| Chinese herbs rich in Aconitum alkaloids are commonly used in current clinical practice for the treatment of cardiovascular and cerebrovascular diseases, rheumatic arthritis and rheumatoid arthritis, digestive disorders and gastrointestinal diseases, and others. They may cause a high incidence of adverse effects including poisoning due to their high toxicity. In addition, potential drug-drug interactions may occur when such herbs are administered concomitantly with other drugs or even with Western medicine. However, few reports are available on the drug-drug interactions between Western medicine and Chinese herbs rich in Aconitum alkaloids.Therefore, in this study we firstly developed a highly efficient and simple UPLC-MS/MS method for the rapid determination of 7 Aconitum alkaloids in the blood plasma of rats, then attempted to construct a model of conscious rats orally administered aconitine, and based on this model, we analyzed the pharmacokinetics of the toxic dose of aconitine. We also investigated the effects of aconitine on the cytochrome P450 enzymes activities in rat liver microsomes. Finally, drug-drug interactions between aconitine and 3 antihypertensive drugs(losartan, metoprolol and amlodipine) were studied in rat liver microsomes.Part one Development a sensitive UPLC-MS/MS method for simultaneous determination of 7 Aconitum alkaloids in rat plasmaObjective: To investigate plasma metabolites of aconitine orally administered in the rat.Methods: A sensitive, rapid and specific UPLC-MS/MS method was developed for simultaneous determination of 7 Aconitum alkaloids. The sample was purified with liquid-liquid extraction prior to analysis. The mass spectrometer was used in a multiple reaction monitoring mode in the ESI+mode.Results: 1 UPLC-MS/MS method validation The developed UPLC-MS/MS methods for the determination of 7 Aconitum alkaloids were very selective. There was no significant interference from rat plasma at the retention times of Aconitum alkaloids and prazosin. All of the analytes were baseline-resolved. The linearity of calibration curves was evaluated using 1/X2 as the weighing factor and showed satisfactory linearity over the range of 0.1-20 ng/m L for aconine and 0.05-20 ng/m L for aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine, benzoylhypaconine. The limit of detection of aconitine, hypaconitine, mesaconitine, benzoylaconine, benzoylhypaconine, benzoylmesaconine and aconine was 5, 5, 5, 5, 5, 10 and 20 pg/m L, respectively. The limit of quantification of aconitine, hypaconitine, mesaconitine, benzoylaconine, benzoylhypaconine, benzoylmesaconine and aconine was 10, 10, 20, 20, 20, 20 and 50 pg/m L, respectively. The precision and accuracy of the methods were assessed for both intra-day and inter-day determinations and they did not exceed 3.9% and 5.3%. The recoveries and matrix effects ranged from 90.2%-101.3% and 90.3%-99.2%, respectively. After 4 h of short-term storage at room temperature, the content variation of the analytes in the plasma was less than 10%. After three freeze-thaw cycles, and 30 days of long-term storage at-20 °C, the determined contents of the Aconitum alkaloids were in the range of 90.0%-100.5% and 89.4%-100.1% of the added amount. In addition, for the ready-to-inject samples(after extraction) in the autosampler at 4 °C for 4 h or 24 h, the content variation of the analytes in the plasma was less than 4.4%. 2 Sample determination The content of mesaconitine, hypaconitine, benzoylaconine were around the limit of quantification of the assay. Other expected metabolites, including benzoylmesaconine, benzoylhypaconine and aconine, were lower than the limit of detection of the established range. Moreover, by product ion confirmation(PIC) scanning, an unexpected metabolite M was also firstly found in rat plasma and its structure might be 16-O-demethylaconitine.Part two Cardiovascular toxicity and toxicokinetics of aconitine in conscious rat after oral administration of aconitineObjective: A model of aconitine-induced bradycardia and hypotension was constructed in conscious rats by oral administration, and the toxicokinetics of aconitine was investigated.Methods: Blood pressure and heart rate in conscious rats were measured using a volume pressure recording system. The pharmacokinetics of the toxic dose of aconitine and its metabolites were analyzed using UPLC-MS/MS, and the pharmacokinetic parameters