Research Of Atypical Antipsychotics Represented By Quetiapine Fumarate For Personalized Therapy In Chinese Schizophrenics

Schizophrenia is a group of serious mental illnesses with disturbances in perception,thinking,emotion,and behavior.According to the WHO statistics,there are approximately 23 million patients with schizophrenia in the world until 2018,and about half of the patients will experience lifelong mental disability.Its high hospitalization rate,high recurrence rate and high disability rate bring serious mental and economic burdens to patients,their families and society.According to the“Chinese Guideline for the Prevention and Treatment of Schizophrenia",the current main treatment for schizophrenia is drug therapy.Among them,atypical antipsychotics(AAs)are selected as the first-line treatment drugs.Representative drugs include olanzapine and quetiapine,risperidone and aripiprazole,etc.According to the different clinical symptom groups of patients,it is often difficult to obtain the best effect with a single drug.Especially for patients with refractory schizophrenia and recurrent and worsening conditions,the combined use of antipsychotics has become the norm.In addition,because of the patients’ "stigma"and unbearable drug adverse reactions,the medication compliance of patients with schizophrenia is generally poor.Therefore,therapeutic drug monitoring(TDM)is required in clinical practice to evaluate patient compliance and improve the safety and effectiveness of medication.Patients with schizophrenia have the characteristics of long treatment cycle,poor treatment compliance,complex medication conditions,and multiple adverse reactions,which make the demand for the clinical individualized medication increasingly prominent.Combining existing research and clinical TDM practice,we have found that quetiapine(QTP)has a wide range of blood drug concentration variations,and its pharmacokinetics are affected by individual differences such as age,weight,metabolic enzyme gene polymorphisms,and combination medication.In addition,we also found that hyperprolactinemia and neutropenia caused by AAs also have individual response differences.Therefore,this project chooses Chinese patients with schizophrenia as the research object,selects QTP as the representative drug of AAs,selects genes involved in QTP metabolism and transport to investigate its polymorphism,and comprehensively analyzes the three aspects of non-genetic factors,genetic factors and comedication.to exploring the determinants of QTP blood concentration.And quantifying each influencing factor through the NONMEM method.At the same time,investigating the correlation of DRD2 and HLA gene polymorphisms with hyperprolactinemia and neutropenia,and it is planned to be an AAs clinical individual precise medication provides a reference basis.The main research contents and results are summarized as follows:1.Determination of the concentration of QTP in human plasma by LC-MS/MS double internal standard methodEthyl-quetiapine and levmepromazine were selected as dual internal standards for QTP quantitative analysis.Using 50%methanol-water(containing 6 mM ammonium acetate)and acetonitrile(containing 0.2%formic acid)as mobile phases,the gradient elution program was performed for chromatographic separation.The detection of mass spectrometry was carried out with an electrospray-ionization(ESI)source in positive mode,and MRM scanning mode was used for further quantitative analysis.Moreover,validation of this method was performed as well.And in order to get convincing data,self-made quality control samples with three concentration levels were tested before daily analysis.The results show that this method has good specificity and requires a small amount of sample(only 100μL).The internal standard normalized matrix effect factor of normal plasma,high-fat plasma and hemolyzed plasma is less than 10%,indicating that plasma matrices do not affect the sample determination.The established method presents a good linear relationship in the range of 10～1000 ng/mL with R2>0.99.The lower limit of quantification is 10 ng/mL.The intra-day and inter-day precision RSDs of low,medium,and high concentrations are all less than 15%,and the extraction recoveries of the two internal standards are 88.86±7.69～113.86±2.19 and 90.35±5.58～108.47±3.71 respectively.After the high-concentration plasma sample test,there was no obvious residue of the analyte and internal standard