Quantification of pyrazinamide, isoniazid, acetyl-isoniazid, and rifampicin by a high-performance liquid chromatography method in human plasma from patients with tuberculosis.

The aim of this study was to establish and validate an alternative high-performance liquid chromatography method for simultaneous quantification of pyrazinamide, isoniazid, acetyl-isoniazid and rifampicin in plasma of patients under treatment for tuberculosis. The performed method was lineal (r2  > 0.99) in the range of 2.00-50.00 µg/mL for pyrazinamide, 0.50-20.00 µg/mL for both acetyl-isoniazid and isoniazid, and 1.20-25.00 µg/mL for rifampicin. Precision and trueness were demonstrated with coefficient of variation < 15% and deviations < 15%, respectively, for quality controls samples. The lower limits of quantification were 2.00, 0.50, 0.50, and 1.20 µg/mL for pyrazinamide, isoniazid, acetyl-isoniazid and rifampicin, respectively. The method was applied for the analysis of plasma from patients with tuberculosis. This method allowed ensuring reliable quantification of the target compounds and their pharmacokinetics parameters. In general, the mean values of maximum concentration of each antituberculosis drug were located within their respective reference therapeutic ranges. However, patients with sub-therapeutic plasma concentrations of isoniazid and rifampicin were detected. This is the first analytical technique that simultaneously quantifies isoniazid, acetyl-isoniazid, rifampicin, and pyrazinamide concentrations from plasma samples by high-performance liquid chromatography with ultraviolet/visible. The proposed method could be applied for therapeutic drug monitoring and pharmacokinetics studies of the four compounds throughout the treatment of tuberculosis patients.

Standardization and validation of a novel UPLC-MS/MS method to quantify first line anti-tuberculosis drugs in plasma and dried blood spots.

Tuberculosis (TB) is a high-burden infectious disease with high prevalence and mortality rates. The first-line anti-TB drugs include isoniazid (INH), rifampicin (RMP), pyrazinamide (PZA), and ethambutol (EMB). At present, the standard method of blood sampling for therapeutic drug monitoring (TDM) analysis is venipuncture. Dried blood spots (DBS) are a minimally invasive method for collecting small quantities of whole blood from fingertips. The aim of the current study was to develop an ultrahigh-performance liquid chromatography technique coupled to tandem mass spectrometry (UPLC-MS/MS) for simultaneous quantification of the first-line anti-TB drugs in human plasma and DBS as a sampling alternative. The separation and detection conditions were optimized to quantify INH, RMP, PZA, and EMB in both matrices in an ACQUITY UPLC H Class system coupled to a XEVO TQD detector. Chromatographic separation was performed through an Acquity HSS T3 column (2.1 × 100 mm, 1.8 µm) with 0.1% formic acid in water and acetonitrile as the mobile phase. The total run time was 7 min for both methods, with retention time in plasma of 0.85, 1.22, 3.16, and 4.04 min and 0.74, 0.87, 0.97, and 4.16 min for EMB, INH, PZA, and RMP in DBS, respectively. The bioanalytical methods developed were proved selective, linear, precise, and accurate (inter- and intra-assay); the matrix effect was demonstrated to be within the established limits. Short- and long-term stability, freeze-thaw cycles for plasma, and short-term stability for DBS were established. A total of 15 patients with 46 ± 17 (mean ± SD) years old were included, and anti-TB drug concentrations were quantified on plasma and DBS as proof of concept. Based on RMP and INH plasma concentrations (Cp), and Bayesian estimation of individual pharmacokinetic parameters, a dose adjustment was necessary for 93% of patients. The slopes of the correlation lines between plasma and DBS concentrations of RMP, EMB, INH, and PZA were 0.5321, 0.8125, 0.5680, and 0.6791, respectively. Finally, significant correlations (p < 0.05) were observed between DBS and plasma concentrations for RMP (r2 = 0.6961), EMB (r2 = 0.4369), INH (r2 = 0.8675) and PZA (r2 = 0.7363). A simple, fast, and reliable UPLC-MS/MS method was developed to quantify first-line anti-TB drugs in plasma and DBS, which provides an easy sampling and storage to be applied as a new strategy for TDM in patients with TB.

Disease activity and therapeutic drug monitoring of polyglutamates of methotrexate after daily or weekly administration of low-dose methotrexate in patients recently diagnosed with rheumatoid arthritis.

