Activities Of Bactrian Camel CYP3A Enzyme In Vitro And Its Influence On Metabolism Of Probe Drugs

CYP3A enzyme participates in the biotransformation of endogenous and exogenous substances, and plays an important role in regulating the interaction of body and external environment to maintain homeostasis. Bactrian camel is the national secondary protected animals, but its unique biological characteristics about adapting to the bad surroundings, as well as the ability to metabolize exogenous drugs and poisons, and other researches were relatively backward. The experiment was carried out to study activities of Bactrian camel hepatic CYP3A enzyme and its influence on exogenous drug metabolism.Firstly, the hepatic microsomes were prepared by the Ca2+precipitation method, and Cytochrome P450enzymes were preliminarily studied by the biochemical methods. The results showed that protein contents of hepatic microsomes detected by BCA method were1.936±0.052mg/mL, the contents of Cytochrome b5and Cytochrome P450detected by differential spectroscopy were0.248±0.070nmol/mg and0.397±0.080nmol/mg, and the activities of CYP3A enzyme evaluated by erythromycin-N-demethylase were1.375±0.062nmol·mg-1·min-1Secondly, midazolam (MDZ) was used as probe drug and incubated with Bactrian camel hepatic microsomes for15min, and then the reaction was terminated by ice-cold methanol mixed with the internal standard diazepam (DZP). The dynamic contents of reaction product1’-Hydroxymidazolam (l’-OHMDZ) were detected by the high performance liquid chromatography with UV method (HPLC-UV). Mobile phase are acetonitrile:PBS (0.01mol, pH7.0)=40:60, elution last20min with the same concentration. Detection wavelength is254nm, and a flow rate is0.8mL/min. Column temperature was30℃, and injection volume is20μL. The retention times of l’-OHMDZ, MDZ and DZP were6.710min,11.383min, and15.263min, chromatographic peaks of each component were completely separated. Bactrian camel hepatic microsomal incubation system was optimized by the above method, and the results showed the optimum concentration of substrate MDZ was7.073μmol/L, optimal concentration of hepatic microsomal protein was0.050mg/mL, and optimal incubation time was15min. And then some pharmacokinetic parameters were detected, and the results showed the maximum reaction speed was0.380±0.028pmol-min-1·mg-1, michaelis constant was9.603±3.229 μmol/L, intrinsic metabolic clearance rate was0.041±0.014μL-min-1·mg-1.At last, the experiment of ketoconazole (KET) influence on the activities of Bactrian camel hepatic microsomes CYP3A was carried out, and found that KET inhibited Bactrian camel hepatic microsomes CYP3A, which was intense and irreversible competitive inhibition, and50%inhibiting concentration was0.168±0.068μmol/L.Bactrian camel hepatic microsomes were prepared successfully for further study of drug metabolism in vitro. The HPLC-UV methods were established for detecting the midazolam, the specific probe drug of CYP3A enzyme, and its metabolic product, as well as, the metabolic activities and characteristics of CYP3A enzyme in vitro were preliminary studied. All of these results would lay a good foundation for further research of activities of Bactrian camel CYP3A enzyme in vivo and the other CYP subfamilies in vitro, and filled the scientific gap of this study field.