Relationship of anti-tuberculosis drug-induced liver injury and genetic polymorphisms in CYP2E1 and GST

ABSTRACT Setting: Treatment of tuberculosis (TB) can result in Drug-Induced Liver Injury (DILI) since hepatotoxic metabolites are formed during the biotransformation of isoniazid (INH).DILI can be related to the genetic profile of the patient. Single nucleotide polymorphisms in the CYP2E1 gene and GSTM1 and GSTT1 deletion polymorphisms have been associated with adverse events caused by INH. Objective: To characterize the genetic polymorphisms of CYP2E1, GSTT1 and GSTM1 in TB carriers. Design: This is an observational prospective cohort study of 45 patients undergoing treatment of TB. PCR-RFLP and multiplex-PCR were used. Results: The distribution of genotypic frequency in the promoter region (CYP2E1 gene) was: 98% wild genotype and 2% heterozygous. Intronic region: 78% wild genotype; 20% heterozygous and 2% homozygous variant. GST enzyme genes: 24% Null GSTM1 and 22% Null GSTT1. Patients with any variant allele of the CYP2E1 gene were grouped in the statistical analyses. Conclusion: Patients with the CYP2E1 variant genotype or Null GSTT1 showed higher risk of presenting DILI (p = 0.09; OR: 4.57; 95% CI: 0.75-27.6). Individuals with both genotypes had no increased risk compared to individuals with one genotype.

Relationship of anti-tuberculosis drug-induced liver injury and genetic polymorphisms in CYP2E1 and GST.

SETTING: Treatment of tuberculosis (TB) can result in Drug-Induced Liver Injury (DILI) since hepatotoxic metabolites are formed during the biotransformation of isoniazid (INH). DILI can be related to the genetic profile of the patient. Single nucleotide polymorphisms in the CYP2E1 gene and GSTM1 and GSTT1 deletion polymorphisms have been associated with adverse events caused by INH. OBJECTIVE: To characterize the genetic polymorphisms of CYP2E1, GSTT1 and GSTM1 in TB carriers. DESIGN: This is an observational prospective cohort study of 45 patients undergoing treatment of TB. PCR-RFLP and multiplex-PCR were used. RESULTS: The distribution of genotypic frequency in the promoter region (CYP2E1 gene) was: 98% wild genotype and 2% heterozygous. Intronic region: 78% wild genotype; 20% heterozygous and 2% homozygous variant. GST enzyme genes: 24% Null GSTM1 and 22% Null GSTT1. Patients with any variant allele of the CYP2E1 gene were grouped in the statistical analyses. CONCLUSION: Patients with the CYP2E1 variant genotype or Null GSTT1 showed higher risk of presenting DILI (p=0.09; OR: 4.57; 95% CI: 0.75-27.6). Individuals with both genotypes had no increased risk compared to individuals with one genotype.

Estimating the genetic component (RGC) in pharmacokinetic variability of the antiretroviral didanosine among healthy Brazilians.

BACKGROUND: We estimate the variance between between- and within-subjects, using mixed effect models, as a way to assess the genetic component in explaining the observed heterogeneity of ddl kinetics among healthy individuals. Our work expands on a previous reported method known as RGC. METHODS: Repeated measurements of ddl concentration in the serum were obtained from 48 healthy adult volunteers enrolled in two bioequivalence study. We use the NONMEM program (Non-linear Mixed Effect Model) to estimate the between- and within-subject variability and the corresponding pharmacokinetic parameters. We assess the genetic contribution to the variability of each pharmacokinetic parameter through the RGC method. RESULTS: Pharmacokinetic parameters, expressed as functions of covariates gender and creatinine clearance (CLCR), were: Oral clearance (CL = 55.1 + 240 * CLCR + 16.6 l/h for male and CL = 55.1 + 240 * CLCR for female), central volume (V2 = 9.82), inter-compartmental clearance (Q = 40.90/h), peripheral volume (V3 = 62.7 + 22.90 for male and V3 = 62.70 for female), absorption rate constant (Ka = 1.51 h(-1)) and duration of the dose administration (D = 0.44 h). The RGC of CL, Q, V3, Ka and D were 0.58, 0.97, 0.60, 0.53 and 0.88, respectively. CONCLUSION: We estimated parameter-specific RGC indices and rank them according to the potential genetic contribution as an explanation for the observed variance. Our study design improved precision by decreasing background noise and, thus, improved the chances that indices such as the RGC are in fact describing genetic variability.

A rapid and sensitive method for simultaneous determination of lamivudine and zidovudine in human serum by on-line solid-phase extraction coupled to liquid chromatography/tandem mass spectrometry detection.