obtained from the result.The processed plasma samples can be stored stably at room temperature for 24 hours and at 4℃ for 1 week.The self-made quality control sample helps to control the error of the whole detection process.This method was applied to determine 900 blood samples from 433 patients.The result was shown that 70.67%patients with the QTP concentration in the treatment range(100～500 ng/mL),17.11%below 100 ng/mL and 12.22%exceeding 500 ng/mL.The blood drug concentration level of patients varies greatly.In order to achieve safe and effective QTP application,it is necessary to explore the factors that affect the blood drug concentration of QTP.2.Research on the polymorphism of QTP metabolism and transport genesIt can be seen from the results of the previous part that about 30%of patients have QTP blood concentrations outside the treatment range.Considering that genetic polymorphism is one of the important factors for individual differences in pharmacokinetics,433 patients’ blood samples were collected for DNA extraction,and MassArray or KASP methods were used to analyze 15 SNPs polymorphism of the CYP3A4,CYP3A5,CYP2D6 and ABCB1 genes.To lay the foundation for exploring the influence of gene polymorphism on the process of QTP in vivo.The genotype and allele frequency statistics were performed on the sequencing results,and the Hardy-Weinberg balance test was performed using the Chi-square or Fisher test.As a result,only the rs16947 genotype distribution did not meet the balance.The SHEsis online program was used to perform linkage disequilibrium test on the 4 SNP sites of gene ABCB1.The results showed that r2 between each site was less than 0.5,indicating that there is no strong linkage disequilibrium relationship between the 4 SNP sites.Continue to perform star allelic typing and phenotyping of metabolic enzymes.The results showed that the proportions of CYP2D6 normal metabolizers,intermediate metabolizers,and slow metabolizers were 65.9%,31.8%,and 2.4%,respectively,of which alleles*10 had the highest frequency,46.82%,followed by*1 and*2,respectively 30.00%and 15.06%;the most common alleles of CYP3A4 are*1 and*1G,accounting for approximately 73.97%and 24.70%,respectively;CYP3A5*3/*3 slowed metabolizers account for about 50%of the total population,suggesting that a high proportion of CYP3A5 poor metabolizers may reflect individual differences in QTP pharmacokinetics.The results provide useful information for continuing to explore the relationship between genetic polymorphism and QTP plasma concentration.3.Analysis of influencing factors of QTP plasma concentrationStatistics the information of dosage and frequency,blood drug concentration,laboratory biochemical test results,and combination medications of the 405 hospitalized patients,and combine the patient’s genotype and phenotype determination results.The statistical method used to preliminarily investigate the influencing factors of QTP-CDR from three aspects:genetic factors,non-genetic factors,and drug combination.The results showed that the age,BMI,ALT,AST/ALT,ALB,hs-CRP,WBC and GLU have a significant effect on QTP-CDR(P<0.005).Among them,age,BMI,ALT,hs-CRP,WBC and GLU were positively correlated with QTP-CDR;AST/ALT and ALB were negatively correlated with QTP-CDR.The combined use of mirtazapine,oxcarbazepine,sertraline,buspirone and escitalopram had the most significant effect on QTP-CDR(P<0.0001).Among them,mirtazapine,oxcarbazepine and sertraline could reduce the blood concentration of QTP;and buspirone,escitalopram could increase the blood concentration of QTP.To the genetic factors,no variants were found that have an effect on QTP plasma concentration,and some variants with low mutation frequency and important effects on metabolic enzymes need to be further investigated by expanding the sample size.4.Population pharmacokinetic modeling of QTP by nonlinear mixed effects model in Chinese schizophrenia patientsIn order to quantitatively investigate the influencing factors of QTP plasma concentration,we used the non-linear mixed effects model method(NONMEM)to establish a QTP population pharmacokinetic model.The one-compartment model of oral administration was used as the structural model to describe the pharmacokinetic characteristics of QTP;the inter-individual and intra-individual variability was described by the exponential model and the proportional addition model respectively;the fixed-effect model was described by