine and hypaconitine were calculated individually, and the difference values from six dogs were averaged at each time point. The Cmax and Tmax represent means of values from six dogs. All Data is represented as mean 7SD (n¼ 6). n
P o 0.05. Po 0.01 vs. control.
nn
3. Results 3.1. QTcV interval prolongation in conscious dog Typical ECG waveforms before and 2 h after oral administrations of 450 μg/kg hypaconitine are shown in Fig. 2. The QTcV interval after hypaconitine administration was substantially longer than vehicle. Changes in QTcV interval in response to the hypaconitine and positive control, quinidine are summarized in Table 1. All doses prolonged the QTcV interval compared to the vehicle group in a dose-dependent manner. The maximum QTcV prolongation from time-matched vehicle was observed at 0.5 h after administration of quinidine and 2 h after administration of hypaconitine respectively. The maximum prolongation value for hypaconitine was 23% or 67 ms for the 50 μg/kg of group, 37% or 108 ms for the 150 μg/ kg of group and 61% or 174 ms for the 450 μg/kg of group. 3.2. Hypaconitine exposure and toxicity-concentration correlation A validated LC-MS/MS method was used for the determination of hypaconitine in plasma. The mass spectrometer was operated in
positive ion mode with m/z 616.40 for hypaconitine (Fig. 3A). A typical chromatograph of hypaconitine and phenacetin, a frequently-used internal standard owing to its good stability and high response, is shown in Fig. 3B. The concentrations of hypaconitine corresponding to the time points of QTcV measurement were analyzed and the average concentrations were calculated (Fig. 4A and B). The maximum percent prolongation of QTcV from six dogs was plotted in relation to plasma concentrations of hypaconitine. These results showed a strong plasma concentration-response correlation (R²¼0.789) (Fig. 4C). The maximum value of QT interval corrected according to the Van de Water formula (QTcV) prolongation after oral administration of quinidine (0.5 h) and hypaconitine (2 h) in conscious dogs (A). Serum concentration of hypaconitine at each time-point for electrocardiogram recording (B).The relationship between plasma concentration and the percentage of QTcV prolongation in Conscious Dog (C). All Data is represented as mean 7SD (n ¼ 6); n Po 0.05, nnP o0.01 vs. control.
378
S. Xie et al. / Journal of Ethnopharmacology 166 (2015) 375–379
3.3. Inhibition of hypaconitine on hERG currents Hypaconitine (1, 3, 10, and 30 nM) inhibited KCNH2 currents by 5.597 2.56%, 32.30 73.34%, and 58.95 73.87%, and 73.64 73.51%, respectively. Table 2 shows the percent inhibition in the individual cells and the statistics of the percent inhibition at different hypaconitine concentrations. The representative KCNH2 voltageclamp current recordings acquired at the steady state before and after application of hypaconitine are superimposed in Fig. 5A. Hypaconitine inhibited KCNH2 currents with an IC50 of 8.1 nM (Fig. 5B). Table 2 summarizes the effect of application of hypaconitine and terfenadine on KCNH2 currents.
4. Discussion Traditional Chinese medicines (TCM) are widely used around the world. However, with the increasing cases of adverse drug reactions (ADRs), the ADRs induced by TCM are becoming more widely recognized (Zeng and Jiang, 2010). A number of the ADRs are cardiovascular including arrhythmias, myocardial damage, heart failure, increased or decreased blood pressure, and cardiac arrest. Up to date, most studies are limited to the isolation and identification Fig. 3. The mass spectrometry of hypaconitine (A) and the typical chromatograph of hypaconitine (RT: 2.98) and phenacetin (RT: 2.47) as the internal standard(B).
Table 2 Percentage inhibition of KCNH2 currents by hypaconitine and terfenadine. Concentration (nM)
Hypaconitine
Terfenadine
Fig. 4. Correlation of hypaconitine serum concentration and QT prolongation.
1 3 10 30 3 10 30 100
Inhibition (%)
Mean (%)
Cell 1
Cell 2
Cell 3
4.14 28.78 54.50 71.11 22.78 30.23 72.10 94.90
4.09 35.43 60.79 72.16 14.28 37.16 61.98 83.55
8.55 32.69 61.56 77.64 16.57 32.69 63.46 80.58
5.59 32.30 58.95 73.64 17.88 33.36 65.85 86.34
SD
2.56 3.34 3.87 3.51 4.40 3.51 5.47 7.58
Fig. 5. The representative KCNH2 current traces before and after hypaconitine application (A) and the concentration-response relationship of hypaconitine on KCNH2 currents (B). Percentage inhibition of KCNH2 currents at each concentration of hypaconitine (n¼ 3) is shown. The IC50 value was 8.1 nM.
