Pyrazinamide Concentrations Research Articles

Optimization of biotransformation process for the synthesis of pyrazine-2-carboxylic acid hydrazide using acyltransferase activity of Bacillus smithii IITR6b2 in batch and fed-batch mode

The synthesis of pyrazine-2-carboxylic acid hydrazide, a heterocyclic hydrazide compound, with negligible production of the by-product (pyrazine-2-carboxylic acid) in a solvent-free reaction condition was developed using the acyltransferase activity of amidase of Bacillus smithii strain IITR6b2. This study involves a greener approach with no hazardous chemicals and one-step biotransformation of pyrazinamide to its acid hydrazide (through hydrazinolysis). The Central Composite Design of the Response Surface Methodology was used to find the factors affecting the synthesis of the pyrazine-2-carboxylic acid hydrazide and pyrazine-2-carboxylic acid to find its true optimum. The four factors, namely pyrazinamide, hydrazine dihydrochloride, temperature, and cell concentrations, were considered during optimization. The optimized reaction conditions from the Central Composite Design for achieving the 32.26 mM of pyrazine-2-carboxylic acid hydrazide involve 40 mM of pyrazinamide, 1000 mM of hydrazine dihydrochloride with 2.5 mg/mL cell concentration at 20 °C. It resulted in an optimum production of pyrazine-2-carboxylic acid hydrazide utilizing the acyltransferase activity of amidase of Bacillus smithii IITR6b2, and the results obtained through the Design of Experiments were validated. The fed-batch biotransformation was carried out with alginate-entrapped whole-cell enzyme with the following parameters: agitation at 200 rpm, a pyrazinamide concentration of 40 mM, and 1000 mM of hydrazine dihydrochloride. The final pyrazine-2-carboxylic acid hydrazide concentration of 126 mM was obtained, corresponding to a 63% molar conversion.

Studying the dynamics of the drug processing of pyrazinamide in Mycobacterium tuberculosis.

Pyrazinamide (PZA) is a key drug in the treatment of Mycobacterium tuberculosis. Although not completely understood yet, the bactericidal mechanism of PZA starts with its diffusion into the cell and subsequent conversion into pyrazinoic acid (POA) after the hydrolysis of ammonia group. This leads to the acidification cycle, which involves: (1) POA extrusion into the extracellular environment, (2) reentry of protonated POA, and (3) release of a proton into the cytoplasm, resulting in acidification of the cytoplasm and accumulation of intracellular POA. To better understand this process, we developed a system of coupled non-linear differential equations, which successfully recapitulates the kinetics of PZA/POA observed in M. tuberculosis. The parametric space was explored, assessing the impact of different PZA and pH concentrations and variations in the kinetic parameters, finding scenarios of PZA susceptibility and resistance. Furthermore, our predictions show that the acidification cycle alone is not enough to result in significant intracellular accumulation of POA in experimental time scales when compared to other neutral pH scenarios. Thus, revealing the need of novel hypotheses and experimental evidence to determine the missing mechanisms that may explain the pH-dependent intracellular accumulation of POA and their subsequent effects.

Effect of Metformin on Plasma Exposure of Rifampicin, Isoniazid, and Pyrazinamide in Patients on Treatment for Pulmonary Tuberculosis.

To evaluate the effect of metformin on the plasma levels of rifampicin, isoniazid, and pyrazinamide in patients with drug-sensitive pulmonary tuberculosis being treated with first-line antituberculosis treatment (ATT) and to assess the influence of gene polymorphisms on the metabolic pathway of metformin and plasma levels of antitubercular drugs. Nondiabetic adults aged 18-60 years with pulmonary tuberculosis were randomized to either the standard ATT (ATT group) or ATT plus metformin (METRIF group) groups in a phase IIB clinical trial. An intensive pharmacokinetic study with blood collection at 0 hour (predosing), followed by 1, 2, 4, 6, 8, and 12 hours after dosing was conducted during the first month of treatment in a subset of 60 study participants after a minimum of 14 doses. Plasma concentrations of rifampicin, isoniazid, pyrazinamide, and metformin were measured by high-performance liquid chromatography using validated methods, and pharmacokinetic parameters and OCT1 and MATE1 gene polymorphisms were compared between the groups. Significant increases in the clearance of rifampicin, isoniazid, and pyrazinamide were observed in patients in the METRIF group (n = 29) compared with those in the ATT group (n = 31). The AA genotypes of the single-nucleotide polymorphism of rs2289669 ( MATE1 ) in the METRIF group showed a significantly decreased area under the concentration-time curve to the last observation point and increased clearance of rifampicin. Metformin altered rifampicin and isoniazid plasma concentrations in patients receiving antituberculosis treatment for pulmonary tuberculosis with little effect on sputum conversion at the end of treatment. Studies with larger sample sizes are needed to understand host drug-drug interactions.