the exponential model for continuous variables,Categorical variables were described using Piece-wise model.The fixed effects were screened by graphical method,forward inclusion method and backward elimination method to obtain a full regression model:#12The bootstrap method and goodness-of-fit diagram were used to verify the final model,and to simulate typical dosing regimens.The results showed that age and hs-CRP was inversely proportional to QTP clearance,and the AST/ALT was directly proportional to QTP clearance,and combined with oxcarbazepine or sertraline can increase the CL of QTP.Among the five covariates that had a significant impact on the pharmacokinetic parameters of QTP,oxcarbazepine has a large effect on the plasma concentration of QTP.The AUC0-24,ss and Cmin2,ss of QTP when combined with oxcarbazepine are 59%and 18%of the non-combined use,respectively.The final model can better reflect the distribution characteristics of the measured value of QTP blood drug concentration,and the parameter estimation of the model is relatively robust,which can provide an effective basis for the individualized medication of QTP.5.Study on the correlation between DRD2 gene polymorphism and AAs-induced hyperprolactinemiaIn addition to the pharmacokinetics,individual differences in drug treatment also include pharmacodynamics differences.Especially for some serious adverse reactions.the mechanism of individual response differences is also worthy of attention.For this reason,we selected two serious adverse reactions during the treatment of AAs to explore the reasons for their individual differences.Patients were screened and grouped according to the results of prolactin monitoring during treatment.MassArray method was used to detect the 6 SNPs’polymorphisms of DRD2(rs1076560,rs1079596,rs4274224,rs6277,rs6275 and rs6278).SPSS 26.0,Plink software and SNPStats online program were used for corresponding statistical analysis.Chi-square or Fisher’s exact test and allele logistic regression(Logistic Regression,LR)were used to investigate the distribution differences of alleles at each SNP in the Case/Control group.Genotype LR was performed based on the five genetic models provided by SNPStatsThe results showed that the alleles’ distribution of 6 SNP was not statistically different between the two groups of patients;and the genotypes of each SNP under different genetic models were not associated with hyperprolactinemia caused by AAs.The mechanism of individual differences of hyperprolactinemia remains to be further studied.6.Study on the correlation between HLA-B,HLA-DQB1 gene polymorphisms and AAs-induced neutropeniaPatients were screened and grouped according to the results of neutrophil monitoring during the treatment period.The MassArray method was used to analyze rs2523591 polymorphism of HLA-B and rs1130375,rs3828789,rs3828802,rs4993986,rs9273417,rs9273436,rs9274617 polymorphisms of HLA-DQB1.Using the same statistical analysis method as the fifth part,to explore the relationship between the HLA-B,HLA-DQB1 gene polymorphisms and the neutropenia caused by AAs.The results showed that rs1130375,rs3828789,rs3828802,rs4993986,rs9273417,rs9274617 were not associated with neutropenia;but rs2523591 and rs9273436 were significantly associated with this adverse reaction.Among them,the recessive genetic model of rs2523591 is the best model(P=0.021,ORAA=0.32,95%CIAA:0.11-0.91).The risk of neutropenia in GG/GA genotype carriers is 3.13 times that of AA genotype patients;rs9273436 takes the over-dominant genetic model as the best model(P=0.0094,ORGT=2.36,95%CIGT:1.23-4.53),the risk of neutropenia in patients with GT genotype is 2.36 times that of patients with GG/TT genotype.In summary,this study comprehensively investigated the influencing factors of blood concentration of QTP in the Chinese patients with schizophrenia.And by constructing a QTP-PPK model for Chinese patients with schizophrenia,the valuable influencing factors were quantitatively analyzed.This is the first time add the hs-CRP,oxcarbazepine and sertraline in the model.At the same time,a preliminary exploration was made on the differences in individual responses of AAs induced hyperprolactinemia and neutropenia.rs2523591 and rs9273436 were found for the first time to be the risk factors for AAs induced neutropenia.The research work will provide a theoretical basis for the precise personalized therapy of QTP and to prevent AAs induced neutropenia.