S. Xie et al. / Journal of Ethnopharmacology 166 (2015) 375–379
of active ingredients and their pharmacological aspects, toxicological research is clearly insufficient (Lv et al., 2012). Our study on the toxicity of hypaconitine will help us understand its toxic mechanism, which is also the basis for the control and treatment of corresponding indication. In this study, the non-invasive EMKA jacket system was used to detect QT prolongation in conscious dogs. The data indicated that the ECG signal from the EMKA jacket system was robust and sensitive. The analysis revealed significant prolongation of QTcV interval (23% greater than the vehicle control) by a single dose of hypaconitine in a dose dependent manner. There was also a strong correlation between plasma concentration of hypaconitine and prolongation QTcV interval (Fig. 4C, R²¼0.789). The maximum plasma concentration (Cmax) of single-dose hypaconitine was 10.11 ng/mL, and time to maximum plasma concentration (Tmax) was 1.0 h. The maximum QTcV prolongation was observed with different doses of hypaconitine at 2 h. This delayed peak effect on QT prolongation (compared to peak plasma concentration) was consistent with other studies (Toyoshima et al., 2005). One reason may be the delayed distribution of hypaconitine from the blood to the myocardial cells, the other reasons for the delayed maximal effect may be the formation of active metabolites of hypaconitine and mixed ion channel blocking properties. It is clear that potassium efflux through KCNH2 is a major determinant of repolarization and APD in ventricular cardiomyocytes. Blockade of IKCNH2 could lengthen APD and thus lead to QT prolongation. In this study, hypaconitine directly inhibited IKCNH2 in a concentration-dependent manner, with a maximum blockade of 73.6473.51% at the highest concentration tested (30 nM). This suggested that hypaconitine produced QT interval prolongation mainly through KCNH2 channel inhibition. The ratio of Cmax (10.11 ng/mL ) to the IC50 (8.1 nM) was far lower than 30, which usually means the compound can be concerned with toxicity (Redfern et al., 2003). Thus, the data in the present study may indicate a safety margin for hypaconitine-induced arrhythmogenes is that could be useful in guiding future clinical applications. Compared with two other toxic Acotinum alkaloids, aconitine and mesaconitine, the content of hypaconitine in aconites was much higher (Lu et al., 2010). Therefore, the therapeutic index of hypaconitine was lowest among the three toxic alkaloids (Zhang, 2007). In the present study, the pharmacological effects of hypaconitine on the QT interval were examined along with the inhibitory effect on the cloned KCNH2 potassium channel. These results may make the mechanism of cardiovascular risk of hypaconitine clearer. QT prolongation studies in vivo showed that the non-invasive system used was quantitatively comparable to invasive telemetry. The noninvasive system could be used successfully to acquire continuous ECG data from conscious freely moving dogs for at least 6 h (Prior et al., 2009). For the studies on the KCNH2 channel, the percent inhibition by hypaconitine of KCNH2 currents was evaluated using manual patch clamp studies. In previous studies, inhibition of KCNH2 inwardly rectifying potassium channels closely predicted the cardiac action potential prolongation by non-cardiac drugs (Yap and Camm, 2000). Such cardiac action potential prolongation may lead to a potentially lethal form of cardiac arrhythmia called Torsades de pointes. KCNH2 potassium channels were expressed in a human embryonic kidney (HEK293) cell line that does not have any endogenous inwardly rectifying potassium channels. In summary, this study shows that oral administration of hypaconitine can induce QT prolongation in a dose-dependent manner. Hypaconitine also blocked KCNH2 channels expressed in HEK293 cells and this may be the molecular mechanism for the QT interval prolongation in conscious dogs. However, further studies are required to determine the effect of hypaconitine on other cardiac ion channels to fully elucidate the mechanism of hypaconitineinduced QT prolongation.
379
Acknowledgments This study was supported by the Fundamental Research Funds for the Central Universities (2014ZM0067) and National Natural Science Foundation of China (81202585).
