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The Correlation Research between the Gene Polymorphisms of Five CYP450 Isoforms of Enzymes and the Differences of Metabolic Ratio of Cocktail Probes

Author: LiNa
Tutor: ShiXiaoJin
School: Fudan University
Course: Pharmacy
Keywords: Cytochrome P450 High performance liquid chromatography - tandem mass spectrometry Mixed probe Substrate method Gene polymorphism
CLC: R969.1
Type: Master's thesis
Year: 2011
Downloads: 157
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Cytochrome P450 enzymes (cytochrome P450, CYP450), mainly in the liver microsomes, plays a very important role in the biotransformation of exogenous compounds and endogenous substances, according to the amino acids in the primary structure of the enzyme protein homologous degrees, CYP is divided into 18 families and 44 subfamilies, oxidative metabolism of more than 90% of the drug in the clinical mainly mediated by five isozymes CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4's. There are many factors affect drug metabolism activity, environmental factors such as food and environmental toxicants; physiological factors such as age and gender; pathological factors such as liver, kidney or heart disease, as well as genetic factors. Important factors affecting the activity of genetic factors, gene polymorphism is the main source of causing of drug metabolism racial and individual differences. Therapeutic doses of drugs in poor metabolizers possible occurrence of serious adverse reactions; metabolizers who may be invalid, would not achieve the treatment of the disease. Therefore, the clinical need to predict a patient's drug-metabolizing enzyme activity, implementation of individualized treatment programs. The study predicts that activity There are two main methods: the probe substrates Law and gene analysis. More mixed probe substrate method of application, the advantage of multiple metabolic pathways can be obtained in a single test process, ask and intra-individual variability of drug-metabolizing enzyme activity can reduce individual shorten the analysis cycle, reduce experimental costs However, this method can not be applied to the liver and kidney dysfunction, adverse reaction of the crowd on the probe substrates, and there are ethical and moral requirements of the problem, analytical methods. Genotyped Just a few plasma, cheap, easy to operate, can be more easily used in clinical, but the need to pre-identification of genotyping affect metabolism. It is necessary to carry out personalized medicine genotype, can reduce the incidence of adverse drug reactions, medication effects, and save resources. The various subtypes of enzymes having a plurality of mutant alleles need to explore the relationship between gene polymorphism and the enzyme activity, and determine the functional changes of the enzyme caused by a variety of mutations, by detecting the individual's genotype to predict alkylene type enzyme on drug metabolism, thus optimizing the dosing regimens, so that the individual administration. Subjects of clinical drug trials screening provides a feasible method to the exclusion of clinical trials in poor metabolizers, can improve the safety and effectiveness of clinical drug trials. The first part of the present study to establish a simultaneous determination of the probe substrate and its metabolites in plasma CYP450 enzymes toluene of tolbutamide / 4 - hydroxy tolbutamide (CYP2C9), omeprazole / 5 - hydroxy Ogilvy omeprazole (CYP2C19), dextromethorphan / the right brown alkyl (CYP2D6), midazolam midazolam / 1'-hydroxy midazolam alprazolam (CYP3A4) concentration the LC-MS/MS analysis methods. Plasma sample pretreatment methods blown dry with nitrogen the acetonitrile directly precipitated protein reorganization. The chromatographic conditions were as follows: Column: CAPCELL PAK C18 column (MG Ⅲ, 100mm × 2.0mmID, 5μm) Mobile phase A: water / formic acid (100:0.05, v / v), mobile phase B: acetonitrile / formic acid (100:0.05 v / v), using gradient elution program: 0 for 5 min, B% (10% to 90%); 5 to 5.5 min, B% (90%); B, 5.5 to 6 min (90% -10% ); 6-11 min, B% (90%). The flow rate of 0.3 mL / min, column temperature was room temperature, and the injection volume was 10μL electrospray ion source (ESI) triple quadrupole tandem mass spectrometry, a multistage reaction monitoring quantitative