Promethazine Aggravates Proarrhythmia Effect Induced By Chinese Herbal Intravenous Injection Of Shuanghuanglian

Objective:The experiments were carried out using adapted techniques from ICH （International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use）. Promethazine, an anti-allergic drug, is commonly used for the treatment of adverse drug reactions caused by Chinese herbal intravenous injection of Shuanghuanglian （SHL）. However, the proarrhythmia effects induced by both promethazine and SHL are reported in clinics. The study was to investigate the aggravated effect of promethazine on proarrhythmia induced by SHL using pro-clinical cardiac safety evaluation methods.Chemicals:1. Promethazine:Promethazine hydrochloride, batch number1001154582, which were ordered from Sigma-Aldrich.2. SHL:lyophilized type; batch number0910019was approved by SFDA with registration number Z10960058, and purchased from a local hospital in accordance with relevant localregulations.Methods:1. In vivo electrocardiogram （ECG） recording: II lead ECG was recorded by conventional in vivo ECG technique. The heart rate, the P-R interval, the QRS interval and the rate-corrected QT （QTc） intervals were analyzed.2. Ex vivo electrocardiogram （ECG） recording: Langendoff isolated heart perfusion technique was used to record ECG. The positive electrode was placed at the cardiac apex, the negative electrode at right atrium and the reference electrode at the root of the aorta. The heart rate, the P-R interval, the QRS interval and the rate-corrected QT （QTc） intervals were analyzed.3. Recording of action potentials from guinea pig heart papillary muscle: The hearts were taken from anesthetized guinea pig and placed in ice cold water. The guinea pig left ventricular papillary muscles were separated and placed in a recording chamber with perfusion solution at35.0±0.5℃gased with95%O2plus5%CO2（pH7.4with NaOH）. The action potential duration was analyzed from papillary muscle using conventional intracellular recording technique.4. Recording of L-type Ca2+current from rat left ventricular cardiomyocyte. Patch clamp technique was used to record the L-type Ca2+current from rat left ventricular cardiomyocyte. The recording external solution （room temperature） contained （in mmol/L）: NaCl138, KC19, CaCfel, MgCl21, TEA-Cl10, HEPES10,pH7.4with NaOH. The internal solution contained （in mmol/L）:CsCl130, MgCl22, EGTA11, Glucose10, HEPES20, Na2ATP2, GTP0.1, pH7.2with CsOH. To record L-type Ca2+current, depolarizing pulses applied from a holding potential of-80mV were stepped to-40mV for20ms （to inactivate Na+current） and then to0mV for300ms.5. Recording of hNavl.5current from HEK cell line cloned with hNavl.5cDNA: The sequence of the hNav1.5cDNA （NM₀00335.4） was synthesized and cloned into the expression vector pcDNA3.1with the G418resistance. The plasmid was transfected into the HEK cell line by lipofectamine2000. During the screening process, G418at500μg/mL was used. The cell lines, which showed good and stable current by manual patch clamp, were used for the further experiments. The recording external solution （room temperature） contained （in mmol/L）:NaCl25, TEACl122, MgCl21, Glucose10, HEPES10, CaCl21.8, pH7.4with NaOH. The internal solution contained （in mmol/L）:CsF129, MgCl22, EGTA11, HEPES10, Na2ATP3, pH7.2with CsOH. To record hNavl.5current, depolarizing pulses applied from a holding potential of-80mV were stepped to-20mV for20ms.6. Recording of hERG current from HEK cell line cloned with hERG cDNA: The sequence of the hERG cDNA （NM₀00238.2） was synthesized and cloned into the expression vector pcDNA3.1with the G418resistance. The plasmid was transfected into the HEK cell line by lipofectamine2000. During the screening process, G418at500μg/ml was used. The cell lines, which showed good and stable current by manual patch clamp, were used for the further experiments. The recording external solution （room temperature） contained （in mmol/L）:NaCl137, MgCl21.2, KCl5.4, Glucose10, HEPES10, CaCl22, pH7.4with NaOH. The internal solution contained （in mmol/L）:KCl140, MgCl21, EGTA5, Na2ATP5, HEPES10, pH7.2with KOH. The tail currents were evoked in room temperature once every30s by a5000ms-50mV repolarizing pulse following a4800ms +50mV depolarizing pulse with a hold voltage of-80mV. A50ms depolarized pulse to-50mV at the beginning of the voltage protocol served as a baseline for calculating the amplitude of the peak