LC-QToF-MS method for quantification of ethambutol, isoniazid, pyrazinamide and rifampicin in human plasma and its application.

In this research, we developed and validated a liquid chromatography coupled to mass spectrometry (LC-QToF-MS) method for simultaneous quantification of the anti-tuberculosis drugs ethambutol, isoniazid, pyrazinamide and rifampicin in human plasma. Plasma samples spiked with cimetidine (internal standard) were extracted using protein precipitation with acetonitrile containing 1% formic acid. Separation was performed using a C18 column under flow gradient conditions with water and acetonitrile, both containing 5 mm ammonium formate and 0.1% formic acid. The method was validated according to the ANVISA and US Food and Drug Administration guidelines for bioanalytical method validation. The calibration curve was linear over a concentration range of 0.2-5 µg ml-1 for ethambutol, 0.2-7.5 µg ml-1 for isoniazid, 1-40 µg ml-1 for pyrazinamide and 0.25-2 µg ml-1 for rifampicin, all with adequate precision and accuracy. The method was reproducible, selective and free of carryover and matrix effects. The validated LC-QToF-MS method was successfully applied to real samples and shown to be applicable to future therapeutic and pharmacokinetic monitoring studies.

Rapid and highly sensitive quantification of the anti-tuberculosis agents isoniazid, ethambutol, pyrazinamide, rifampicin and rifabutin in human plasma by UPLC-MS/MS.

With the increased cases of multidrug- or rifampicin-resistant tuberculosis and co-infection with HIV globally, it is difficult to achieve ideal clinical responses because of poor drug absorption and drug-drug interactions. Herein, a bioanalytical UPLC-MS/MS method was developed and validated to quantify five anti-TB agents in human plasma samples for detecting blood drug concentrations to improve therapeutic effects. To overcome the matrix effects, stable isotope labeled analogue of each analyte was used for internal standardization. A simple single-step protein precipitation by acetonitrile was employed for the sample preparation, then the analytes including rifampicin, rifabutin, pyrazinamid, ethambutol, isoniazid and their isotope labeled internal standards (ILISs) were implemented on an HILIC silica column with a gradient mode. The linear range for each analyte was covering the peak drug concentration (Cmax) in the 20 times diluted plasma samples. The coefficient of variation of intra- and inter-day precision was less than 17.0 %, and the accuracy ranged between 91.5 and 110.0 %. The extraction recoveries of all agents were ≥90.2 %, and the matrix effects with internal standard-normalization for all agents were 97.1-110.0 %. The optimal blood sampling time was designed basing on the results of stability validation. This UPLC-MS/MS method with a run time of 3.5 min was successfully applied to routine therapeutic monitoring of the five anti-TB agents in patient plasma.

Nonparametric Population Pharmacokinetic Modeling of Isoniazid in Colombian Patients With Tuberculosis.

BACKGROUND: Isoniazid (INH) is a first-line antituberculosis (TB) agent with a pharmacokinetic profile characterized by high interindividual variation; however, population pharmacokinetic studies in patients with TB are scarce. The aim was to develop a population model for INH in Colombian patients with TB suitable for predicting drug exposure and assessing the probability of target attainment of pharmacodynamic goals. METHODS: Ten hospitalized adult patients with TB undergoing INH treatment were recruited. After an 8-hour fasting, subjects took 300 mg of INH, and 10 samples were taken from 0 to 12 hours. INH was quantified by high-performance liquid chromatography-UV, and data were analyzed with the Pmetrics R package software. A Monte Carlo simulation with the model parameters was run to determine the probability of target attainment for optimal efficacy. RESULTS: The best model included 2 compartments, first-order absorption (Ka), delayed absorption (Tlag), and linear clearance (CL). Median Tlag was 0.25 hours, 5.54 hour for Ka, (Equation is included in full-text article.)for CL, (Equation is included in full-text article.)for the volume of the central compartment (Vc), 1.04 L/h for intercompartmental clearance (Q), and 788 L for the volume of the peripheral compartment (Vp). CL and Vc were allometrically scaled on basis of the normalized body weight. CONCLUSIONS: The Monte Carlo simulation indicated that 300 mg of INH per day is appropriate for Mycobacterium tuberculosis strains with minimal inhibitory concentration (MIC) up to 0.03 mg/L (target: area under the concentration-time curve/MIC >597); however, to cover strains with MIC up to 0.125 mg/L (80% of clinical isolates), a dose of 900 mg per day would be required.