positive and negative ions separate scanning. Omeprazole / 5 - hydroxy-omeprazole, dextromethorphan / right brown alkyl, midazolam / 1'-hydroxy midazolam alprazolam concentrations of positive ion monitoring, internal standard non-phenacetin; toluene tolbutamide / 4 - hydroxy tolbutamide anion monitoring internal standard chlorzoxazone. Results of omeprazole in the 0.4-40 ng / mL, 5 - hydroxy omeprazole, dextromethorphan, 1'-hydroxy midazolam, 4 - hydroxy toluene sulfonamide Ding urea 0.2 ~ 20 ng / mL, right brown alkyl tolbutamide O.1-10 ng / mL, midazolam in the 0.3-30 ng / mL range linear relationship was good (r gt; 0.999). Analyte batch and batch asked were less than 15% precision and accuracy were in the range of 85% -115%. The method is simple, rapid, accurate, sensitive, simultaneous determination of CYP450 subtypes probe substrate of the enzyme and its metabolites, may promote mixed probe substrate method widely used to meet the requirements of high-throughput analysis. The second part of the mixed probe substrate method of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 enzyme activity. Select caffeine, omeprazole, dextromethorphan, tolbutamide and midazolam midazolam as a probe substrate, respectively, for CYP1A2, CYP2C19, CYP2D6, CYP2C9, CYP3A4 activity and phenotype analysis. 3h screened out in 50 healthy subjects and healthy subjects, while giving the above-mentioned five kinds of probe substrates, 5h, 24h blood samples were collected, and then with a first portion established by LC-MS/MS analysis method in the plasma was measured EXPLORATION needle substrate concentration and its metabolites to predict drug metabolizing enzyme activity. Results no interaction between these five drugs, subjects are within the normal range throughout the trial period of the monitoring indicators, no adverse events occurred. In addition to the caffeine and the sub xanthine not accurate quantitative determination of the blank plasma interference large blood sample measurement, the rest of the analyte were accurate determination of the measured plasma concentration of the 50 subjects the body of the analyte, lay the foundation for the next step of the research. Probe substrates used in this study a combination of caffeine omeprazole River dextromethorphan toluenesulfonamide Ding urea midazolam alprazolam simultaneous determination of the body of CYP1A2, CYP2C19, of CYP2D6, CYP2C9, CYP3A4 activity, sampling convenient, can be used to co-workers enzyme activity and phenotypic studies as well as for the evaluation of the interaction between the drug. Third part CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A (?) Gene polymorphism with probe substrate metabolism differences. CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 subtypes enzyme sites of major mutations in the Chinese population genotyping study of 50 healthy subjects by direct sequencing. Investigated the frequency distribution of these mutation sites in the study population, and research the of these subtypes enzyme mutation sites in different genotypes of each probe substrate metabolism in the body. Mutant gene frequency distribution of the study of gene polymorphisms (SNPs) in the study population were CYP1A2 * 1B (10%), CYP1A2 * 1C (31.9%), CYP1A2 * 1D (16.4%), CYP1A2 * 1F (59 %), CYP2C9 * 3 (6%) CYP2C9 * 13 (0%), CYP2C19 * 2 (20.8%), CYP2C19 * 3 (8.2%), CYP2D6 * 10 (64.1%), CYP3A4 * 1G (26.7%), CYP3A4 * 15 (0%), CYP3A4 * 18 (2%). CYP2C9 * 3 of CYP2C19 * 2, CYP2D6 * 10 mutated sites can cause the reduction of the corresponding activity of, carrying these loci mutant gene populations in clinical medicine to reduce the dose, in order to reduce the occurrence of adverse reactions. CYP1A2 * 1B, CYP1A2 * 1C, CYP1A2 * 1D, CYP1A2 * 1F, CYP2C19 * 3, of CYP3A4 * 1G of CYP3A4 * 18 locus mutations in genes for each activity of no effect, CYP2C9 * 13, CYP3A4 * 15 was not detected to, unable to determine its impact on the activity of the mutant gene. The research results will achieve clinical genotype individual administration to provide the basis for, and help to promote the rational use of drugs in clinical, to ensure the safety of medication.

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CLC: > Medicine, health > Pharmacy > Pharmacology > Journal of Clinical Pharmacology > Pharmacokinetic
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