Pharmacokinetics and safety of first-line tuberculosis drugs rifampin, isoniazid, ethambutol, and pyrazinamide during pregnancy and postpartum: results from IMPAACT P1026s.

Physiological changes during pregnancy may alter the pharmacokinetics (PK) of antituberculosis drugs. The International Maternal Pediatric Adolescent AIDS Clinical Trials Network P1026s was a multicenter, phase IV, observational, prospective PK and safety study of antiretroviral and antituberculosis drugs administered as part of clinical care in pregnant persons living with and without HIV. We assessed the effects of pregnancy on rifampin, isoniazid, ethambutol, and pyrazinamide PK in pregnant and postpartum (PP) persons without HIV treated for drug-susceptible tuberculosis disease. Daily antituberculosis treatment was prescribed following World Health Organization-recommended weight-band dosing guidelines. Steady-state 12-hour PK profiles of rifampin, isoniazid, ethambutol, and pyrazinamide were performed during second trimester (2T), third trimester (3T), and 2-8 of weeks PP. PK parameters were characterized using noncompartmental analysis, and comparisons were made using geometric mean ratios (GMRs) with 90% confidence intervals (CI). Twenty-seven participants were included: 11 African, 9 Asian, 3 Hispanic, and 4 mixed descent. PK data were available for 17, 21, and 14 participants in 2T, 3T, and PP, respectively. Rifampin and pyrazinamide AUC0-24 and C max in pregnancy were comparable to PP with the GMR between 0.80 and 1.25. Compared to PP, isoniazid AUC0-24 was 25% lower and C max was 23% lower in 3T. Ethambutol AUC0-24 was 39% lower in 3T but limited by a low PP sample size. In summary, isoniazid and ethambutol concentrations were lower during pregnancy compared to PP concentrations, while rifampin and pyrazinamide concentrations were similar. However, the median AUC0-24 for rifampin, isoniazid, and pyrazinamide met the therapeutic targets. The clinical impact of lower isoniazid and ethambutol exposure during pregnancy needs to be determined.

Correlations among the plasma concentrations of first-line anti-tuberculosis drugs and the physiological parameters influencing concentrations.

Background: The plasma concentrations of the four most commonly used first-line anti-tuberculosis (TB) drugs, isoniazid (INH), rifampicin (RMP), ethambutol (EMB), and pyrazinamide (PZA), are often not within the therapeutic range. Insufficient drug exposure could lead to drug resistance and treatment failure, while excessive drug levels may lead to adverse reactions. The purpose of this study was to identify the physiological parameters influencing anti-TB drug concentrations. Methods: A retrospective cohort study was conducted. The 2-h plasma concentrations of the four drugs were measured by using the high-performance liquid chromatography-tandem mass spectrometry method. Results: A total of 317 patients were included in the study. The proportions of patients with INH, RMP, EMB, and PZA concentrations within the therapeutic range were 24.3%, 31.5%, 27.8%, and 18.6%, respectively. There were positive associations between the concentrations of INH and PZA and RMP and EMB, but negative associations were observed between the concentrations of INH and RMP, INH and EMB, RMP and PZA, and EMB and PZA. In the multivariate analysis, the influencing factors of the INH concentration were the PZA concentration, total bile acid (TBA), serum potassium, dose, direct bilirubin, prealbumin (PA), and albumin; those of the RMP concentration were PZA and EMB concentrations, weight, α-l-fucosidase (AFU), drinking, and dose; those of the EMB concentration were the RMP and PZA concentrations, creatinine, TBA and indirect bilirubin; and those of the PZA concentration were INH, RMP and EMB concentrations, sex, weight, uric acid and drinking. Conclusion: The complex correlations between the concentrations of the four first-line anti-TB drugs lead to a major challenge in dose adjustment to maintain all drugs within the therapeutic window. Levels of TBA, PA, AFU, and serum potassium should also be considered when adjusting the dose of the four drugs.