References Bisset, N.G., 1981. Arrow poisons in China. Part II. Aconitum—botany, chemistry, and pharmacology. J. Ethnopharmacol. 4, 247–336. Bouchaud, O., Imbert, P., Touze, J.E., Dodoo, A.N., Danis, M., Legros, F., 2009. Fatal cardiotoxicity related to halofantrine: a review based on a worldwide safety data base. Malar. J. 8, 281–289. Chan, T.Y., 2002. Incidence of herb-induced aconitine poisoning in Hong Kong: impact of publicity measures to promote awareness among the herbalists and the public. Drug Saf. 25, 823–828. Chan, T.Y., 2012. Contributory factors in herb-induced fatal aconite poisoning. Forensic Sci. Int. 223, 40–43. Chen, R.C., Sun, G.B., Zhang, Q., Ye, Z.G., Sun, X.B., 2013. Advances in studies on toxicity of aconite. Zhongguo Zhong Yao Za Zhi 38, 1126–1129. Chen, S.P., Ng, S.W., Poon, W.T., Lai, C.K., Ngan, T.M., Tse, M.L., Mak, T.W., 2012. Aconite poisoning over 5 years: a case series in Hong Kong and lessons towards herbal safety. Drug Saf. 35, 575–587. Cubeddu, L.X., 2003. QT prolongation and fatal arrhythmias: a review of clinical implications and effects of drugs. Am. J. Ther. 10, 452–457. Food and Drug Administration, HHS, 2005. The nonclinical evaluation of the potential for delayed ventricular repolarization (QT interval prolongation) by human pharmaceuticals. Fed. Regist. 70 (202), 61133–61134. Hu, R., Zhao, J., Qi, L.W., Li, P., Jing, S.L., Li, H.J., 2009. Structural characterization and identification of C(19)- and C(20)-diterpenoid alkaloids in roots of Aconitum carmichaeli by rapid-resolution liquid chromatography coupled with time-offlight mass spectrometry. Rapid Commun. Mass Spectrom. 23, 1619–1635. Li, Y., Tu, D., Xiao, H., Du, Y., Zou, A., Liao, Y., Dong, S., 2010. Aconitine blocks HERG and Kv1.5 potassium channels. J. Ethnopharmacol. 131, 187–195. Lu, G., Dong, Z., Wang, Q., Qian, G., Huang, W., Jiang, Z., Zhao, Z., 2010. Toxicity assessment of nine types of decoction pieces from the daughter root of Aconitum carmichaeli (Fuzi) based on the chemical analysis of their diester diterpenoid alkaloids. Planta Med. 76, 825–830. Lv, W., Piao, J.H., Jiang, J.G., 2012. Typical toxic components in traditional Chinese medicine. Expert Opin. Drug Saf. 11, 985–1002. Olivier, N.B., Eyster, G.E., Sanders, R., Cheng, J., Bohart, G., Girand, M., Bailie, M., 2003. Atrioventricular nodal ablation and His-bundle pacing: an acute canine model for proarrhythmic risk assesment. J. Cardiovasc. Electrophysiol. 14, 1356–1360. Prior, H., McMahon, N., Schofield, J., Valentin, J.P., 2009. Non-invasive telemetric electrocardiogram assessment in conscious beagle dogs. J. Pharmacol. Toxicol. Methods 60, 167–173. Redfern, W.S., Carlsson, L., Davis, A.S., Lynch, W.G., MacKenzie, I., Palethorpe, S., Siegl, P.K., Strang, I., Sullivan, A.T., Wallis, R., Camm, A.J., Hammond, T.G., 2003. Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: evidence for a provisional safety margin in drug development. Cardiovasc. Res. 58, 32–45. Soloviev, M.V., Hamlin, R.L., Barrett, R.M., Chengelis, C.P., Schaefer, G.J., 2006. Different species require different correction factors for the QT interval. Cardiovasc. Toxicol. 6, 145–157. Spence, S., Soper, K., Hoe, C.M., Coleman, J., 1998. The heart rate-corrected QT interval of conscious beagle dogs: a formula based on analysis of covariance. Toxicol. Sci. 45, 247–258. Testai, L., Calderone, V., Salvadori, A., Breschi, M.C., Nieri, P., Martinotti, E., 2004. QT prolongation in anaesthetized guinea-pigs: an experimental approach for preliminary screening of torsadogenicity of drugs and drug candidates. J. Appl. Toxicol. 24, 217–222. Toyoshima, S., Kanno, A., Kitayama, T., Sekiya, K., Nakai, K., Haruna, M., Mino, T., Miyazaki, H., Yano, K., Yamamoto, K., 2005. QT PRODACT: in vivo QT assay in the conscious dog for assessing the potential for QT interval prolongation by human pharmaceuticals. J. Pharmacol. Sci. 99, 451–471. Poon, W.T., Lai, C.K., Ching, C.K., et al., 2006. Aconite poisoning in camouflage. Hong Kong Med. J. 12, 456–459. Xiao, P.G., Wang, F.P., Gao, F., Yan, L.P., Chen, D.L., Liu, Y., 2006. A pharmacophylogenetic study of Aconitum L. (Ranunculaceae) from China. Acta Phytotaxon. 44, 1–46. Yap, Y.G., Camm, A.J., 2003. Drug induced QT prolongation and torsades de pointes. Heart 89, 1363–1372. Yap, Y.G., Camm, J., 2000. Risk of torsades de pointes with non-cardiac drugs. Doctors need to be aware that many drugs can cause qt prolongation. BMJ 320, 1158–1159. Zeng, Z.P., Jiang, J.G., 2010. Analysis of the adverse reactions induced by natural product-derived drugs. Br. J. Pharmacol. 159, 1374–1391. Zhang, S.F., 2007. The relationship between toxicity and curable effect induced by diester diterpenoid alkaloids and intervention of liquiritin. Dissertation of Beijing University of Chinese Medicine.