were analyzed using Win Nonlin software, version 5.1.Results: 1 UPLC-MS/MS method validation The results were the same as those in the Part one. 2 Effect of aconitine on blood pressure and heart rate in conscious rats The SBP, DBP and HR did not change significantly 2, 4, 6, 8, and 12 h after solvent administration in control rats(P>0.05). The HR in rats treated with 200 μg/kg aconitine significantly decreased 2 h(200±13 bpm) after administration compared with the pre-drug baseline level(283±10 bpm), which was reduced to 71%, and then recovered at 4 h(P>0.05). The HR in rats treated with 400 μg/kg aconitine significantly decreased 2 h(190±11 bpm) and 4 h(228±26 bpm) after administration compared with the pre-drug baseline level(283±11 bpm; P<0.05, P<0.01), which was reduced to 67% and 81%, respectively. In the rats treated with 400 μg/kg aconitine significant effects on the SBP(102.4±4.5 mm Hg) and DBP(69.1±3.7 mm Hg) were observed 2 h after administration compared with the pre-drug baseline level(115.4±3.3 mm Hg and 78.9±2.7 mm Hg)(P<0.05), which was reduced to 89% and 88%, respectively. However, there was no significant change in the SBP and DBP after 200 μg/kg aconitine administration(P>0.05). 3 Pharmacokinetic behavior of aconitine under different toxic doses In the 200 μg/kg group, aconitine reached the maximum plasma concentration(C max) of 7.5±0.8 ng/m L at 25.5±5.8 min; while the drug concentration-time curve showed a double-peak phenomenon at 14.5±1.9 min and 131±10 min at the dose of 400 μg/kg, and the C max was 7.7±0.9 ng/m L(the second peak). Compared to the 200 μg/kg group, the plasma concentration of aconitine in the 400 μg/kg group was significantly higher at 120, 180, 240, 360 and 480 min(P<0.05); however, there was no significant difference in C max(P>0.05). Compared to the 200 μg/kg group, the AUC and V d values of aconitine at 400 μg/kg were increased 0.8- and 1.2-fold, respectively(P<0.01), and the t1/2 and MRT were all increased 0.7-fold(P<0.01); but the Cl value of aconitine did not change significantly(P>0.05). 4 Determination and identification of metabolites of aconitine The concentration of benzoylaconine was 0.239 ng/m L(90 min) and 0.421 ng/m L(120 min) in the plasma after the administration 400 μg/kg aconitine, which was only quantitatively determined in one animal. The content of mesaconitine and hypaconitine were around the limit of quantification of the assay. The others expected metabolites, including benzoylmesaconine, benzoylhypaconine and aconine, were lower than the detection limits of the established range. Additionally, we compared the ratio of the peak area between compound M and aconitine of the two groups, it was found that the largest ratios of peak area after the administration of aconitine at 200 μg/kg and 400 μg/kg were 37.2%±1.7% and 40.8%±2.3%, respectively. No significant differences were observed between 200 μg/kg group and 400 μg/kg group(P>0.05).Part three Inhibitory effects of aconitine on probe substrates for cytochrome P450 enzyme of rat liver microsomes in vitroObjective: To observe the inhibitory effects of aconitine on pharmacokinetic parameters of 5 probe substrates for cytochrome P450 enzyme in the rat liver microsomes.Methods: Rat liver microsomes were prepared by using differential-velocity centrifugation. Potein contents of the rat liver microsomes were determined using a bicinchoninic acid(BCA) protein assay kit. UPLC-MS/MS method was used for the determination of 5 probe substrates: phenacetin(CYP1A2), chlorzoxazone(CYP2E1), tolbutaminde(CYP2C9), dextromethorphan(CYP2D6) and testosterone(CYP3A4), which was incubated with rat liver microsomes.Results: 1 UPLC-MS/MS method validation The developed UPLC-MS/MS methods for the determination of 5 probe substrates were very selective. There was no significant interference at the retention times of 5 probe substrates and prazosin. All of the analytes were baseline-resolved. The linearity of calibration curves was evaluated using 1/X2 as the weighing factor and showed satisfactory linearity over the range of 0.5-1000 nmol/L for tolbutaminde, dextromethorphan and testosterone; 1-1000 nmol/L for phenacetin and 5-1000 nmol/L for chlorzoxazone. The precision and accuracy of the methods were assessed for both intra-day and inter-day determinations and they did not exceed 5.7% and 5.9%. The recoveries and matrix effects ranged from 90.7%-102.4% and 91.2%-105.6%. 