Determination Of The CYP1A2 CYP3A4 And CYP2C9 Activities And The Effects Of Chinese Medicine Injection On The Drug-metabolizing Enzymes In Rat Hepatic Microsomes

Objective: At first,a simple and specific high performance liquid chromatography(HPLC) method was established for determination of acetaminophen 、 1-hydroxy midazolam and 4-hydroxy diclofenac.The next, phenacetin、midazolam and diclofenac were chosen as the specific probe drugs of CYP1A2 CYP3A4 and CYP2C9 in hepatic microsomes incubation system, respectively, and thereby to measure the kinetics parameters of the three enzymes in rats.In the end,the effects of five Chinese Medicine Injections,Salviae Miltiorrhizae and Ligustrazine Hydrochloride Injection、Safflower Extract and Aceglutamide Injection、Extract of Ginkgo Biloba Leaves Injection、Shenmai injection 、 Shen fu injection,on the activities of CYP1A2 CYP3A4 and CYP2C9 in rats were also evaluated, through adding a series of different volume percent concentrations of injection to hepatic microsomes incubation systems, determining the generated quantity of acetaminophen 、 1-hydroxy midazolam and 4-hydroxy diclofenac,and calculating their half inhibitory concentrations(IC50), respectively.Methods:Acetaminophen was eluted on a Hypersil BDS C18 analytical column(150mm?4.6mm,5?m)with a linear gradient mobile phase composed of(A)CH3CN-(B)50.0 mmol·L-1 disodium hydrogen phosphate buffer(pH4.5) running at flow rate of 0.8m L·min-1 at 30℃ and the UV detector was set at 245 nm.To 1-hydroxymidazolam,it employed a Hypersil BDS C18 column(150mm×4.6mm,5μm) running at flow rate of 1.0m L·min-1 at 30℃ and the UV detector was set at 230 nm.The mobile phase consisted of(A)Acetonitrile-(B)10.0mmol/L dipotassium hydrogen phosphate buffer(pH8.4).As for 4-hydroxy diclofenac, the chromatographic separation was achieved on a reversed-phase Hypersil BDS C18 analytical column(250mm?4.6mm, 5?m)using an isocratic mobile phase consisting of(A)CH3CN-(B)50.0mmol·L-1 disodium hydrogen phosphate buffer(pH4.5) running at flow rate of 0.8 mL·min-1 at 30℃ and the UV detector was set at 276 nm. After optimizing incubating time and protein concentration of hepatic microsomes incubation system,the Vmax and Km values were obtained from Lineweaver-Burk polt, respectively. A series of different volume percent concentrations of Salviae Miltiorrhizae and Ligustrazine Hydrochloride Injection、Safflower Extract and Aceglutamide Injection、Extract of Ginkgo Biloba Leaves Injection、Shenmai injection or Shen fu injection,were added into hepatic microsomes incubation systems, and the generated quantity of acetaminophen 、1-hydroxy midazolam and 4-hydroxy diclofenac were detected, and the IC50 values of the five injections for inhibiting CYP1A2 CYP3A4 and CYP2C9 metabolism of the specific probe drugs phenacetin 、 midazolam and diclofenac were calculated, respectively. Results: The established liver microsomal incubation system in this paper was verified by positive inhibitor and was well for study. Under the optimum chromatographic conditions, the phenacetin、midazolam、diclofenac and their respective internal standards and metabolites acetaminophen、1-hydroxy midazolam and 4-hydroxy diclofenac were well separated from each other and free from interference from other endogenous substances in the reaction system.The method showed good linearity over range of 0.10~49.90μmol·L-1 for acetaminophen、0.10~29.90μmol·L-1 for 1-hydroxy midazolam and 0.20~29.90μmol·L-1 for 4-hydroxy diclofenac. The enzyme kinetics parameters Km were 102.0 57.0 and 16.9μmol·L-1, and Vmax were 1.05 1.05 and 1.26 nmol·min-1·mg protein-1 for acetaminophen 、 1-hydroxy midazolam and 4-hydroxy diclofenac, respectively. For three compounds, the recovery was greater than 85%, the intra and inter-day precision was less than 10%, the sample stability was less than 10%,and the assay accuracy was between 85%~115%. Both Salviae Miltiorrhizae and Ligustrazine Hydrochloride Injection and Safflower Extract and Aceglutamide Injection had no inhibition effect on CYP1A2 CYP3A4 and CYP2C9.The IC50 values of Extract of Ginkgo Biloba Leaves Injection for inhibiting CYP3A4 and CYP2C9 metabolism of midazolam and diclofenac respectively, were 11.48% and 12.91%。The IC50 value of Shenmai injection for inhibiting CYP2C9 metabolism of diclofenac,was 3.68%.The IC50 values of Shen fu injection for inhibiting CYP1A2 CYP3A4 and CYP2C9 metabolism of phenacetin、midazolam and diclofenac respectively, were 4.27%、6.65% and 5.23%.Conclusions: The HPLC method developed in the present study for determination of the metabolic products acetaminophen、1-hydroxy midazolam and 4-hydroxy diclofenac is simple, reliable, and specific, and is validated to fully meet requirements for enzyme kinetics study of drug-metabolizing P450 enzymes CYP1A2 CYP3A4 and CYP2C9. The results demonstrate that: Salviae Miltiorrhizae and Ligustrazine Hydrochloride Injection doesn’t show any inhibitory effect on CYP1A2 CYP3A4 and CYP2C9 in vitro,obviously.Safflower Extract and Aceglutamide Injection has the same effect.Extract of Ginkgo Biloba Leaves Injection shows an slight inhibition effect on CYP3A4 and CYP2C9 in vitro. And Shen Mai Injection in vitro also hasn’t be found in obvious inhibition effect on CYP1A2 and CYP3A4,while has inhibition effect on CYP2C9.But Shen Fu Injection has inhibition effect on CYP1A2、CYP3A4 and CYP2C9 in vitro.