A physiologically-based pharmacokinetic model for tuberculosis drug disposition at extrapulmonary sites.

Tuberculosis (TB) is a leading cause of mortality attributed to an infectious agent. TB primarily targets the lungs, but in about 16% cases can affect other organs as well, giving rise to extrapulmonary TB (EPTB). However, an optimal regimen for EPTB treatment is not defined. Although the recommended treatment for most forms of EPTB is the same as pulmonary TB, the pharmacokinetics of EPTB therapy are not as well studied. To address this gap, we formulate a whole-body physiologically-based pharmacokinetic (PBPK) model for EPTB that for the first time includes the ability to simulate drug concentrations in the pleura and lymph node, the most commonly affected sites of EPTB. Using this model, we estimate the time-dependent concentrations, at potential EPTB infection sites, of the following four first-line anti-TB drugs: rifampicin, ethambutol, isoniazid, and pyrazinamide. We use reported plasma concentration kinetics data to estimate model parameters for each drug and validate our model using reported concentration data not used for model formulation or parameter estimation. Model predictions match the validation data, and reported pharmacokinetic parameters (maximum plasma concentration, time to reach maximum concentration) for the drugs. The model also predicts ethambutol, isoniazid, and pyrazinamide concentrations in the pleura that match reported experimental values from an independent study. For each drug, the predicted drug concentrations at EPTB sites are compared with their critical concentration. Simulations suggest that although rifampicin and isoniazid concentrations are greater than critical concentration values at most EPTB sites, the concentrations of ethambutol and pyrazinamide are lower than their critical concentrations at most EPTB sites.

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.

Development of a population pharmacokinetic model of pyrazinamide to guide personalized therapy: impacts of geriatric and diabetes mellitus on clearance.

Objectives: This study was performed to develop a population pharmacokinetic model of pyrazinamide for Korean tuberculosis (TB) patients and to explore and identify the influence of demographic and clinical factors, especially geriatric diabetes mellitus (DM), on the pharmacokinetics (PK) of pyrazinamide (PZA). Methods: PZA concentrations at random post-dose points, demographic characteristics, and clinical information were collected in a multicenter prospective TB cohort study from 18 hospitals in Korea. Data obtained from 610TB patients were divided into training and test datasets at a 4:1 ratio. A population PK model was developed using a nonlinear mixed-effects method. Results: A one-compartment model with allometric scaling for body size effect adequately described the PK of PZA. Geriatric patients with DM (age >70years) were identified as a significant covariate, increasing the apparent clearance of PZA by 30% (geriatric patients with DM: 5.73L/h; others: 4.50L/h), thereby decreasing the area under the concentration-time curve from 0 to 24h by a similar degree compared with other patients (geriatric patients with DM: 99.87μgh/mL; others: 132.3μgh/mL). Our model was externally evaluated using the test set and provided better predictive performance compared with the previously published model. Conclusion: The established population PK model sufficiently described the PK of PZA in Korean TB patients. Our model will be useful in therapeutic drug monitoring to provide dose optimization of PZA, particularly for geriatric patients with DM and TB.

Predictions of Bedaquiline and Pretomanid Target Attainment in Lung Lesions of Tuberculosis Patients using Translational Minimal Physiologically Based Pharmacokinetic Modeling.