2 NADPH-dependent metabolisms of 5 probe substrates in rat liver microsomes Five probe substrates were not metabolized obviously in the rat liver microsomes without NADPH-generating system, but they were metabolized at least 20% in the rat liver microsomes with NADPH-generating system, suggesting that the metabolism of each of the 5 probe substrates is NADPH-dependent. 3 K m value determination of 5 probe substrates The K m values for dextromethorphan, testosterone, chlorzoxazone, phenacetion and tolbutaminde in rat liver microsomes were 7.1, 63.3, 85.3, 163.1 and 75.9 μmol/L, respectively. 4 Effects of aconitine on the metabolism of 5 probe substrates in the rat liver microsomes. Aconitine inhibited the metabolism of dextromethorphan, tolbutaminde, phenacetion, testosterone and chlorzoxazone in rat liver microsomes, and the IC50 values for the 5 agents were 13.4, 23.5, 65.7, 66.1 and >200 μmol/L, respectively. 5 Inhibitory profile of tolbutaminde and dextromethorphan in rat liver microsomes Aconitine competitively inhibited the metabolism of the CYP2C9 probe drug tolbutamide and the CYP2D6 probe drug dextromethorphan in the rat liver microsomes with Ki values of 12.5 and 4.9 μmol/L, respectively.Part four Drug-drug interaction between aconitine and antihypertensive drugs in rat liver microsomesObjective: To observe drug-drug interactions between aconitine and 3 antihypertensive drugs in rat liver microsomes.Methods: Rat liver microsomes were prepared by using differential-velocity centrifugation. Potein contents of the rat liver microsomes were determined using a bicinchoninic acid(BCA) protein assay kit. UPLC-MS/MS method was used for the determination of losartan, metoprolol and amlodipine in rat liver microsomes.Results: 1 UPLC-MS/MS method validation The developed UPLC-MS/MS methods for the determination of 3 antihypertensive drugs were very selective. There was no significant interference at the retention times of losartan, metoprolol, amlodipine and prazosin. All of the analytes were baseline-resolved. The linearity of calibration curves was evaluated using 1/X2 as the weighing factor and showed satisfactory linearity over the range of 5-1000 nmol/L for losartan, 1-1000 nmol/L for amlodipine and 1-5000 nmol/L for metoprolol. The precision and accuracy of the methods were assessed for both intra-day and inter-day determinations and they did not exceed 4.8% and 5.3%. The recoveries and matrix effects ranged from 89.1%-104.4% and 87.0%-100.9%. 2 NADPH-dependent metabolisms of 3 antihypertensive drugs in rat liver microsomes Three antihypertensive drugs were not metabolized obviously in the rat liver microsomes without NADPH-generating system, but they were metabolized at least 20% in the rat liver microsomes with NADPH-generating system, suggesting that the metabolism of each of the 3 antihypertensive drugs is NADPH-dependent. 3 K m value determination of 3 antihypertensive drugs The K m values for losartan, metoprolol and amlodipine in rat liver microsomes were 5.5, 16.7 and 3.0 μmol/L, respectively. 4 Effects of aconitine on the metabolism of 3 antihypertensive drugs in the rat liver microsomes. Aconitine inhibited the metabolism of metoprolol, losartan and amlodipine in rat liver microsomes, and the IC50 values for the 3 agents were 16.3, 30 and >200 μmol/L, respectively. 5 Inhibitory profile of losartan and metoprolol in rat liver microsomes Aconitine competitively inhibited the metabolism of losartan and metoprolol in the rat liver microsomes with Ki values of 9.54 and 4.13 μmol/L, respectively.Conclusion: Mesaconitine, hypaconitine, benzoylaconine and 16-O-demethylaconitine are detected in rat plasma for the first time after oral administration of aconitine, and 16-O-demethylaconitine might be a major metabolite of aconitine.Not only bradycardia but also hypotension is induced in conscious rats by a toxic dose of aconitine(400 μg/kg), and the pharmacokinetic parameters t1/2and V d increase significantly without changing C max when compared to a 200 μg/kg dose. Aconitine is a moderate competitive inhibitor of CYP2C9(tolbutaminde) and CYP2D6(dextromethorphan), and it weakly inhibits CYP1A2(phenacetion) and CYP3A4(testosterone) in rat liver microsomes. Aconitine moderately and competitively inhibits the metabolism of losartan and metoprolol in rat liver microsomes. |
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