Quality Assessment of Dried Blood Spots from Patients With Tuberculosis from 4 Countries.

BACKGROUND: Dried blood spot (DBS) sampling is a blood collection tool that uses a finger prick to obtain a blood drop on a DBS card. It can be used for therapeutic drug monitoring, a method that uses blood drug concentrations to optimize individual treatment. DBS sampling is believed to be a simpler way of blood collection compared with venous sampling. The aim of this study was to evaluate the quality of DBSs from patients with tuberculosis all around the world based on quality indicators in a structured assessment procedure. METHODS: Total 464 DBS cards were obtained from 4 countries: Bangladesh, Belarus, Indonesia, and Paraguay. The quality of the DBS cards was assessed using a checklist consisting of 19 questions divided into 4 categories: the integrity of the DBS materials, appropriate drying time, blood volume, and blood spot collection. RESULTS: After examination, 859 of 1856 (46%) blood spots did not comply with present quality criteria. In 625 cases (34%), this was due to incorrect blood spot collection. The DBS cards from Bangladesh, Indonesia, and Paraguay seemed to be affected by air humidity, causing the blood spots not to dry appropriately. CONCLUSIONS: New tools to help obtain blood spots of sufficient quality are necessary and environmental specific recommendations to determine plasma concentration correctly. In addition, 3% of the DBS cards were rejected because the integrity of the materials suggesting that the quality of plastic ziplock bags currently used to protect the DBS cards against contamination and humidity may not be sufficient.

Anthropometric and Genetic Factors Associated With the Exposure of Rifampicin and Isoniazid in Mexican Patients With Tuberculosis.

BACKGROUND: Tuberculosis (TB) remains a critical infectious, contagious disease worldwide with high prevalence and mortality rate. The directly observed treatment short-course therapy includes rifampicin (RMP) and isoniazid (INH) for at least 6 months. The purposes of this scheme are to interrupt the transmissibility of the Mycobacterium tuberculosis complex and to avoid secondary complications. Low plasma concentrations of these anti-TB drugs have been associated with extended treatment duration, therapeutic failure, and relapse. The determination of anthropometric, genetic, and clinical variables that may affect plasma concentrations of RMP and INH might facilitate the detection of patients at increased risk of therapeutic failure. METHODS: A prospective observational study was performed in patients with TB diagnosis. A fixed-dose combined formulation was administered following clinical guidelines, and 12 venous blood samples were collected within 24 hours after dose for the quantification of plasma levels of RMP and INH by high-performance liquid chromatography-ultraviolet. The plasma concentrations versus time for each drug in each patient were assessed by a noncompartmental approach to obtain Cmax, and the area under the concentration-time curve to the last observation point (AUC0-24 h) was calculated by the linear trapezoidal rule. Genetic polymorphisms of the enzyme involved in INH metabolism (NAT2) and proteins involved in RMP transport (glycoprotein-P and OATP1B1) were determined. RESULTS: A total of 34 patients aged between 18 and 72 years with the diagnosis of TB were included in the current study. A multivariate analysis was performed to determine the anthropometric and genetic characteristics that modified the Cmax and AUC0-24 h of RMP and INH. Results indicated that RMP Cmax and AUC0-24 h were affected by sex, dose/weight, and single nucleotide polymorphism of MDR1. In addition, age, body mass index, and NAT2 acetylator genotype were shown to determine the Cmax and AUC0-24 h for INH. CONCLUSIONS: Anthropometric, genetic, and dosage characteristics of Mexican patients with TB are an important source of risk for subtherapeutic plasma concentrations of anti-TB drugs. Factors such as lower-than-recommended RMP dose, male patients with TB, and MDR1 3435 genotype, in addition to age group, body mass index, and INH acetylator phenotype based on NAT2 genotype, should be considered during treatment.

Combined effect of CYP2B6 and NAT2 genotype on plasma efavirenz exposure during rifampin-based antituberculosis therapy in the STRIDE study.

In STRIDE, slow metabolizer CYP2B6 and NAT2 genotypes were each associated with increased plasma efavirenz concentrations during antituberculosis therapy. Concentrations were greater on therapy than off therapy in 58% with CYP2B6 and 93% with NAT2 slow metabolizer genotypes. Individuals with slow metabolizer genotypes in both genes had markedly elevated concentrations.