Site-of-action concentrations for bedaquiline and pretomanid from tuberculosis patients are unavailable. The objective of this work was to predict bedaquiline and pretomanid site-of-action exposures using a translational minimal physiologically based pharmacokinetic (mPBPK) approach to understand the probability of target attainment (PTA). A general translational mPBPK framework for the prediction of lung and lung lesion exposure was developed and validated using pyrazinamide site-of-action data from mice and humans. We then implemented the framework for bedaquiline and pretomanid. Simulations were conducted to predict site-of-action exposures following standard bedaquiline and pretomanid, and bedaquiline once-daily dosing. Probabilities of average concentrations within lesions and lungs greater than the minimum bactericidal concentration for non-replicating (MBCNR) and replicating (MBCR) bacteria were calculated. Effects of patient-specific differences on target attainment were evaluated. The translational modeling approach was successful in predicting pyrazinamide lung concentrations from mice to patients. We predicted that 94% and 53% of patients would attain bedaquiline average daily PK exposure within lesions (Cavg-lesion) > MBCNR during the extensive phase of bedaquiline standard (2 weeks) and once-daily (8 weeks) dosing, respectively. Less than 5% of patients were predicted to achieve Cavg-lesion > MBCNR during the continuation phase of bedaquiline or pretomanid treatment, and more than 80% of patients were predicted to achieve Cavg-lung >MBCR for all simulated dosing regimens of bedaquiline and pretomanid. The translational mPBPK model predicted that the standard bedaquiline continuation phase and standard pretomanid dosing may not achieve optimal exposures to eradicate non-replicating bacteria in most patients.

Enteropathogen spectrum and effect on antimycobacterial pharmacokinetics among children with tuberculosis in rural Tanzania: a prospective cohort study

SummaryBackgroundEnteropathy is prevalent in tuberculosis-endemic areas, and it has been shown to impair intestinal absorptive function; therefore, enteropathogen burden might negatively affect antimycobacterial pharmacokinetics, particularly among malnourished children. We sought to quantify enteropathogen burden among children initiating tuberculosis treatment in rural Tanzania and determine the effect of enteropathogen burden on serum antimycobacterial pharmacokinetics.MethodsWe performed a prospective cohort study at one site in rural Tanzania as an exploratory substudy of a large multicountry cohort study. We included children younger than 15 years of age with confirmed or probable tuberculosis undergoing treatment with first-line tuberculosis therapy; children were excluded from the study if they were unable to undergo sample collection. Participants were consecutively recruited from the inpatient paediatric wards or the outpatient tuberculosis clinic at Haydom Lutheran Hospital, Tanzania. The main outcome was to quantify symptomatic enteropathogen burden and the effect on serum antimycobacterial pharmacokinetics. We quantified enteropathogen burden (defined as the sum of distinct enteropathogens detected in stool) using a multipathogen PCR capable of simultaneous detection of 37 bacterial, viral, and parasitic species or species groups from stool collected within 72 h of treatment initiation. Comprehensive clinical assessment, including presence of gastrointestinal symptoms, was performed at baseline, and serum was collected approximately 2 weeks after treatment initiation at steady state and throughout the dosing interval with concentrations of isoniazid, rifampicin, pyrazinamide, and ethambutol measured by liquid chromatography with a tandem mass spectrometry assay to quantify peak (Cmax) and total area under the concentration curve (AUC0–24), as determined by non-compartmental analysis. Enteropathogen burden was compared with pharmacokinetic measurements using bivariable and multivariable linear regression.Findings58 children were assessed for eligibilty and enrolled between June 25, 2016, and Feb 6, 2018; 44 had complete stool testing and serum pharmacokinetic data, and they were included in the analyses. 20 (45%) were female, and 24 (55%) were male. 37 (84%) had moderate or severe malnutrition. A mean of 2·1 (SD 1·3) enteropathogens were detected per participant. Target peak concentrations of rifampicin were reached in eight (18%) of 44 participants, isoniazid in 24 (54%) of 44 participants, pyrazinamide in 28 (74%) of 38 participants, and ethambutol in six (15%) of 39 participants. Compared with controlled comparisons, each summative additional bacterial enteropathogen detected was associated with a 40% lower rifampicin Cmax (95% CI −62 to −5) and a 36% lower ethambutol Cmax (−52 to −14), while viral pathogens were associated with a 51% lower isoniazid Cmax (−75 to −7). The combination of gastrointestinal symptoms and detection of an additional enteropathogen was associated with a 27% reduction in rifampicin AUC0–24 (95% CI −47 to −1).InterpretationTanzanian children undergoing tuberculosis treatment rarely attained pharmacokinetic targets; enteropathogen carriage was common and enteropathogen burden was associated with significant reductions in the concentrations of some antimycobacterial drugs. Further research should explore mechanistic relationships of individual pathogens and antimycobacterial pharmacokinetics in larger cohorts, or determine if screening for and treating enteropathogens at tuberculosis treatment initiation improves pharmacokinetic target attainment.FundingNational Institute of Allergy and Infectious Diseases, National Institutes of Health.

