Development Of Analytical Method Of Clozapine Metabolites And Its Metabolites Profiling In Rat And Human Liver Microsomes

In recent years, drug metabolism analysis after rapid development has become an important part of kinetic studies in drug metabolism, research and fluid constantly improve the hyphenated technique is widely used in drug metabolism in the analysis. Compared to the traditional liquid chromatography technology, has the advantages of high sensitivity, high throughput and high accuracy. Method for analysis of drug metabolites in this paper aims to establish a new sensitive, high-throughput, high accuracy, and is used for the analysis of metabolites in liver microsomes of clozapine in human and rat. At the same time, CYP enzyme produced main metabolites for catalytic clozapine were further research.The first chapter outlines the development of drug metabolism and its analysis method, reviewed the existing detection methods of MRM and MIM, then discusses the application of MRM and MIM method in the detection of drug metabolites, and the current research situation of CYP enzyme leading clozapine metabolism.The second chapter analysis method using the new DE-DD (dynamic exclusion and data dependent scanning), metabolites of propranolol, Vera Pammy and amlodipine in rat liver microsomes, and has reported the literature comparing the accuracy, the detection method; analysis of metabolite of clozapine in rat and human liver microsomes. Using the new method, and infer the possible metabolic pathway, metabolites of newly discovered. All the test drugs were incubated in liver microsomes, in the condition of 37℃ by oscillating water bath, the use of methanol at incubation time after termination of the reaction, the supernatant of centrifuged sample, sample concentration under nitrogen, water -0.1% formic acid water, acetonitrile -0.1% formic acid as the organic phase, the flow rate was 0.6 mL/min, Thermo Hypersil-C18 (2.1 X,50 mm,1.9μm) column with gradient elution. The Thermo LTQ XL linear ion trap mass spectrometer, model the electrospray ionization source (HESI), a full scan monitoring (Full scan), at the same time two pieces and three pieces of scanning. The DE-DD method results, and the results associated with the reported literature comparison, determine the reliability and accuracy of the results. This method can find out the simulation of the metabolites produced in the metabolism experiment in vitro drug comprehensively, and also can obtain the metabolites of multistage fragmentation information, save the sample analysis times; can quickly, the metabolite analysis for unknown compounds in accurate and efficient access to metabolism of spectrum fragmentation information, provide rich information for mass spectrometry metabolite identification step.In the third chapter, we use the DE-DD analysis method analysis of clozapine in human and rat liver microsomes incubated samples, samples were incubated with experimental operation and propranolol, Vera Pammy and amlodipine in the same. According to the results, we do analysis the differences in liver microsomes of clozapine in human and rat. The results infer the possible metabolic pathway, compared with clozapine in different metabolic differences among genera. According to the experimental results, there are great difference in rat and human liver microsoems of clozapine. One of the major metabolite of clozapine in rat liver microsomes is demethylation of clozapine. And clozapine in human liver microsomes the major metabolites is N-oxide clozapine. At the same time, glucuronidation metabolite of clozapine is unique in human liver microsome; metabolism to methyl oxidation of clozapine and clozapine as unique open-loop oxidation in rat liver microsomes.In the fourth chapter, we selected human liver microsomes in vitro metabolism as a model, simulation experiment, to find the catalysis of clozapine in human liver microsomes in the main metabolites of CYP isoforms. So the CYP enzyme on catalytic clozapine metabolism screening experiment, select 1A2,2B6,2C8,2C9, 2D6,2C19, and 3A4 as the representative of the CYP isoforms of incubation, the 7 subtypes of basic accounting for more than 92% of CYP450, thus to obtain more reliable results can be. At the same time, considering two cases containing inhibitors and containing no inhibitor. Through the sample analysis, summarize the obtained experimental data, show that in the single subtypes of CYP enzyme incubation system, the main enzyme leading clozapine metabolism in human liver microsomes was 1A2. 2D6 and 3A4 are also involved in the metabolism of clozapine, but not clozapine metabolism key enzyme catalysis.1A2 mainly catalyzed demethylation of clozapine, 2D6 is involved in the catalytic demethylation of clozapine, and 3A4 is involved in the catalytic oxidation of clozapine.In the fifth chapter, we treat all the experimental results are summarized and discussed, draw a conclusion. Metabolites established in this theory analysis method is sensitive, efficient, fast, can be used to judge the metabolites in vitro drug and metabolite analysis of structure, metabolic abundant information for the drug development process. At the same time through the analysis of the metabolites of clozapine in vitro, found that clozapine in human liver microsome metabolism pathway leading to methylation and N-oxidation, the demethylation as the main way, through screening using CYP enzyme found the demethylation pathway is catalyzed by CYP1A2; the main enzyme CYP3A4 and CYP2D6 not catalytic clozapine metabolism, but CYP3A4 is involved in the catalytic oxidative metabolism of N-, CYP2D6 in the catalyzed demethylation.