The Correlation Research Between The Gene Polymorphisms Of Five CYP450 Isoforms Of Enzymes And The Differences Of Metabolic Ratio Of Cocktail Probes

Cytochrome P450 enzymes (CYP450) are mainly in the liver microsome. They play an important role in the biotransformation of exogenous and endogenous compounds. CYP450 are divided into 18 families and 44 subfamilies, including CYP1,CYP2,CYP3 families which have 8 to 10 isozymes, respectively. More than 90% drugs in clinical are metabolismed by CYP1A2,CYP2C9,CYP2C19,CYP2D6,CYP3A4 isozymes. There are many factors influence the activities of drug metabolism enzymes, such as drug, toxins and other environmental factors; age, gender and other physiological factors; liver, kidney or heart disease pathological factors and genetic factor.The most important factor is genetic factor. The gene polymorphisms are the main source for the differences of drug metabolism among the ethnics and individuals. CYP450 gene polymorphisms cause the differences of drug metabolisms among the individuals, the identical dose of the same drug may cause serious adverse effects in poor metabolizers, but in the extensive metabolizers the concentration of drug is so low that does not reach the effective therapeutic concentration. As a result, the activity of drug metabolism enzyme of patient in clinic should be predicted to realize the individual treatment. There are two research methods to predict the enzyme activity. One is the "cocktail" probe drugs method and the other is the gene analysis method. The advantages of the "cocktail" probe drugs method are more than one metabolic pathway information can be obtained in a single test process, reducing the variations of drug metabolizing enzymes among individuals, reducing time and cost. However, the people of liver and kidney dysfunction, and people who have adverse reactions to drugs limited the application of probe drugs. In addition, the problems of ethics and the high analytical methods requirement also should be considered. However, the gene analysis method is cheap and little plasma will meet the need of gene analysis, moreover, the method has small individual variability. Gene analysis method can be more convenient for clinical application.The individual treatment base on the genotype is necessary. It can be reduce the adverse drug reaction, improve the drug treatment and conserve the resource. Each isoenzyme has many mutation alleles, the relationship between the genetic polymorphism and enzyme activity need to be explored to determine the change of enzyme function which caused by the gene mutations. The individual administration can be realized by predicting the enzyme activity base on the individual genotype test. The gene analysis method is also an effective method for screening the healthy volunteer in clinical trial, that the exclusion of the poor metabolizers in clinical trial can improve the safety and efficiency of the trial. The part one developed a LC-MS/MS analytic method to simultaneously determine the five probe parent drugs and their metabolites of cytochrome P450 (CYP450) including tolbutamide/4-hydroxytolbutamide(C YP2C9),omeprazole/5-hydroxyomeprazole(C YP2C19), dextro-methorphan/dextrorphan (CYP2D6) and midazolam/1'-hydroxymidazolam (CYP3A4) in human plasma. The pretreatment of sample is directly protein precipitated with acetonitrile then the analytes are restituted after being dried by nitrogen. An aliquot (10μL) was injected into a CAPCELL PAK C18column (MGⅢ,100 mm×2.0 mm ID,5μm) which were kept at room temperature. The flow rate was 0.3 mL/min. The mobile phase consisted of 0.05% formic acid in acetonitrile (B) and 0.05% formic acid in water (A). During the gradient elution step (0～5 min), the solvent composition of mobile phase B was increased linearly from 10% to 90%, then maintained to 90% for 0.5 min, and reduced from 90%(B) to 10%(B) in 0.5 min, then maintained the 10% (B) until the end of the analytic time. The column was then allowed to re-equilibrate at initial conditions. The complete analytic run was lasted for 11 min. A 3200 QTrap triple-quadrupole mass spectrometer, equipped with an electrospray ionization (ESI) source, was used for the mass analysis and detection. The positive and negative ions were respectively determined by the multiple reaction monitoring (MRM).Omeprazole/5-hydroxyomeprazole, dextromethorphan/dex-trorphan, midazolam/1'-hydoxymidazolam were determined in positive ionization mode with the internal standard phenacetin; whereas, tolbutamide/4-hydroxytolbutamide were determined in negative ionization mode with the internal standard chlorzoxazone. The result indicated that the calibration curves for each analyte were linear (r>0.999) over the range of 0.4～40 ng/mL for omeprazole; 0.2～20 ng/mL for 5-hydroxyomeprazole, dextromethorphan,l'-hydroxy-midazolam and 4-hydroxytolbutamide; 0.1～10 ng/mL for dextrorphan and tolbutamide; 0.3～30 ng/mL for midazolam. The precision of intra and inter batches for each analyte were less than 15%, the accuracy of intra and inter batches for each analyte were in the range of 85%～115%. The method is simple, quick, accurate and sensitive to determine eight parent drugs and metabolites at a time. It can promote the "cocktail" probe drugs widely used in future, and meet the requirements of high-throughput analysis.The part two, we select caffeine, omeprazole, dextromethorphan, tolbutamide and midazolam as the probe drugs for the research activities of CYP1A2, CYP2C9, CYP2C19,C-YP2D6, CYP3A4 enzymes. Fifty healthy volunteers were selected and the plasma samples were drawn at 3,5,24h after administration of five probe drugs simultaneously. The concentration of probe parent drugs and their metabolites in plasma were determined by the LC-MS/MS method developed in the part one. The result indicated that five probe drugs were safe to human and had no drug interactions. All the monitoring indexes for the subjects were in the normal range and no adverse events were happened during the whole research period. Each of analyte was determined precisely except the caffeine and paraxanthine due to the interferences in bank human plasma. The concentration of each analyte in plasma laid the foundation for further research. The "cocktail" probe drugs of caffeine, omeprazole, dextromethorphan, tolbutamide and midazolam determine the activities of five CYP450 enzymes simultaneously, this method is quick and convenient. This cocktail has the potential to become a useful tool to determine the activity of enzyme and research the phenotype, as well as in the detection of clinically important drug-drug interactions during drug development.The part three mainly studied the relations between the gene polymorphisms of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 enzymes and the differences of probe drugs metabolisms. The DNA direct sequencing of single nucleotide polymorphisms (SNPs) of the important mutation alleles of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 enzymes in the fifty Chinese healthy volunteers. The genotype frequencies and allelic frequencies of the important mutation alleles of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 enzymes and the difference of probe drugs metabolisms among the different genotypes were investigated in the the above mentioned volunteers. The frequencies of distribution of mutation alleles of the CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 enzymes are 10% for CYP1A2*1B,31.9% for CYP1A2*1C,16.4% for CYP1A2*1D,59% for CYP1A2*1F, 6% for CYP2C9*3,0% for CYP2C9*13,20.8% for CYP2C19*2,8.2% for CYP2C19*3, 64.1% for CYP2D6*10,0% for CYP3A4*15,26.7% for CYP3A4*1G and 2% for CYP3A4*18. The mutation sites of CYP2C9*3, CYP2C19*2, CYP2D6*10 can reduce the activities of the enzymes. The patients who carry these mutation alleles should reduce the dose of drug in clinical in order to avoiding the drug adverse reactions. There are no relation between the activity of drug enzymes and mutation alleles of CYP1A2*1B, CYP1A2*1C, CYP1A2*1D, CYP1A2*1F, CYP2C19*3, CYP3A4*1G, CYP3A4*18. The influence of CYP2C9*13, CYP3A4*15 on the activity of enzyme was not determined because no mutation alleles were found in the subjects. The result will provide the basis for the individual drug administration base on the genotype, and promote the rational use of drug in clinical to ensure the safety of drug.