Expression of NAT2 in immune system cells and the relation of NAT2 gene polymorphisms in the anti-tuberculosis therapy in Mexican mestizo population.

Arylamine N-acetyltransferase 2 (NAT2) metabolizes isoniazid (INH) and Single Nucleotide Polymorphisms (SNP) responsible for its activity has been reported. The aim of this study in the Mexican mestizo population was to evaluate NAT2 expression at the protein level in immune cells, as well as the distribution and frequency of six NAT2 SNPs and their association with anti-TB therapy, by measuring the plasma levels of INH and Acetyl-INH (AcINH). We performed genotyping assays of NAT2 SNPs in 40 TB patients and 121 healthy volunteers by real-time PCR. A method for detecting NAT2 in immune cells using flow cytometry was developed. Plasma concentrations of INH and AcINH were obtained by HPLC in TB patients and the Metabolic Ratio (MR) was calculated. The phenotypes obtained in the healthy volunteers were as follows; 18.87 % of subjects had the rapid acetylator phenotype, 45.45 % had the intermediate phenotype and 39.66 % exhibited the slow acetylator phenotype. In the TB patient group, 35 % of patients had the rapid acetylator phenotype, 32.5 % were intermediate and 32.5 % showed the slow acetylator phenotype. A higher expression level of NAT2 in innate immune cells from TB patients compared to those from healthy volunteers was detected (P < 0.013). In TB patients the MR showed a bimodal distribution with an antimode of 0.7, which was used as a threshold value for acetylator classification. A high correspondence between the rapid and slow acetylator phenotype with MR was demonstrated. In conclusion, the 282C>T, 341T>C, 481C>T, 590G>A, 803A>G, 857G>A SNPs of NAT2 gene provides accurate for prediction of the acetylator phenotype in Mexican mestizo population. A statistical difference was found in frequency of rapid metabolizer phenotype, which was higher in TB patients. In addition, the expression of NAT2 protein in immune cells can lead to further studies related to its functional role in the innate immune response against M. tuberculosis and other xenobiotics metabolized by this enzyme.

Relative bioavailability of isoniazid in a fixed-dose combination product in healthy Mexican subjects.

SETTING: Subtherapeutic plasma isoniazid (INH) concentrations and the development of bacterial resistance may be attributed to poor quality and reduced bioavailability of fixed-dose combination (FDC) formulations. The bioavailability of INH from a generic and that of a branded FDC formulation had not been compared in the Mexican population. OBJECTIVE: To evaluate the bioequivalence of a generic three-drug FDC formulation (3FDC) in comparison with a 3FDC reference with doses of 300 mg INH in 20 healthy Mexican adults, and to generate data regarding the oral relative bioavailability of the drug in this population. DESIGN: A single-dose, randomised-sequence, open-label, two-period crossover study. RESULTS: Both formulations were well tolerated. The pharmacokinetic parameters of INH showed wide inter-individual variability. The average relative bioavailability calculated for maximum serum concentration area under the concentration-time curve (AUC), AUC(0-24h) and AUC(0-∞) of the test 3FDC formulation vs. the 3FDC reference were respectively 64.84% (90%CI 56.01-75.06), 59.05% (90%CI 50.27-69.36) and 57.26% (90%CI 46.93-69.84). CONCLUSIONS: The 3FDC test and reference formulations were not bioequivalent because the 90%CI for the geometric mean ratios did not meet the regulatory requirements for bioequivalence (range 80-125%) based on the rate and extent of absorption.

Pharmacokinetics of rifampin in Peruvian tuberculosis patients with and without comorbid diabetes or HIV.

