Study On In Vivo Bactrian Camel's CYP3A Enzyme With Specific Probe Drug

Bactrian camel is an unique animal species living in desert and Gobi desert.Due to the long-term volutionary process in harsh and extreme environments,the Bactrian camel is given a lot of unique biological characteristics,such as properties of salt tolerance,resistance to hunger and thirst,resistance to low air pressure and scorching hot-day.As well as,Bactrian camels like to eat plants with a few protein content and rich cellulose,also like to eat the plants with high salty content and some kind of poisonous plants.CYP enzymes are superfamily enzymes responsible for metabolism of many xenobiotics.endogenous substance and drugs.CYP3A plays important role in metabolism of about 50%of drugs.In order to study the characteristics of enzyme kinetics and its sensitivity to the specific inhibitors.the Bactrian camel CYP3A enzyme was investigated by specific substrate method in viivo.Firstly,To provide methodological and technical support for studying the in vivo activities of Bactrian camel CYP3A enzyme,the high performance liquid chromatography-Ultra Violet(HPLC-UV)method for determination of Midazolam concentration in Bactrian camel plasma was established in this study.Analyses were performed on Shimadzu LC-2010 AHT HPLC system fitted with a C18 column(4.6mm×150mm.5?m.Sigma),using the mobile phase consisting of acetonitrile:0.01M PBS=60:40(v/v)at a flow rate of 0.5 mL/min.maintained at 30?.The UV detection wavelength was set at 254 nm and all injection volumes were 10?L.A linear relationship was found in the concentration range of 0.39?25?g/mL(R2=0.999,n=7).Both the relative and absolute recovery were met the requirements of HPLC detection under the high,medium and low concentrations of Midazolam.The established HPLC detection method for determination of Midazolam concentration in Bactrian camel plasma possess a characteristics of high sensitivity.good stability and good separation with no miscellaneous peaks.Secondly,To study on the activity of CYP3A enzyme in Bactrian Camels by investigating the pharmacokinetics of CYP3A-specific probe drug Midazolam in Bactrian Camel,and studying the effect of Itraconazole on the pharmacokinetic behaviors of Midazolam.Five healthy adult Bactrian Camels in group specific substrate only were injected intramuscularly the Midazolam with single dose of 0.1mg/kg,then the blood samples were collected at time 0 min,5 min,15 min.30 min,1 h,2 h,4 h,6 h,12 h,and samples were collected at time 0 min,5 min,15 min,30 min,1h,2 h,4 h,6 h,12 h,and 24 h following the drug administration.After 7 days drug clearance periods,each camel in group enzyme inhibitor plus specific substrate is injected intramuscularly of single dose of 0.1mg/kg Itraconazole for 4 consecutive days,once a day.2 h after the last injection,Bactrian Camels were received the Midazolam intramuscularly with a single dose of 0.1mg/kg.Then blood samples were collected at same time intervals as above.The plasma Midazolam concentration was determined by high performance liquid chromatography-Ultra Violet detection,and the pharmacokinetic parameters of Midazolam were analyzed by Phoenix WinNonLin 7.0 analysis.The main pharmacokinetic parameters of Midazolam were as follow:for group specific substrate only,T1/2 is 2.5±0.073 h,Tmax is 0.85±0.09 h,Cmax is 0.62±0.12 ?/ml,AUC0-t is 1,47±0.35 h?g/mL,Vd is 259.17±41.29 mL/kg,CL is 53.46±14.25 mL/h/kg,and MRT is 3.71±0.16 h,respectively;for group enzyme inhibitor plus specific substrtate,T1/2 is 3.674±0.29h,Tmax is 0.54±0.06h,Cma,is 0.8±0.06?g/mL,AUC0-t is 2.15±0.15h·?g/mL,Vd is 152.09±22.49mL/kg,CL is 34.38±5.13mL/h/kg.and MRT is 4.60±0.52 h,respectively.The CYP3A enzyme of Bactrian Camels has a strong degradation activity against Midazolam.After intramuscular administration of Itraconazole,finding suggest that the injection of CYP3A enzyme inhibitor Itraconazole were significantly inhibited CYP3A activity by administration of Bactrian Camels.Therefore,a stable,reliable,sensitive and specific HPLC for CYP3A enzyme-specific substrate was successfully established.The first time,through the effect of Itraconazole on pharmacokinetics of Midazolam in bactrian camel,and studying on in vivo activities of bactrian camel's CYP3A Enzyme with specific probe drug.The results that can fill the gaps in the in vivo activity of CYP3A in bactrian camel,and provide a basis for the study of toxic plant metabolism in the subsequent bactrian camel.