| The stereoselective metabolism of THP enantiomer was studied by applying animal liver microsomes, human liver microsomes and human recombinant CYP450, through which the phase I metabolism enzymes mainly mediating the stereoselective metabolism of THP were found. The inhibitory and inductive effect of (-)-THP and (+)-THP on main CYP450 isoforms in mouse liver microsomes were also studied in order to predict the possible drug-drug interaction clinically.Stereoselective metabolism of tetrahydropalmatine enantiomer in animal liver microsomesObjective:To investigate stereoselective metabolism of THP enantiomer in animal liver microsomes and to elucidate CYP450 isoforms mainly mediating stereoselective metabolism of THP enantiomer.Methods:Liver microsomes from monkeys, dogs, mice and rats (untreated or induced by Dex/β-NF) were incubated with THP enantiomer and the concentration of (+)-THP and (-)-THP were determined by RP-HPLC; to determine and compare the enzyme kinetic parameters of (+)-THP and (-)-THP in animal liver microsomes; to investigate the inhibitory effect of main CYP450 isoforms'inhibitors on metabolism of THP enantiomer in RLM in order to elucidate CYP450 isoforms mainly mediating metabolism of THP enantiomer; to investigate the inhibitory effect of CYP450 inhibitors with series of concentration-ketoconazole((0.1～1μmol/L) and fluvoxamine(2～20μmol/L) on the (+)-THP and (-)-THP in Dex-RLM andβ-NF-RLM.Results:Among the animal liver microsomes in this research,the metabolism of (+)-THP were significantly higher than that of (-)-THP (P<0.05 or P<0.01 or P<0.001), moreover, the metabolism of (+)-THP and (-)-THP in Dex andβ-NF RLM were increased significantly compared to the untreated RLM (P<0.001); in Dex-RLM, the increased metabolism of (+)-THP was more than that of (-)-THP,while inβ-NF RLM, the increased metabolism of (-)-THP and (+)-THP were near; 20μmol/L fluvoxamine and ketoconazole had comparatively obvious inhibitory effects on the metabolism of THP enantiomer in mixed-HLM. In MLM, DLM, mLM, Untreated-RLM, Dex-RLM andβ-NF RLM, the Km of (+)-THP and (-)-THP were 33.88,22.41,63.52,47.75, 52.60,31.22,30.39,41.73,31.43,13.31,6.560 and 20.55μmol/L, Clint were 42.99, 71.69,7.570,32.07,31.64,90.23,29.01,77.24,107.6,617.2,274.0 and 222.1μL/min/mg protein; The metabolism of THP enantiomer was primarily mediated by CYP1A2 and CYP3A1/2; ketoconazole and fluvoxamine dose dependently inhibited the metabolism of (+)-THP or (-)-THP in Dex orβ-NF-RLM. The inhibitory rate of THP enantiomer by ketoconazole were higher in Dex-RLM than that inβ-NF-RLM and the inhibitory rate of (+)-THP were higher than (-)-THP,while the inhibitory rate of THP enantiomer by fluvoxamine higher inβ-NF-RLM than that in Dex-RLM and the inhibitory rate of (-)-THP were higher than (+)-THP.Conclusion:THP enantiomer can be stereoselectively metabolized by CYPs in animal liver microsomes and the clearance of (+)-THP were significantly higher than that of (-)-THP. In RLM, the metabolism of THP enantiomer was primarily mediated by CYP1A2 and CYP3A1/2, in addition, CYP3A1/2 was much specialized on metabolism of (+)-THP, while CYP1A2 on (-)-THP.Stereoselective metabolism of tetrahydropalmatine enantiomer in HLM and human recombinant CYP450Objective:To study stereoselective metabolism of THP enantiomer in HLM and rCYP, and to elucidate human CYP450 isoforms mainly mediating stereoselective metabolism of THP enantiomer.Methods:Mixed-HLM, individual HLM with high CYP1A2 activity(IANO-HLM), rCYP3A4 and rCYP1A2 were incubated respectively with THP enantiomer and the concentration