For drug-compliant patients, poor responses to tuberculosis (TB) treatment might be attributable to subtherapeutic drug concentrations. An impaired absorption of rifampin was previously reported for patients with diabetes mellitus (DM) or HIV. The objectives of this study were to determine whether TB drug pharmacokinetics differed in Peruvian TB patients with DM or HIV. In this cross-sectional study, TB patients, recruited from health centers in Lima, Peru, had blood samples taken at 2 and 6 h after directly observed TB drug ingestion, to determine plasma concentrations of rifampin. Of 105 patients, 50 had TB without a comorbidity, 26 had coexistent DM, and 29 had coexistent HIV. Unexpectedly, the overall median 2- and 6-h levels of rifampin were 1.6 and 3.2 mg/liter, respectively, and the time to the peak concentration was 6 h (slow absorber) instead of 2 h (fast absorber) for 61 patients (62.2%). The geometric mean peak concentration of drug in serum (C(max)) was significantly higher in fast absorbers than in slow absorbers (5.0 versus 3.8 mg/liter; P = 0.05). The rifampin C(max) was significantly lower in male patients than in female patients (3.3 versus 6.3 mg/liter; P < 0.001). Neither slow nor fast absorbers with comorbidities (DM or HIV) had significantly different C(max) results compared to those of TB patients without comorbidities. An analysis of variance regression analysis showed that female gender (P < 0.001) and the time to maximum concentration of drug in serum (T(max)) at 2 h (P = 0.012) were independently correlated with increased exposure to rifampin. Most of this Peruvian study population exhibited rifampin pharmacokinetics different from those conventionally reported, with delayed absorption and low plasma concentrations, independent of the presence of an HIV or DM comorbidity.

Serum concentrations of rifampin, isoniazid, and intestinal absorption, permeability in patients with multidrug resistant tuberculosis.

This study evaluates the serum concentrations of rifampin (RMP), isoniazid (INH), and intestinal barrier function in patients with multidrug-resistant tuberculosis (MDR-TB), drug susceptible tuberculosis (DS-TB), and health volunteers (HC; controls). Peak serum concentrations of RMP were significantly lower in MDR-TB and DS-TB as compared with HC (odds ratio [OR] = 3.125, confidence interval [CI] [1.037-9.418] and OR = 4.025, CI [1.207-13.418], respectively). The INH peak serum concentration was not significantly different between MDR-TB versus DS-TB or DS-TB versus HC. The percent of mannitol excretion was significantly lower in the MDR-TB group compared with DS-TB (13.18 versus 16.03, analysis of covariance [ANCOVA], P = 0.0369) and compared with HC (13.18 versus 16.61, ANCOVA, P = 0.0291) the other study groups. These data suggested a lower peak serum concentration of RMP for both MDR-TB and DS-TB as compared with the HC group. The data also showed a lower intestinal area of absorption in patients with tuberculosis and even worse in MDR-TB.

Intestinal barrier function and serum concentrations of rifampin, isoniazid and pyrazinamide in patients with pulmonary tuberculosis

Intestinal barrier function and serum concentrations of rifampin, isoniazid and pyrazinamide were studied in healthy controls and patients with active pulmonary tuberculosis. A case-control study of 29 controls and 30 cases attending at the Health Center, July, 2004 to December, 2005 was conducted. The body mass index was significantly reduced in cases compared to controls (p < 0.001). The intestinal paracellular transport of lactulose was significantly (p = 0.019) reduced in cases compared to controls. The transcellular transport of mannitol and the lactulose:mannitol ratio were not significantly (p = 0.0698) reduced in cases compared to controls. Low serum concentrations of rifampin, isoniazid and pyrazinamide were observed in 81 percent (48/59), 92 percent (54/59) and 28 percent (12/59), respectively, in all individuals. The results demonstrated a marked decrease on intestinal paracellular transport in patients with active pulmonary tuberculosis and reduced serum concentrations of rifampin and isoniazid in both groups.

Intestinal barrier function and serum concentrations of rifampin, isoniazid and pyrazinamide in patients with pulmonary tuberculosis.

Intestinal barrier function and serum concentrations of rifampin, isoniazid and pyrazinamide were studied in healthy controls and patients with active pulmonary tuberculosis. A case-control study of 29 controls and 30 cases attending at the Health Center, July, 2004 to December, 2005 was conducted. The body mass index was significantly reduced in cases compared to controls (p < 0.001). The intestinal paracellular transport of lactulose was significantly (p = 0.019) reduced in cases compared to controls. The transcellular transport of mannitol and the lactulose:mannitol ratio were not significantly (p = 0.0698) reduced in cases compared to controls. Low serum concentrations of rifampin, isoniazid and pyrazinamide were observed in 81% (48/59), 92% (54/59) and 28% (12/59), respectively, in all individuals. The results demonstrated a marked decrease on intestinal paracellular transport in patients with active pulmonary tuberculosis and reduced serum concentrations of rifampin and isoniazid in both groups.