Analysis of influencing factors on the plasma concentration of first-line anti-tuberculosis drugs-a single-center retrospective cohort study.

BackgroundThe four most commonly used first-line anti-tuberculosis (TB) drugs in clinical practice are isoniazid (INH), rifampicin (RFP), ethambutol (EMB), and pyrazinamide (PZA). The plasma concentration of anti-TB drugs is an important factor influencing the effectiveness of TB treatment. Factors affecting blood concentration of antituberculosis drugs have not been elaborated clearly. The purpose of this study is to investigate the status of plasma concentration of anti-TB drugs, explore the factors influencing anti-TB drug plasma concentration, and guide the rational use of clinical drugs.MethodsThis is a single-center retrospective cohort study. Patients with pulmonary TB received first-line anti-TB drugs in the 309th Hospital of the PLA from June 2014 to September 2018 were investigated. The primary endpoint was factors affecting the anti-tuberculosis drug plasma concentration which were determined by high performance liquid chromatography-mass spectrometry. The factors influencing plasma concentration analyzed by the multiple linear regression model.ResultsA total of 205 patients were included in the study. The rates of patients with substandard 2-hour plasma concentrations of INH, RFP, EMB, and PZA were 45.8%, 54.4%, 37.7%, and 52.9%, respectively. Intravenous administration of INH (P<0.001) significantly increased plasma concentrations compared with oral administration, and its plasma concentration was negatively correlated with blood uric acid levels (P=0.001). RFP 2-hour plasma concentrations were positively correlated with serum albumin levels (P=0.04). EMB 2-hour plasma concentrations were positively correlated with age (P=0.01), dose (P<0.001), and serum creatinine levels (P<0.001). PZA 2-hour plasma concentrations were positively correlated with dose (P<0.001) and total bilirubin levels (P<0.001), and were negatively correlated with blood urea nitrogen levels (P=0.001).ConclusionsAge, gender, dose, intravenous administration, retreatment, blood uric acid level, serum albumin level, serum creatinine level, total bilirubin level, and blood urea nitrogen level were independent influencing factors of anti-TB drug plasma concentration. During anti-TB treatment, plasma concentration monitoring is essential and helpful to optimize the drug dose and carry out individualized treatment regimen.

Neurodevelopmental toxicity of pyrazinamide to larval zebrafish and the restoration after intoxication withdrawal

To investigate the neurotoxicity of pyrazinamide (PZA) to larval zebrafish, the PZA effects were assessed followed by its mechanism being explored. Same as isoniazid (INH), this compound is a first-line anti-tuberculosis drug and is suggested to be a risk that inducing nerve injury with long-term intoxication. Our findings indicated that zebrafish larvae obtained severe nerve damage secondary to constant immersion in various concentrations of PZA (i.e., 0.5, 1.0, and 1.5 mM) from 4 hpf (hours post fertilization) onwards until 120 hpf. The damage presented as dramatically decrease of locomotor capacity and dopaminergic neuron (DAN)-rich region length in addition to defect of brain blood vessels (BBVs). Moreover, PZA-administrated zebrafish showed a decreased dopamine (DA) level and downregulated expression of neurodevelopment-related genes, such as shha, mbp, neurog1, and gfap. However, secondary to 48-h restoration in fish medium (i.e., at 168 hpf), the neurotoxicity described above was prominently ameliorated. The results showed that PZA at the concentrations we tested was notably neurotoxic to larval zebrafish, and this nerve injury was restorable after PZA withdrawing. Therefore, this finding will probably provide a reference for clinical medication.