A method based on solid-phase extraction (SPE) coupled to high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed for the simultaneous determination of lamivudine (3TC) and zidovudine (AZT) in human serum, using didanosine (ddI) as internal standard. The acquisition was performed in multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 230.0 --> 111.8 for 3TC, m/z 268.1 --> 126.8 for AZT, and m/z 237.2 --> 136.8 for ddI. The limits of detection and quantitation were 3 and 10 ng/mL for 3TC, and 5 and 15 ng/mL for AZT. The method was linear in the studied ranges (10-1500 ng/mL for 3TC and 15-3000 ng/mL for AZT), with r(2) > 0.99 for each drug, and the run time was 4 min. The intra-assay precisions (%) were in the ranges 1.9-8.7 (3TC) and 2.2-8.9 (AZT), the inter-assay precisions were in the ranges 2.6-9.0 (3TC) and 4.2-8.1 (AZT), and the intra- and inter-assay accuracies were >97% for both drugs. The absolute recoveries were 95-99% for 3TC (45, 600 and 1200 ng/mL) and 104-112% for AZT (45, 1000 and 2400 ng/mL). The analytical method was applied to a bioequivalence study in which 24 healthy adult volunteers received single oral doses of the reference formulation and two test combined AZT/3TC tablets, in an open, three-period, balanced, randomized, crossover protocol. Based on the 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak serum concentration) and AUC(0-inf) (extrapolated area under the serum concentration vs. time curve from time zero to infinity), it was concluded that the two test formulations are bioequivalent to the reference formulation with respect to the rate and extent of absorption of both 3TC and AZT.

Prednisolone: limited sampling strategies for estimating pharmacokinetic parameters.

To develop limited-sampling strategy (LSS) models for estimating prednisolone's area under plasma concentration versus time curve (AUC(0-infinity)), its maximum concentration in plasma (C(max)), and total clearance (CL/F). Healthy subjects (n = 24), enrolled in a bioequivalence study, received 20 mg PO of the prodrug prednisone as reference and test tablets, and plasma prednisolone concentrations (n = 576) were measured by a validated HPLC assay. A linear regression analysis of AUC(0-infinity), C(max), CL/F, and log(CL/F) against the plasma prednisolone concentrations for the reference formulation was carried out to develop LSS models to estimate these parameters. The LSS models were validated on the test formulation data sets and on simulated sets generated by the software ADAPT II. LSS models based on a single [1.5 hours for C(max) and 7 hours for AUC(0-infinity), CL/F, and log(CL/F)] plasma sample, accurately estimated (R2 = 0.84-0.97, mean bias < 1%; mean precision < 10%) these pharmacokinetic parameters. Validation tests indicated that the most informative single-point LSS models developed for the reference formulation provide precise estimates (R(2) > 0.83; mean bias < 3%; mean precision < 10%) of the corresponding pharmacokinetic parameters for the test formulation. LSS models based on the two most informative sampling points (1.5 and 7 hours) were required for accurate estimates (R(2) > 0.87; mean bias < 6%; mean precision < 8%) of prednisolone's C(max), AUC(0-infinity), CL/F, and log(CL/F) for the simulated data sets. Finally, bioequivalence assessment of the prednisone formulations, based on LSS-derived AUC(0-infinity) and C(max) values provided results identical to those obtained using the original values for these parameters. One- and 2-point LSS models provided accurate estimates of prednisolone's C(max), AUC(0-infinity), and CL/F, following single oral doses of prednisone, and allowed correct assessment of bioequivalence between two prednisone formulations.

Determination of didanosine in human serum by on-line solid-phase extraction coupled to high-performance liquid chromatography with electrospray ionization tandem mass spectrometric detection: application to a bioequivalence study.

A method based on solid-phase extraction coupled to liquid chromatography with positive ion electrospray ionization and tandem mass spectrometric detection was developed for the determination of didanosine in human serum, using lamivudine as internal standard. The acquisition was performed in the multiple reaction monitoring mode, monitoring the transitions m/z 237 --> 136.7 for didanosine and m/z 230 --> 111.7 for lamivudine. The method was linear over the range studied (10-1500 ng ml(-1)), with r(2) > 0.98, and the run time was 5 min. The intra- and inter-assay precisions were < or =10% and the intra- and inter-assay accuracies were >95%. The absolute recoveries were 99.8% (10 ng ml(-1)), 98.4% (30 ng ml(-1)), 91.5% (700 ng ml(-1)) and 94.7% (1200 ng ml(-1)). The limits of detection and quantitation were 5 and 10 ng ml(-1), respectively. The method was applied to a bioequivalence study, in which 24 healthy adult volunteers (12 men) received single oral doses (200 mg) of reference and test didanosine formulations (buffered powder for oral solutions), in an open, two-way, randomized, crossover protocol. The 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak serum concentration) and AUC(0-inf) (area under the serum concentration versus time curve from time zero to infinity) were within the range 80-125%, which supports the conclusion that the two formulations are bioequivalent regarding the rate and extent of didanosine absorption.