Browsing by Author "Wiesner, Lubbe"
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- ItemOpen AccessA pharmacokinetic and antimalarial efficacy evaluation of pyridodibemequines and their metabolites(2021) Redhi, Devasha; Wiesner, Lubbe; Egan, Timothy JohnThe recurring challenge of the emergence of drug resistance necessitates the continual development of improved antimalarial treatments, which can target parasite strains that display reduced susceptibility towards current therapy. Consequently, a novel series of dual functioning pyridodibemequine (PDBQ) compounds were designed with the intention of reversing resistance in chloroquine-resistant (CQR) strains of Plasmodium falciparum. These hybrid molecules integrate a 4-amino-7-chloroquinoline antiplasmodial core with a modified dibenzylmethylamine side chain, which interacts with the CQR mutant P. falciparum chloroquine resistance transporter (PfCRT) to hinder the efflux of chloroquine (CQ) from its site of action, thereby reversing CQ resistance. The parent compounds of the PDBQ series, which differ in the ortho-, meta-, and para-orientation of the dibenzylmethylamine side chain, displayed favourable in vitro potency against chloroquine-sensitive (CQS) and CQR strains of P. falciparum; however, they were shown to be metabolically labile. Structure elucidation demonstrated that all formed PDBQ metabolites retained the 4-amino-7-chloroquinoline pharmacophore of the parent. Thus, the major metabolites, M1 and M2, generally conserved the in vitro antimalarial activity and selectivity of the parent compounds. Mechanistic studies revealed that the antiplasmodial activity of the PDBQ parent compounds and major metabolites primarily results from the inhibition of haemozoin formation, culminating in a toxic accumulation of ferriprotoporphyrin IX. The parents and major metabolites exhibited minimal toxicity against a mammalian cell line, and the metabolites are proposed to display reduced systemic toxicity, and human ether-a-go-go-related gene liability compared to the parent compounds. Furthermore, the major metabolites generally exhibited similar or improved in vitro solubility, permeability, lipophilicity, and metabolic stability compared to the parent compounds. Therefore, given their favourable in vitro characteristics, the major active metabolites of each parent PDBQ compound were further evaluated in this project to determine their potential as early preclinical antimalarial lead candidates. The proof of concept study presented herein investigated the in vivo pharmacokinetics (PK) of the PDBQ series of parents and major metabolites in a healthy murine model to allow the rational selection of candidates to be evaluated for their in vivo antimalarial efficacy and PK in a P. falciparum-infected murine model. For the PK studies in healthy and malaria-infected mice, analyte detection from whole blood was achieved using high-performance liquid chromatography coupled to tandem mass spectrometry. The bioanalytical methods were developed and partially validated based on a fit-for-purpose approach, which ensured that the generated PK concentration data was reliable and accurate. Lastly, given the significance of combination therapy in delaying the onset of drug resistance, fixed-dose ratio isobologram analyses were performed to probe the potential of the lead candidates to be used synergistically with an antimalarial partner drug possessing a distinct mechanism of action to haemozoin inhibition. Additionally, the ability of the lead candidates to reverse CQ resistance was evaluated in a CQR strain of P. falciparum. A comparative PK study was performed in a healthy murine model to determine whether the parent PDBQ compound should be used as a strategy to deliver the active metabolites or whether the active major metabolite should be directly administered. This was achieved by oral administration of the parent PDBQ compound and subsequent quantification of the parent compound and formation of the major metabolites. In addition, each pre-synthesised major metabolite was orally administered, at the equivalent parent dose, to characterise the PK profile of the individual active metabolite. These PK studies revealed that the overall oral exposure of the antimalarial pharmacophore was markedly greater after direct administration of