tail current.Results:1. The combined application of SHL and promethazine significantly slowed down the guinea pig heart rate and prolonged the in vivo ECG P-R and QRS interval:Promethazine15.35mg/kg （two times clinical dose） significantly prolonged the QRS interval （P<0.05）; Promethazine38.33mg/kg （five times clinical dose） significantly slow down the heart rate and prolonged the P-R, QRS and QTc intervals （P<0.05）. SHL1380mg/kg （five times clinical dose） significantly slowed down the heart rate and prolonged the P-R and QRS intervals （P<0.05）. The combined application of SHL （1380mg/kg） and promethazine （7.67mg/kg） further slow down the heart rate and prolonged the in vivo ECG P-R and QRS intervals.2. The combined application of SHL and promethazine significantly slowed down the heart rate and prolonged the in vitro ECG P-R, QRS and QTc intervals:Promethazine （10μmol/L） significantly slowed down the in vitro ECG heart rate （P<0.05）; Promethazine （50μmol/L） significantly slowed down the heart rate and prolonged the the QRS and QTc intervals （P<0.05）. SHL3g/L significantly slowed down the heart rate and prolonged the P-R interval （P<0.05）. The combined application of SHL （3g/L） and promethazine （50μmol/L） further slowed down the heart rate and prolonged the in vitro ECG P-R, QRS and QTc intervals.3. The combined application of SHL and promethazine partially abolished action potential firing recorded from left ventricle papillary muscles of guinea pig:Promethazine26μmol/L had no significantly effect on action potential. The combined application of SHL （3g/L） and promethazine （26μmol/L） abolished action potential firing recorded from left ventricle papillary muscles of guinea pig without prolonging the durations of action potentials. Washout with SHL （3g/L） restored the action potential firing.4. The combined application of SHL and promethazine further suppressed the L-type Ca2+current from left ventricular cardiomyocyte of rats: Promethazine suppressed the L-type Ca2+current from left ventricular cardiomyocyte of rats in a dose-dependent manner with an IC50of12.6±4.7μmol/L. The combined application of SHL （1.5g/L） and promethazine further suppressed the L-type Ca2+current from left ventricular cardiomyocyte of rats in a dose-dependent manner. IC50of promehtazine for L-type Ca2+current was6.7±2.8μmol/L in the presence of SHL （1.5g/L）.5. The combined application of SHL and promethazine further suppressed the hNavl.5current:Promethazine suppressed the hNav1.5current in a dose-dependent manner with an IC506.8±0.7μmol/L. The combined application of SHL （0.3g/L） and promethazine further suppressed the hNav1.5current in a dose-dependent manner. IC50of promehtazine for hNav1.5current was5.1±0.6μmol/L in the presence of SHL （0.3g/L）.6. The combined application of SHL and promethazine further suppressed the hERG current:Promethazine suppressed the hERG current in a dose-dependent manner with an IC50of1.5±0.1μmol/L. The combined application of SHL （0.3g/L） and promethazine further suppressed the hERG current in a dose-dependent manner. IC50of promehtazine for hERG current was0.9±0.1μmol/L in the presence of SHL （0.3g/L）.Conclusion:1. Promethazine significantly reduced the guinea pig heart rate in both in vivo and in vitro ECG recordings.Also promethazine significantly prolonged the the QRS and QTc interval of both in vivo and in vitro ECGPromethazine （50μmol/L） partially abolished action potential firing from left ventricle papillary muscles of guinea pig.2. Promethazine suppressed the L-type Ca2+current, hNavl.5current and hERG current in a dose-dependent manner. IC50S of promehtazine for L-type Ca2+, hNavl.5and hERG currents were12.6±4.7μmol/L,6.8±0.7μmol/L and1.5±0.1μmol/L, respectively.3. SHL caused brady arrhythmias of both in vivo and in vitro ECG in a dose-dependent manner. Promethazine aggravated bradycardia induced by SHL in the guinea pig hearts. SHL combined with promethazine further slowed down the heart rate and proloned the P-R, QRS and QTc intervals which was induced by SHL alone in both in vivo and in vitro ECG.The combined application of SHL （3g/L） and promethazine （26μmol/L） abolished action potential firing from left ventricle papillary muscles of guinea pig.4. Promethazine further suppressed the L-type Ca2+current, hNavl.5and hERG currents in a dose-dependent manner with their IC50s of6.7±2.8μmol/L,5.1±0.6μmol/L,0.9±0.1μmol/L, respectively in the presence of SHL.