of (+)-THP and (-)-THP were determined by RP-HPLC; to determine and compare the enzyme kinetic parameters of (+)-THP and (-)-THP in mixed-HLM and IANO-HLM; to investigate the inhibitory effect of main CYP450 isoforms' inhibitors with the final concentration of 20μmol/L on the metabolism of THP enantiomer in mixed-HLM in order to elucidate CYP450 isoforms mainly mediating metabolism of THP enantiomer; to investigate the inhibitory effect by inhibitors-fluvoxamine and ketoconazole on the metabolism of (+)-THP and (-)-THP in mixed-HLM and IANO-HLM on the basis of preliminary screening.Results:Both in mixed-HLM and IANO-HLM, the metabolism of (+)-THP were significantly higher than that of (-)-THP(P<0.01 or P<0.001); the metabolism of (+)-THP and (-)-THP in IANO-HLM increased significantly compared to mixed-HLM but the increased metabolism of (+)-THP was more than that of (-)-THP; 20μmol/L fluvoxamine and ketoconazole had comparatively obvious inhibitory effects on the metabolism of THP enantiomer in mixed-HLM. the inhibitory rate of THP enantiomer by fluvoxamine were higher in IANO-HLM than that in mixed-HLM and the inhibitory rate of (+)-THP were higher than that of (-)-THP,while the inhibitory rate of THP enantiomer by ketoconazole were higher in mixed-HLM than that in IANO-HLM and the inhibitory rate of (-)-THP and (+)-THP were similar. The metabolism of (+)-THP in rCYP1A2 was more than that of (-)-THP, while the metabolism of (+)-THP and (-)-THP did not show markedly difference in rCYP3A4.Conclusion:THP can be stereoselectively metabolized by CYPs in HLM. In HLM,the metabolism of THP enantiomer was primarily mediated by CYP1A2 and CYP3A4, and that CYP1A2 showed much specialized on metabolism of (+)-THP, while CYP3A4 showed no preference which inferred that the stereoselective metabolism by CYP1A2 and CYP3A were different between HLM and RLM.Inhibitory and inductive effects of (-)-and (+)-tetrahydropalmatine on CYP450 in miceObjective:To investigate the inhibitory and inductive effect of (-)-tetrahydropalmatine (THP) and (+)-THP on main CYP450 isoforms in mouse liver microsomes.Methods:The in vitro inhibitory effect was evaluated by incubating (-)-THP or (+)-THP with the probe substrates of main phaseⅠmetabolic enzymes in mouse liver microsomes, and the remaining substrates were determined by HPLC or LC-MS/MS method. Mice were administrated with (-)-THP or (+)-THP at dosage of 240 mg-kg-1 or 60mg-kg-1 by gastric lavage for successive 7 days, then the cocktail-LC-MS method was applied to assess the activities of main CYP450 isoforms in mouse liver microsomes.Results:The IC50 values of both (-)-THP and (+)-THP on isoforms studied were higher than 100μmol/L except that IC50 value of (+)-THP on CYP2C was 43.89μmol.L-1, indicating weak inhibition of (-)-THP and (+)-THP on CYP1A2, CYP2D22, CYP2E1 and CYP3A11 in vitro. Compared with the vehicle group, the activities of CYP2D22, CYP2E1 and CYP3A11 were not increased significantly in (-)-THP and (+)-THP treatment groups, while the activities of CYP1A2 in 60mg/kg and 240 mg/kg (-)-THP groups were 68.7% and 73.0% higher than that of the vehicle group (P<0.05, P<0.01, respectively), the activity of CYP2C37 in 240 mg/kg (-)-THP treatment group was 80.4% higher than that of the vehicle group (P<0.05). Conclusion:There is negligible or weak inhibition on main CYP450 in mouse liver microsomes by (-)-THP and (+)-THP in vitro. (+)-THP can not induce main CYP450 in mouse liver microsomes while (-)-THP induces CYP1A2 and CYP2C37 to some extent. |
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