| Background and AimAccording to the report of World Health Organization,tuberculosis(TB)is the second largest killer among all the single infectious diseases,only second to AIDS.China is a high TB burden country with more than 9 million new cases per year.Tuberculous meningitis(TBM)is the most serious disease form among all types of tuberculosis.Both the diagnosis and treatment of TBM are very challenging,and consequently,incur high mortality and disability.Early diagnosis,timely and effective treatment are essential for the control of tuberculosis,and for reducing mortality and disability.Isoniazid,rifampicin,ethambutol and pyrazinamide are recommended by WHO as the first-line drugs for TB treatment,which together constitute the chemotherapy regimen for newly diagnosed tuberculosis.Isoniazid is currently known as the anti-TB drug with the strongest early bactericidal activity.Isoniazid is of vital importance for tuberculous meningitis chemotherapy because of its strong bactericidal activity and good meningeal permeability.With limited drug choice for TBM treatment,ensure that optimal efficacy of isoniazid for TBM treatment is critical.In realistic condition,although patients taking the standard doses of isoniazid 5 mg/kg·day,their plasma drug concentrations can have 3-7 times difference.The main cause of this phenomenon is the dramatically viarable metabolic rates of isoniazid for different individuals.Isoniazid is mainly metabolized by genetically polymorphic N-acetyltransferase 2(NAT 2)to acetylisoniazid(AcINH).A large number of literatures and our previous study have confirmed that NAT2 polymorphism incur the isoniazid concentration difference of plasma and cerebrospinal fluid.Patients might suffer treatment failure or drug resistance when they have very low blood concentration of isoniazid,while high risk of hepatotoxicity might happen due to high blood concentration.Therefore,monitoring the concentrations of isoniazid in plasma and cerebrospinal fluid concentration,identifying the NAT2 genotype of the patients are useful to achieve therapeutic effect and avoid adverse reactions.This study analyzed the population pharmacokinetic characteristics of isoniazid in PTB and TBM patients,and evaluated the effects of different factors such as NAT2 gene polymorphism on INH metabolism,and provide evidence for individualized dosage of isoniazid.MethodsIsoniazid and its main metabolite acetylisoniazid were detected by high-performance chromatography-mass spectrometry.Acetaminophen was used as an internal standard and the clinical samples were processed with methanol.The specificity,standard curve,quantitative range,accuracy,stability,precision and extraction recovery rate,etc.of the method were performed and validated accordingly.During 2017-2018,100 pulmonary tuberculosis(PTB)patients and 46 TBM patients were enrolled in Beijing Chest Hospital and the Affiliated Hospital of Zunyi Medical University.Blood samples and/or cerebrospinal fluid samples hwere collected,and the patients’ mdical records were investigated.Population pharmacokinetic models of isoniazid in patients with PTB and TBM were established separately by using a nonlinear mixed-effects model based on 89 PTB and 38 TBM patients’ data,respectively.The models were validated to analyze the effects of gender,body weight and NAT2 genotype on the population pharmacokinetic characteristics of patients.According to the model and literature reports,the individualized dosage of isoniazid for patients with different NAT2 genotypes were simulated with proposed effective plasma concentration 3-6 μg/mL.ResultA quantitative method for detection of isoniazid and acetylisoniazid in plasma and cerebrospinal fluid by high performance chromatography-mass spectrometry was established,which needs only 100 μL volume of plasma or cerebrospinal fluid;the standard curve ranges from 0.1 to 10 μg/mL and the lowest limit of quanlification is 0.1 μg/mL,which can basically cover the plasma and cerebrospinal fluid concentration ranges of isoniazid and acetylisoniazid;the accuracy deviation is within±15%(1.3%～7.9%).The relative standard deviations were below 0.3%and 10%for intra-and interday precision analyses,respectively.The variation of placing at room temperature,freeze-thaw and dilution were within±15%.The extraction recovery rate and matrix effects were 82.9%～88.3%and 88.9%～90.3%,respectively.The method had enough accuracy and specificity to be used in this study.195 plasma samples from 89 PTB patients were used for population pharmacokinetic model establishment and validaiton in PTB patients.The concentrations of isoniazid ranged from 0.1 to 7.2 μg/mL and the concentrations of acetylisoniazid ranged from 0.2 to 16.6 μg/mL.The number of patients of fast acetylation,intermediate acetylation and slow acetylation was 32(36.0%),38(42.7%)and 19(21.3%).The study of tuberculous meningitis included 83 plasma samples and 81 cerebrospinal fluid samples from 38 TBM patients.For TBM patients,the plasma and cerebrospinal fluid concentrations of isoniazid ranged from 0.1 to 9.7 μg/mL and 0.1 to 7.8 μg/mL,respectively.The number of patients of fast acetylation,intermediate acetylation and slow acetylation was 14(36.8%),18(47.4%)and 6(15.8%).A two-compartment model of primary absorption and elimination was conducted as the basic model,the population pharmacokinetic model of isoniazid in pulmonary tuberculosis and tuberculous meningitis patients was established,respectively.Covariate analysis showed that body weight and NAT2 genotype had significant effects on isoniazid clearance of pulmonary tuberculosis,especially the NAT2 genotype.The clearance of fast acetylators was 47.5 L/h,was 32.0 L/h for intermediate acetylators,12.6 L/h for slow acetylators.The population pharmacokinetic model of isoniazid in TBM patients showed that only NAT2 genotype had significant effect on isoniazid clearance.The clearance of fast acetylators was 47.5 L/h,was 28.8 L/h for intermediate acetylators,5.8 L/h for slow acetylators.Both models were proved stable and reliable through model verification.Among 47 PTB patients taking the regular dose of 300 mg/day,the 2 h isoniazid plasma concentrations of all the fast-acetylators(16/16)were below 3 μg/mL,and 95%(19/20)of intermediate acetylators were below 3 μg/mL,and 27.3%(3/11)of slow acetylators were below this standard.Dose simulations of PTB patients were performed by model with propsed effective plasma concentration of 3-6 μg/mL,the presumable dosage of isoniazid for fast,intermediate,and slow acetylators PTB patients were 700,500,and 300 mg/day,respectively.By adjusting the dosage according to the NAT2 genotypes,it is possible to achieve the proposed effective plasma concentration for most patients while still have low chance of adverse reaction.ConclusionIn this study,it is the first time to simulate the effect of NAT2 genotypes on the population pharmacokinetic of isoniazid in Chinese adult PTB patients.The result shows that the NAT2 genotype was the most significant factor affecting the drug clearance.Accordint to our assay,individualized dosage of isoniazid for fast,intermediate,and slow acetylators of PTB patients were presumed to be 700,500,and 300 mg/day,respectively.This study established the population pharmacokinetic model of isoniazid in Chinese TBM patients for the first time.The NAT2 genotype is a significant factor affecting the clearance of isoniazid.This study provides a theoretical basis for individulized dosage of isoniazid in TBM patients based on the NAT2 genotype. |
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