| CYP3A is the main member in Cytochrome P450 famliy, CYP3A4 and CYP3A5 have high homology and belong to CYP3 A. The content of CYP3A4/5 is very rich in liver and they take part in the metabolism of many substances that includes drug and toxic. There are a lot of studies about CYP3A4/5 because this enzyme has a big indivisual difference of metabolic activity, most of these studies are involved in individual difference of CYP3A4/5 metabolic activity. In the early, it is considered that genetic polymorphism could affect enzyme activity and many environmental factors also contribute to individual difference,such as somking, drinking and drug interactions. With the further research, recent studies have found some factors of epigenetics and protein-protein interaction that can affect the enzyme activity of CYP3A4/5. The three-dimensional structure of CYP3A4/5 is flexible and can take part in the metabolism of many drugs, due to the diversity of substrates, thus a sigle substrate does not reflect the metabolic activity of CYP3A4/5. In view of this, FDA recommends that explore the activity of CYP3A4/5 need to use two probe drugs(midazolam and testosterone). But we have not seen that some studies use large sample of normal human liver microsomes to research the activity and correlation of midazolam and testosterone metabolsimed by CYP3A4/5. Most of related research is studied in human body and recombinant enzyme system, the former can’t reflect the process of CYP3A4/5 metabolism in a single environment.The latter and the environment in human body has a certain difference. Therefore, human livermicrosome is a better metabolic platform for vitro research that study the metabolic activity of CYP.This research use the normal human liver microsomes for some related experiments in order to investigate the metabolic activity of CYP3A4/5 metabolism midazolam and testosterone. In order to study the relativity between the two drugs that are the probe drugs of CYP3A4/5. Firstly, we analyze the activity of CYP3A4/5metabolism midazolam and testosterone. And then, we get the polymorphism of CYP2D6 and CYP3A4/5 and analyze the effect of a series of factors includes genetic polymorphism and environment on the enzyme kinetics parameters of midazolam and testosterone in order to provide theoretical basis for clinical use of some drugs that metabolismed by CYP3A4/5.Method1 Collection of human liver samples Human liver samples(n=105) from human donors with hepatic hemangiorma from First Affiliated Hospital of Zhengzhou University and People’s Hospital of Henan Province, including 37 male samples and 68 female samples. The age of patients ranged from 20 to 75 years. The history of medicine showed that drugs can have an effect on CYP enzyme activity had not be taken recently.2 Preparation of human liver microsomes(HLMs)All human liver microsomes were prepared by ultracentrifuge and the protein level of human liver microsomes was determined by Bradford method.3 Determination of enzyme kinetic parameters of midazolam and testosterone in HLMs Firstly, we determine the optimal incubation conditions including the incubation time and microsomal protein. Next, we pre-incubate HLMs incubation mixtures contain phosphate buffer, probe(midazolam or testosterone) for 5 minutes,put NADPH into this mitures to initiate this reaction. After a few minutes, we stop this reaction and take centrifugal supernatant for HPLC.4 Genotyping for CYP3A4/5 and CYP2D6 gene DNA was isolated with DNA extraction kit from liver tissues. The samples were using PCR and direct sequence for analysis of polymorphisms of CYP3A4/5and CYP2D6.5 Statistical analysis Statistical analysis was performed with SPSS 17.0 software and enzyme kinetic parameters was performed by Graph Pad Prism 5.0.Result1 Enzyme kinetic parmeters of CYP3A4/5 in human liver microsomes Use midazolam and testosterone as substrate. The enzyme kinetic parameter of CYP3A4/5 metabolic midazolam ranged from 0.44 to 10.20 μM for Km, from 9.40 to5035.00 pmol·min-1·mg-1 protein for Vmax, from 8.27 to 1673.53 μL·min-1·mg-1protein for CLint in HLMs of 105 normal samples.The data displayed a 23.18, 72.55 and 202.36 fold inter individual difference. The enzyme kinetic parameter of CYP3A4/5 metabolic testosterone ranged from 22.19 to 152.50μM for Km, from265.60 to 10679.00pmol·min-1·mg-1 protein for Vmax, from 1.74 to 278.09μL·min-1·mg-1 protein for CLint in HLMs of 105 normal samples. The data displayed a 6.87, 40.21 and 159.82 fold inter individual difference.2 The correlation of enzyme kinetics between midazolam and testosterone Use Spearman inspection of the enzyme kinetics of midazolam and testosterone,correlation analysis results show that the positive correlation on Vmax and CLint, r is0.643 and 0.245 respectively, and the Km has no obvious correlation.3 The effect of genetic polymorphism on enzyme kinetics of midazolam andtestosterone3.1 The effect of genetic polymorphism of CYP3A4/5 on enzyme kinetics The mutation of CYP3A4 20230 G>A would increase the Km and Vmax of midazolam, however, there is no effect on testosterone.The mutation of CYP3A56986A>G would decrease the Km and Vmax of midazolam,and decrease the Vmax of testosterone.3.2 The effect of genetic polymorphism of CYP2D6 on enzyme kinetics The results showed that the genetic polymorphism of CYP2D6 1661G>C has an effect on Km of midazolam, has no effect on CLint and Vmax. Genetic polymorphism of CYP2D6 100C>T has an effect on Vmax of testosterone, has no effect on CLint and Km.3.3 Effects of smoking, drinking history, sex and age on enzyme kinetic parameters of CYP3 A in human liver microsomes The analysis indicated that smoking, drinking history, sex and age of normal samples had no effect on the enzyme kinetic parameter of CYP3A4/5 in HLMs.Conclusions1.There is an obvious individual difference of CYP3A4/5 activity in HLMs.2.There is a good correlation for Vmax between midazolam and testosterone, and a weak correlation for CLint, however,there is no correlation for Km.3.The mutation of CYP3A4 20230 G>A would increase the Km and Vmax of midazolam, however, there is no effect on testosterone.4. The mutation of CYP3A5 6986A>G would decrease the Km and Vmax of midazolam, and also decrease the Vmax of testosterone. |
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