Study On The Metabolism Of MBZ In Liver Microsomes

The Pre-clinical metabolism is a necessary process for clinical study of a new drug. The in vitro metabolism is very meaningful for drugs, which can clarify the structure of the metabolites, identify the enzymes which involved in the metabolism, evaluate the drug-drug interactions, and judge the safety. MBZ is a candidate for cardiovascular drugs. The preliminary study on pharmacokinetics and pharmacodynamics and toxiclogy for MBZ has been completed. The results were indicated of its unique advantages in cardiovascular effects. This trial clarified the mechanism of action of MBZ, evaluated the drug-drug interactions, which can provide the theory for rational drug use. The author's main contributions were as follows:1. Establish a method for preparation and detection the rat liver microsomes and human liver microsomes. The author made the rat liver microsomes and human liver microsomes both by differential centrifugation and test the protein content of the microsomes by Bradford. The protein content of the rat liver microsomes was 14.58±0.55 mg/mL. The protein content of the rat liver microsomes was 20.09±0.24 mg/mL. And the author detected the enzyme activity by fluorogenic dectection. The result was that when the protein content of the microsomes is from 0.125 mg/ml to 1.5 mg/ml, the reaction time within 60 min, both the rat liver microsomes and human liver microsomes are activity.2. Preliminary study the MBZ metabolism by the in vitro metabolism mode. And established a HPLC method for analysis of MBZ in liver microsomes. The results showed that the linear ranges of MBZ in rat liver microsomes is 0.125μL-50μL. The recovery of low, medium and high concentration were all above 90%. The RSD of precision and stability were all less than 5%.Followed by this HPLC method to evaluate the metabolites in rat liver microsomes and to calculate Km, Vmax and CLint. For M1', the Vmax is 52.87±1.24 AUC/min/nmol, the Km is 26.99±1.02μM, the CLint is 1.95±0.06; For M2',the Vmax is 23.84±0.87 AUC/min/nmol, the Km is 18.82±0.67μM, the CLint is 1.26±0.03; For M3', the Vmax is 114.8±1.56 AUC/min/nmol, the Km is 16.87±0.36μM, the CLint is 6.82±0.07. And evaluated the metabolites in human liver microsomes and calculated Km, Vmax and CLint. For M1, the Vmax is 3.927±0.014 AUC/min/nmol, the Km is 23.634±0.05μM, the CLint is 0.166±0.002; For M2, the Vmax is 1.092±0.006 AUC/min/nmol, the Km is 7.965±0.011μM, the CLint is 0.137±0.004; For M3, the Vmax is 4.486±0.005 AUC/min/nmol, the Km is 28.839±0.03μM, the CLint is 0.156±0.001.3. Identify the enzymes which involved in the metabolism of MBZ in human liver microsomes. Followed by specific inhibitor method to identify the CYP 2C8 is the enzymes which involved in the metabolism of MBZ. Then discuss the safety of MBZ in rational use.4. Preliminary clarify the structure of the metabolites, by the HPLC-MSn method. The result suggested that the molecular weight of the metabolites M1', M2', M3'are all 322.3. The Fragment mass chromatogram showed that three metabolites have the similar structure.