AAC Accepts, published online ahead of print on 20 May 2013 Antimicrob. Agents Chemother. doi:10.1128/AAC.02468-12 Copyright © 2013, American Society for Microbiology. All Rights Reserved.
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Isoniazid, rifampicin and pyrazinamide plasma concentrations in relation to
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treatment response in Indonesian pulmonary tuberculosis patients
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Running title: Antituberculosis drug levels and treatment response
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Authors: Erlina Burhan1*, Carolien Ruesen2*#, Rovina Ruslami3, Arum Ginanjar1, Hadiarto
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Mangunnegoro1, Purwantyastuti Ascobat4, Rogier Donders5, Reinout van Crevel2, Rob
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Aarnoutse6
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*
Shared first authorship
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Affiliations:
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1
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Indonesia, Jakarta, Indonesia
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Netherlands
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Bandung, Indonesia
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Jakarta, Indonesia
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Nijmegen, The Netherlands
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Department of Pulmonology and Respiratory Medicine, Faculty of Medicine, University of
Department of Internal Medicine, Radboud University Medical Centre, Nijmegen, The
Department of Pharmacology and Therapy, Medical Faculty, University of Padjadjaran,
Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Indonesia,
Department of Epidemiology, Biostatistics and HTA, Radboud University Medical Centre,
Department of Pharmacy, Radboud University Medical Centre, Nijmegen, The Netherlands 1
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#
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Medical Centre, and Nijmegen Institute for Infection, Inflammation and Immunity, PO Box 9101,
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6500 HB, Nijmegen, The Netherlands. E-mail:
[email protected]
Corresponding author: Carolien Ruesen, BSc. Department of Medicine, Radboud University
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Word count
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Abstract: 233
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Manuscript (excluding title page, abstract, references, figures and tables): 3603
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Key words: Tuberculosis, plasma concentration, pharmacokinetics, response
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Abstract
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Background.
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Numerous studies have reported low concentrations of antituberculosis drugs in tuberculosis
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(TB) patients, but few studies have examined whether low drug concentrations affect TB
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treatment response.
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Patients and methods.
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We examined steady-state plasma concentrations of isoniazid, rifampicin, and pyrazinamide at
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two hours after administration of drugs (C2h) among 181 patients with pulmonary tuberculosis in
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Indonesia and related these to bacteriological response during treatment.
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Results.
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C2h values below reference values for either isoniazid, rifampicin, or pyrazinamide were found in
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91% of patients; 60% had at least two low C2h concentrations. The isoniazid C2h was noticeably
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lower in fast versus slow acetylators (0.9 mg/L vs. 2.2 mg/L, p70 kg). The anti-TB drugs were administered in fixed dose
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combinations (FDC) from Kimia Farma that are approved by the Indonesian national programme
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based on bio-equivalence studies. Anti-TB drugs were taken without food.
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Basic demographic and clinical information was collected from all participants, including
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age, sex, body weight and length (to calculate body mass index; BMI), co-morbidities (including
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HIV infection and DM) and concomitant drug use. HIV- and DM-status was checked through
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blood samples collected after 4 weeks. AFB and sputum culture were collected at baseline and at
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4, 8, and 24 or 32 weeks (32 weeks for retreatment cases). Spoligotyping was performed at
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baseline. Drug susceptibility testing was performed on all isolates using the proportion method 6
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[19], in a WHO-accredited laboratory. Patients with resistant isolates were excluded from the
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study. Chest X-rays were made at baseline, and after 24 or 32 weeks and extensive lesions were
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categorized by size (area less than 5 vertebrae, 5-9 vertebrae, or more than 9 vertebrae).
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Blood sampling, bio-analysis and pharmacokinetic data analysis
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Patients had refrained from food at least eight hours before drug intake at the pharmacokinetic
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sampling day. Plasma drug concentrations from blood collected 2 hours after administration of
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drugs (C2h) were used as the estimated peak plasma concentration (Cmax). Drug sampling took
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place at 4 weeks after inclusion in the study, because of the expected steady state in the
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pharmacokinetics of the TB drugs at that time. Additionally, six weeks after inclusion in the
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study, full pharmacokinetic curves were obtained in nine subjects. Serial venous blood samples
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were collected just before, and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12 and 24 hours after observed TB
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drug intake. Plasma was separated and stored at -80oC immediately until transport on dry ice to
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The Netherlands for bio-analysis. Isoniazid and acetylisoniazid concentrations were measured
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with a validated method comprising liquid-liquid extraction followed by Ultra Performance
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Liquid Chromatography (UPLC) with ultraviolet (UV) detection. Accuracy was between 97.8%
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and 106.7% for isoniazid and between 98.0% and 108.9% for acetylisoniazid, dependent on the
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concentration level. The intra- and inter-assay coefficients of variation were less than 13.4 % and
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less than 3.2% (dependent on the concentration) over the range of 0.05-15.1 mg/L for isoniazid;
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and less than 4.2% and less than 5.7% over the range of 0.16-16.2 mg/L for acetylisoniazid.
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Lower limits of quantification were 0.05 mg/L for isoniazid and 0.16 mg/L for acetylisoniazid.
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The concentrations of rifampicin and pyrazinamide were assessed with validated high-
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performance liquid chromatographic (HPLC) assays by methods described previously [9]. All
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analytical methods performed well at two concentration levels in the first round of a recently 7
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developed interlaboratory proficiency testing programme for TB drugs [20]. In patients with full
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pharmacokinetic curves, pharmacokinetic parameters were assessed using standard
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noncompartmental methods in WinNonLin Version 5.3 as described before [9].
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Acetylator status for isoniazid was determined phenotypically by calculating the
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metabolic ratio (MR) for isoniazid (acetylisoniazid C2h /isoniazid C2h), with fast acetylators
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having MRs ≥ 1.5 and slow acetylators having MRs < 1.5 [21].
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Statistical analysis
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Correlation analyses were performed for the patients with full PK-curves to check whether C2h
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was a good representative of Cmax or total exposure (AUC0-24h ) for the three drugs. In addition,
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independent samples t-tests and correlation analyses were carried out among all patients to
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identify determinants of pharmacokinetics of the three different antituberculosis drugs.
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The relation between plasma concentrations of antituberculosis drugs and culture results
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at week 4 and 8 was explored among patients with a positive culture at week 0. Treatment
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response was defined as ‘poor’ if patients had a positive culture at 8 weeks of treatment [22]
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(primary analysis), or if patients had any positive culture at 4, 8 or 24/32 weeks of treatment
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(post-hoc analysis). Univariate and multivariate logistic regression analyses were used to detect
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determinants of poor treatment response. Determinants evaluated were demographic variables
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(i.e. gender, age, weight, BMI), smoking, new case or retreatment case, Beijing genotype for M.
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tuberculosis, comorbidities, chest X-ray findings at baseline, sputum microscopy at baseline and
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plasma concentrations of the three TB-drugs. For the plasma concentrations of each of the three
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TB drugs, odds ratios (ORs) for a poor treatment response were assessed for an interquartile
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range increase in C2h, i.e. an increase in C2h values from the 25th percentile to the 75th percentile
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of the observed C2h values (interquartile OR). 8
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C2h values were ln transformed before statistical analysis. The multivariate models
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included C2h of the three drugs separately, any variable with p