Bioequivalence and Pharmacokinetic Evaluation of 2 Pyrazinamide Formulations in Healthy Chinese Adults: A Single-Dose, Open-Label, Randomized-Sequence, 2×2 Crossover Study.

A single‐dose, open‐label, randomized‐sequence, 2×2 crossover study was conducted in healthy Chinese adults, after fasting and postprandial, to evaluate the bioequivalence of 2 pyrazinamide (PZA) formulations. Fasting and postprandial tests were conducted in 24 cases. Test‐reference and reference‐test were randomly divided into 2 sequence groups, with 12 cases in each group. The concentration of PZA in plasma was determined after 0.5 g single oral PZA test and reference formulations by the high‐performance liquid chromatography–tandem mass spectrometry method. In the fasting group, the 90% confidence intervals (CIs) of the 2 formulations maximum plasma concentration (Cmax), area under the plasma concentration–time curve (AUC) from time 0 to last detectable plasma concentration, and AUC from time 0 to infinity after logarithmic conversion were 104.8% to 121.9%, 97.7% to 101.6%, and 97.7% to 101.6%, respectively. In the postprandial group, the 90%CIs of the 2 formulations’ Cmax, AUC from time 0 to last detectable plasma concentration, and AUC from time 0 to infinity after logarithmic conversion were 86.4% to 100.2%, 96% to 102%, 95.8% to 102.3%, respectively. The 90%CIs of the test/reference Cmax ratio and AUC ratio were within the acceptable range of 80.00% to 125.00% for bioequivalence under both fasting and postprandial conditions. No serious adverse events occurred during treatment with the test formulation or the reference formulation.

Simultaneous determination of first-line anti-tuberculosis drugs and one metabolite of isoniazid by liquid chromatography/tandem mass spectrometry in patients with human immunodeficiency virus-tuberculosis coinfection

The incidence rate of tuberculosis (TB) in patients with human immunodeficiency virus (HIV) infection is 26 times higher than that in other patients. Patients with both infections require long-term combination therapy, which increases therapy complexity and might lead to serious adverse reactions and drug-drug interactions. To optimize therapy for patients with HIV and TB coinfection, we developed an ultra-high-performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS) method to simultaneously quantify four anti-tuberculosis drugs and one isoniazid (INH) metabolite. Blood samples (n = 32) from 16 patients with HIV and TB coinfection were collected. Plasma protein precipitation with acetonitrile was followed by a hydrazine reaction between INH and cinnamaldehyde (CA) to produce phenylhydrazone (CA-INH) and dilution with heptafluorobutyric acid. The separation was performed on an Acquity UHPLC HSS T3 1.8 μm column (2.1 × 100 mm, Waters) with a mobile phase consisting of 10 mmol/L ammonium formate (pH = 4) in water (solvent A) and 0.1 % formic acid in methanol (solvent B) in a gradient elution. The compounds were detected using a positive multiple reaction monitoring model. INH, acetyl-INH (AC-INH), rifampicin (RIF), ethambutol (EMB), and pyrazinamide (PZA) showed good linear relationships in their quantitative ranges, with lower limits of quantification of 48, 192, 200, 96, and 480 ng/mL, respectively. The inter- and intraday precision was within 15 %, and the accuracy was between 85 % and 115 %. The mean plasma concentrations of INH, AC-INH, RIF, EMB, and PZA in patients were 1990.23 (24–16 600), 863.06 (96–2880), 3507.05 (229–9800), 808.10 (149–2130), and 18 838.33 (240–34 800) ng/mL, respectively. The plasma concentrations detected in the 16 patients were lower than the targeted concentrations in HIV-negative TB patients. In summary, we developed a simple UHPLC-MS/MS method for simultaneous quantification of first-line TB drugs, and successfully applied it for therapeutic drug monitoring in patients with HIV and TB coinfection. This method will facilitate monitoring of TB drugs in the future.