Low-dose methotrexate can be challenging to treat rheumatoid arthritis due to side effects, lack of adherence and risk of medication errors. The aim of this study was to explore the safety and efficacy of low-dose methotrexate administered daily or weekly in patients with rheumatoid arthritis. Patients were randomized according to a total oral dose of 12.5 mg of methotrexate administered: (A) divided in 5 days/week and (B) once per week. Patients were assessed along 24 weeks after starting treatment. Polyglutamates of methotrexate were quantified by ultrahigh-performance liquid chromatography coupled to tandem mass spectrometer. Patients from groups A and B showed a good response to methotrexate treatment in 29% and 25.5%, respectively, and a global frequency of adverse events of 37%. Methotrexate polyglutamate 3 concentrations were higher in normal weight (body mass index 18.5-24.9 kg/m2 ) than in obese (body mass index 30 kg/m2 ) patients with a median (interquartile range) of 28 (17.95-45.15) and 10.35 (5.22-30.88) nM without differences between dosage groups. Daily dosage regimen represents a therapeutic alternative without compromising the efficacy and safety of methotrexate treatment and with similar adherence patterns than weekly dosage regimen; further, methotrexate polyglutamate 3 concentrations could be a useful tool for therapeutic drug monitoring purposes.

Population pharmacokinetics of isoniazid and dose recommendations in Mexican patients with tuberculosis.

Background The standardized doses of isoniazid in therapy against tuberculosis are determined based on total body weight, without considering genetic polymorphisms of the metabolic enzyme N-acetyltransferase-2 that contribute to the wide pharmacokinetic variability of isoniazid. Objective The aim of this work was to build a population pharmacokinetic model of isoniazid in Mexican patients with tuberculosis to characterize typical estimates of pharmacokinetics, as well as inter-individual and residual variability of isoniazid considering the genetic factors associated with the N-acetyltransferase-2 enzyme. Setting A prospective study was conducted at the Department of Internal Medicine in Hospital Central, San Luis Potosí, México. Methods Plasma concentrations of isoniazid were measured by high performance liquid chromatography. The acetylator phenotype was predicted through single nucleotide polymorphisms in the N-acetyltransferase-2 gene. Genetic, anthropometric and clinical covariates were used to develop a pharmacokinetic model. Main outcome measure Isoniazid plasma concentration. Results A total of 69 patients with tuberculosis were included. Blood samples were drawn from 20 min to 12 h post dose to determinate the isoniazid plasma concentration. Typical pharmacokinetics parameters were characterized through two-compartment open model with first-order absorption and linear elimination. Clearance was different for each predicted N-acetyltransferase-2 phenotype being 11.4, 19.2 and 27.4 L/h for slow, intermediate and rapid acetylators, respectively. Central volume of distribution was determined as 1.5 * body mass index (L). Through the application of the model, external validation was performed and initial dose regimen of isoniazid is proposed based on stochastic simulations. Conclusion A validated population pharmacokinetic model of isoniazid was developed in Mexican patients with tuberculosis. Through the application of the final model, initial dose recommendations were provided considering body mass index and N-acetyltransferase-2 phenotype.

Population Pharmacokinetics and Dosing Recommendations of Levetiracetam in Adult and Elderly Patients With Epilepsy.

The objective was to develop and externally validate a population pharmacokinetic model of levetiracetam in adult and elderly patients with epilepsy, and to perform dosing simulations to propose individualized dosing regimens more likely to achieve therapeutic concentrations. This prospective study included 367 plasma samples from 107 patients receiving oral levetiracetam. Samples were analyzed by HPLC-UV. Pharmacokinetic data, as well as patient demographic, clinical characteristics, other drug therapy, and the use of innovator or generic products of levetiracetam, were collected. Population modeling was performed with NONMEM and included internal and external validations of the final model. Simulations were used to propose optimized dosing regimens. The pharmacokinetics of levetiracetam was described by a one-compartment model with first-order absorption and linear elimination. Body surface area had a significant effect on the apparent volume of distribution, as did creatinine clearance (CrCL) over the drug clearance (p < 0.01). The final model performed adequately during external validation testing. The final model showed a better predictive performance. Dosing simulations support 1000 mg 12-hourly dosing of levetiracetam for patients with CrCL ~60-75 mL/min with higher dose needed for higher values (1500 mg 12-hourly for CrCL ~93-111 mL/min). Dosing regimens should be personalized to the patient's CrCL to maximize the likelihood of therapeutic concentrations.

Genetic polymorphisms of arylamine N-acetyltransferases 1 and 2 and the likelihood of developing pediatric acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is one of the main causes of death in children and is associated with both genetic susceptibility and environmental factors. Genes encoding the arylamine N-acetyltransferases 1 and 2 (NAT1 and NAT2) isoenzymes are highly polymorphic among populations. Single-nucleotide polymorphism analysis was performed by real-time polymerase chain reaction from the genomic DNA of 225 healthy subjects and 57 children with ALL diagnoses. Significant associations were found between the development of ALL and the presence of the haplotypes NAT1*3 (Odds ratio [OR], 2.1), NAT1*4 (OR, 1.92), NAT2*6B (OR, 3.30), NAT2*6J (OR, 3.25) and NAT2*7A (OR, 2.45) and the NAT1 rapid (OR, 6.69) and NAT2 slow phenotypes (OR, 2.95). Our results indicate that haplotypes that provide rapid NAT1 and slow NAT2 acetylating phenotypes may influence the development of ALL in children.