| Objective:To establish a high-performance liquid chromatography?HPLC?method for the determination of voriconazole in human plasma,and to establish a genotyping method for CYP2C19,UGT1A4 and HLA-DRB1*15.The relationship between gene polymorphism,general biological data of patients and voriconazole-related liver injury was studied in order to find out the indexes which could predict the liver injury associated with voriconazole and to provide reference for the safety of voriconazole in clinical use.1.Study on the correlation between Voriconazole-related liver injury and Serum concentration of VoriconazoleMethods:The whole blood samples of patients with invasive fungal disease treated with voriconazole were collected according to the inclusion and exclusion criteria,as well as the general biological data of patients who were admitted to a tertiary hospital from June 2016to December 2018.The plasma concentration of voriconazole was determined by high performance liquid chromatography?HPLC?.Follow up the liver function index of the patients during treatment and refer to Common Terminology Criteria for Adverse Events?CTCAE?Version 4.0 to determine the occurrence and severity of voriconazole-related liver injury in patients.The data were statistically analyzed by nonparametric test and Chi-square test,and Logistic regression was used to analyze the factors affecting the liver injury associated with voriconazole.Under the guidance of blood concentration monitoring,individual drug use advice was put forward.Results:1.A total of 77 patients,56 males and 21 females,aged 23-89 years?mean age 63±21years?and weighing 35-90 kg?mean weight 61.20±11.95 kg?were enrolled in the study.The mean blood concentration was?2.35±2.61?mg·L-1 within the range of 0.29.97 mg·L-1.In 14 cases,the plasma concentration was lower than 1 mg·L-1,55 cases were in the range of 1-5.5 mg·L-1,and 8 cases were higher than 5.5 mg·L-1.2.Of the 77 patients included,22 developed Voriconazole-associated liver injury during treatment.The blood concentration of the patients with liver injury was within the range of0.2-9.97 mg·L-1,the average concentration was 3.09±2.67 mg·L-1,and that of the patients without liver injury was within the range of 0.58-7.28 mg·L-1,the average concentration is2.11±2.63 mg·L-1.3.Univariate analysis showed that body mass,smoking history and drinking history were risk factors for voriconazole-related liver injury?P values were 0.021,0.020 and 0.035,respectively?.Multivariate analysis showed that large body mass?>60 kg?was a risk factor for voriconazole-induced liver injury?P<0.001?.There was no significant difference between patients with and without drug-induced liver injury in sex,age and plasma concentration of voriconazole.4.Liver injury occurred on the 1st to 15th day after administration,and the severity of which was within grade 1 to 3.Liver function of 15 patients was improved or recovered after added liver protectant,reduced the dosage of voriconazole,or discontinued voriconazole use.Seven patients did not receive symptomatic treatment after liver injury,among which,except for 2 patients,the liver function of 5 patients was recovered or improved.2.Development of a method for determination of unbound voriconazole in human plasma and the study of its correlation with liver injuryMethods:Carbamazepine was regarded as internal standard substance,and ultrafiltration procedure was applied to pretreat plasma sample.The concentrations of unbound and total voriconazole were measured by high performance liquid chromatography?HPLC?.This method was validated by studies of its specificity,linearity,lower limit of quantification,accuracy,precision,recovery and stability.The patients who monitored the free concentration of voriconazole in a tertiary hospital from June 2017 to December 2018 were selected,and their liver function were monitored during the treatment of voriconazole.The established method was used to determine the free concentration of voriconazole in these patients.The changes of liver function during voriconazole treatment were observed and recorded.Common Terminology Criteria for Adverse Events?CTCAE?Version 4.0 was referenced to determine whether patients had voriconazole-related liver injury.The correlation between voriconazole-related liver injury and its free plasma concentration was analyzed by nonparametric test.Results:Voriconazole unbound concentration were linear?r=0.9997?over the concentration ranges of 0.32-10.00 mg·L-1.The standard curve equation was y=0.47x-8.16×10-2,and the lower limit of quantification was 0.32 mg·L-1.The precision was less than 15%and the accuracy was in the range of 85-115%.The recovery of ultrafiltration membrane was more than 95%.The plasma sample of voriconazole was stable at room temperature,4?for 24h,and freeze-thaw for 3 times.Twenty-eight patients were enrolled in this study.The mean unbound plasma concentrations were within the range of 0.35-6.96 mg·L-1.And the mean plasma protein binding rate was?49.60±10.95?%.Five patients developed voriconazole related injury,and the average free blood concentration was 1.06±1.79 mg·L-1.There was no adverse reaction in 23 patients,and their average free plasma concentration was 1.36±1.64 mg·L-1.There was no significant difference in free plasma concentration between the two groups?P=0.954?.3.Relationship between Voriconazole-related liver injury and polymorphism of CYP2C19,UGT1A4 and HLA-DRB1*15Methods:Genomic DNA extraction using E.Z.N.A.?Blood DNA Kit was performed from whole blood samples that collected from patients whose total and free plasma concentrations of voriconazole were monitored.The genotypes of UGT1A4 142T>G and HLA-DRB1*15were detected by PCR-RFLP and the genotypes of CYP2C19*2 and*3 were detected by PCR-microarray hybridization.The difference of gene frequency distribution between patients with and without liver injury was analyzed by?2 test,and the correlation between gene polymorphism and voriconazole-related liver injury was analyzed.Results:In this study,CYP2C19,UGT1A4 and HLA-DRB1*15 genotypes were determined in38 patients.The mutation rate of CYP2C19*2?681G>A?was 28.9%,and the mutation rate of CYP2C19*3?636G>A?was 5.3%.The mutation rate of UGT1A4 142T>G was22.4%,and the gene frequency of HLA-DRB1*15 was 28.9%.Ten patients experienced liver injury,and the other 28 patients didn't experience adverse event.The mutation rate of CYP2C19*3 in patients with liver injury was higher than that in patients without adverse reactions?30%vs 3.6%,P=0.048?.There was no significant difference in UGT1A4 142T>G mutation rate and HLA-DRB1*15 gene frequency between patients with and without liver injury.Conclusions:1.In this study,a HPLC method for the determination of total and free concentration of voriconazole in plasma was established.The method is specific and accurate,and can be used to monitor the plasma concentration of voriconazole in clinical patients.2.There was no significant difference in total plasma concentration and free concentration between the patients with voriconazole-related liver injury and those without adverse reactions,indicating that there was no relationship between voriconazole-related liver injury and its plasma concentration.In the clinical use of voriconazole,monitoring the plasma concentration cannot predict and judge the occurrence of voriconazole-related liver injury in patients.The results of univariate analysis showed that smoking and drinking history of patients with body mass larger than 60 kg,were the risk factors of voriconazole-related liver injury.It is suggested that the liver function index should be closely monitored in patients with the above risk factors in the course of using voriconazole.3.The results showed that CYP2C19*2,UGT1A4 and HLA-DRB1*15 were not related to voriconazole-related liver injury.Patients with CYP2C19*3 mutation were more likely to develop voriconazole-associated liver injury.However,the sample size of this study is relatively small,and it needs to be further expanded to confirm the results. |
PDF Full Text Request