Pharmacokinetics and safety/tolerability of isoniazid, rifampicin and pyrazinamide in children and adolescents treated for tuberculous meningitis

ObjectiveTo assess the pharmacokinetics and safety/tolerability of isoniazid, rifampicin and pyrazinamide in children and adolescents with tuberculous meningitis (TBM).DesignProspective observational pharmacokinetic study with an exploratory pharmacokinetic/pharmacodynamic analysis.SettingHasan Sadikin Hospital, Bandung,...

Simultaneous determination of three antituberculosis drugs in the serum of patients with spinal tuberculosis by capillary electrophoresis.

The pharmacokinetic variations of a single drug in an antituberculosis regimen are associated with acquired drug resistance and therapy failure. This study aimed to develop a simple and effective method for monitoring the serum levels of isoniazid (INH), rifampicin (RFP), and pyrazinamide (PZA), three antibiotics used in patients with spinal tuberculosis using capillary electrophoresis (CE). A standard solution of INH, RFP, and PZA was prepared and mixed with serum to prepare the standard curve. The detection limit, quantification limit, precision, stability, repeatability, and sample recovery were determined. Then, INH, RFP, and PZA were measured from the leftover serum samples of all patients with spinal tuberculosis who were treated with 2SHRZ/2.5H2R2Z2 combined with surgery in a tertiary hospital in Qinghai from October 2015 to September 2017. A total of 107 patients with spinal tuberculosis treated using the 2SHRZ/2.5H2R2Z2 regimen combined with surgery were included in this study. All three antibiotics had linear standard curves with high correlation coefficients (R2 = 0.9997, 0.9994, and 0.9986). The recovery rates were 98.1% for INH, 96.5% for PZA, and 97.2% for RFP. The results from the serum samples showed that the plasma concentrations of INH (4.989 ± 1.692 μg mL-1) and RFP (9.400 ± 1.711 μg mL-1) reached effective therapeutic concentrations in all patients, but not PZA (33.860 ± 1.830 μg mL-1). The CE method for measuring INH, RFP, and PZA simultaneously in serum samples of patients with spinal tuberculosis is simple, rapid, and sensitive. This method is suitable for the routine monitoring of INH, RFP, and PZA concentrations in the serum of patients with spinal tuberculosis.

The Impact of First-Line Anti-Tubercular Drugs' Pharmacokinetics on Treatment Outcome: A Systematic Review.

BackgroundTuberculosis remains the major public health problem besides tremendous efforts to combat it. Most tuberculosis patients are treated with a standard dose of first-line anti-TB drugs. The cure rate, however, varies from patient to patient. Various factors have been related to anti-TB treatment failure. In recent years, studies associating lower plasma concentrations of anti-TB drugs with poor treatment outcomes are emerging although the results are inconclusive.ObjectiveInvestigate the impact of first-line anti-tubercular drugs pharmacokinetics on treatment outcome.MethodsA systematic search of Pubmed, EMBASE, Web of Science, and the Cochrane Library for articles published in the English language between January 2010 to June 2020 was conducted to identify eligible studies describing associations of first-line anti-tubercular drug pharmacokinetics with treatment outcomes. The primary outcomes considered were pharmacokinetics parameter results and its association with treatment outcome.ResultsThe search identified 1754 articles of which twelve articles; ten prospective observational studies and two controlled clinical trials fulfilled the eligibility criteria. The majority of the studies showed target concentrations for the first-line anti-tubercular drugs below the current standard range. Among the twelve studies, eleven studies assessed rifampicin pharmacokinetics of which eight reported association of drug concentration and treatment outcomes. Similarly, four out of eight and three out of seven reported drug concentration and treatment outcome association for isoniazid and pyrazinamide, respectively. Despite the low plasma concentration, a favorable treatment outcome was achieved for the bulk of the patients. Irrespective of the inconsistency, an increase in exposure to rifampicin improved the outcome, and lower rifampicin, isoniazid, and pyrazinamide concentration are associated with poor outcome. No data are available for ethambutol associating its pharmacokinetics with treatment outcomes.ConclusionThe pharmacokinetics of first-line antitubercular drugs can influence treatment outcomes. Further controlled clinical studies are, however, required to establish these relationships.

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 2.00-50.00μg/mL for pyrazinamide, 0.50-20.00μg/mL for both acetyl-isoniazid and isoniazid, and 1.20-25.00μg/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 1.20μg/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.

Intrapulmonary Pharmacokinetics of First-line Anti-tuberculosis Drugs in Malawian Patients With Tuberculosis.

BackgroundFurther work is required to understand the intrapulmonary pharmacokinetics of first-line anti-tuberculosis drugs. This study aimed to describe the plasma and intrapulmonary pharmacokinetics of rifampicin, isoniazid, pyrazinamide, and ethambutol, and explore relationships with clinical treatment outcomes in patients with pulmonary tuberculosis.MethodsMalawian adults with a first presentation of microbiologically confirmed pulmonary tuberculosis received standard 6-month first-line therapy. Plasma and intrapulmonary samples were collected 8 and 16 weeks into treatment and drug concentrations measured in plasma, lung/airway epithelial lining fluid (ELF), and alveolar cells. Population pharmacokinetic modeling generated estimates of drug exposure (Cmax and AUC) from individual-level post hoc Bayesian estimates of plasma and intrapulmonary pharmacokinetics.ResultsOne-hundred fifty-seven patients (58% HIV coinfected) participated. Despite standard weight-based dosing, peak plasma concentrations of first-line drugs were below therapeutic drug-monitoring targets. Rifampicin concentrations were low in all 3 compartments. Isoniazid, pyrazinamide, and ethambutol achieved higher concentrations in ELF and alveolar cells than plasma. Isoniazid and pyrazinamide concentrations were 14.6-fold (95% CI, 11.2–18.0-fold) and 49.8-fold (95% CI, 34.2–65.3-fold) higher in ELF than plasma, respectively. Ethambutol concentrations were highest in alveolar cells (alveolar cell–plasma ratio, 15.0; 95% CI, 11.4–18.6). Plasma or intrapulmonary pharmacokinetics did not predict clinical treatment response.ConclusionsWe report differential drug concentrations between plasma and the lung. While plasma concentrations were below therapeutic monitoring targets, accumulation of drugs at the site of disease may explain the success of the first-line regimen. The low rifampicin concentrations observed in all compartments lend strong support for ongoing clinical trials of high-dose rifampicin regimens.

Direct Determination of Pyrazinamide (PZA) Susceptibility by Sputum Microscopic Observation Drug Susceptibility (MODS) Culture at Neutral pH: the MODS-PZA Assay.

Pyrazinamide (PZA) is considered the pivot drug in all tuberculosis treatment regimens due to its particular action on the persistent forms of Mycobacterium tuberculosis However, no drug susceptibility test (DST) is considered sufficiently reliable for routine application. Although molecular tests are endorsed, their application is limited to known PZA resistance associated mutations. Microbiological DSTs for PZA have been restricted by technical limitations, especially the necessity for an acidic pH. Here, for the first time, MODS culture at neutral pH was evaluated using high PZA concentrations (400 and 800 μg/ml) to determine PZA susceptibility directly from sputum samples. Sputum samples were cultured with PZA for up to 21 days at 37°C. Plate reading was performed at two time points: R1 (mean, 10 days) and R2 (mean, 13 days) for each PZA concentration. A consensus reference test, composed of MGIT-PZA, pncA sequencing, and the classic Wayne test, was used. A total of 182 samples were evaluated. The sensitivity and specificity for 400 μg/ml ranged from 76.9 to 89.7 and from 93.0 to 97.9%, respectively, and for 800 μg/ml ranged from 71.8 to 82.1 and from 95.8 to 98.6%, respectively. Compared to MGIT-PZA, our test showed a similar turnaround time (medians of 10 and 12 days for PZA-sensitive and -resistant isolates, respectively). In conclusion, MODS-PZA is presented as a fast, simple, and low-cost DST that could complement the MODS assay to evaluate resistance to the principal first-line antituberculosis drugs. Further optimization of test conditions would be useful in order to increase its performance.