the preformed metabolite compared to the cumulative oral exposure of the parent and formed metabolites after administration of the parent PDBQ compound. Furthermore, the metabolites attained higher maximal concentrations and maintained circulating concentrations which favourably exceeded their respective in vitro half-maximal inhibitory concentration at 24 h post-oral administration compared to the parent compounds at the equivalent oral dose. These findings substantiated the direct administration of the preformed PDBQ major metabolite over the parent compound. From the series of PDBQ metabolite derivatives, compounds 43M1 and 47M1 displayed the highest maximal concentrations of 8 ± 1 and 9.4 ± 0.5 μM, respectively and the greatest oral exposures of 62 ± 3 and 93 ± 9 µM.h, respectively, after single 20 mg/kg oral administrations of either 43M1 or 47M1. Given their encouraging PK profiles, 43M1 and 47M1 were selected to progress to the subsequent phase of the study which evaluated their in vivo antimalarial efficacy and pharmacokinetic/pharmacodynamic (PK/PD) relationship in a P. falciparum-infected humanised murine model. 43M1 and 47M1 were efficacious against asexual intraerythrocytic P. falciparum infection in humanised mice, where both compounds displayed a 98% reduction in parasitaemia after 4 consecutive daily oral administrations of 20 mg/kg of either 43M1 or 47M1 compared to the untreated control. The PK/PD analysis revealed dose-dependent reductions in parasitaemia; and the doses required to produce 90% of the maximal parasiticidal response (ED90) were 12 and 7.7 mg/kg for 43M1 and 47M1, respectively. Additionally, the oral exposures required to achieve the effect at the ED90 were 6.2 and 18.6 µM.h for 43M1 and 47M1, respectively. 43M1 or 47M1 demonstrated overall in vitro antimalarial synergy with dihydroartemisinin or atovaquone and additivity with methylene blue in CQS and CQR strains of P. falciparum. 43M1 or 47M1 with mefloquine or lumefantrine displayed in vitro antimalarial synergy in a CQS strain of P. falciparum; however, in a CQR strain, antagonism and additivity were displayed with mefloquine and lumefantrine, respectively. Additionally, 43M1 and 47M1 were unable to potentiate the in vitro antiplasmodial activity of CQ in a CQR strain of P. falciparum which suggested that, unlike the parent PDBQ compound, the metabolite did not possess the ability to reverse CQ resistance. The promising in vivo antimalarial efficacy of 43M1 and 47M1 against P. falciparum and their prospective in vitro antimalarial synergy with dihydroartemisinin and atovaquone underlines the potential of 43M1 and 47M1 for further development as preclinical antimalarial candidates.
- ItemOpen AccessAntimalarial activity and pharmacokinetic properties of new chemical entities(2013) Dambuza, Ntokozo Shirley; Smith, Peter John; Wiesner, Lubbe; Chibale, KellyIncludes abstract. Includes bibliographical references.
- ItemOpen AccessThe Artemiside-Artemisox-Artemisone-M1 Tetrad: Efficacies against Blood Stage P. falciparum Parasites, DMPK Properties, and the Case for Artemiside(2021-12-03) Gibhard, Liezl; Coertzen, Dina; Reader, Janette; van der Watt, Mariëtte E; Birkholtz, Lyn-Marie; Wong, Ho Ning; Batty, Kevin T; Haynes, Richard K; Wiesner, LubbeBecause of the need to replace the current clinical artemisinins in artemisinin combination therapies, we are evaluating fitness of amino-artemisinins for this purpose. These include the thiomorpholine derivative artemiside obtained in one scalable synthetic step from dihydroartemisinin (DHA) and the derived sulfone artemisone. We have recently shown that artemiside undergoes facile metabolism via the sulfoxide artemisox into artemisone and thence into the unsaturated metabolite M1; DHA is not a metabolite. Artemisox and M1 are now found to be approximately equipotent with artemiside and artemisone in vitro against asexual P. falciparum (Pf) blood stage parasites (IC50 1.5–2.6 nM). Against Pf NF54 blood stage gametocytes, artemisox is potently active (IC50 18.9 nM early-stage, 2.7 nM late-stage), although against the late-stage gametocytes, activity is expressed, like other amino-artemisinins, at a prolonged incubation time of 72 h. Comparative drug metabolism and pharmacokinetic (DMPK) properties were assessed via po and iv administration of artemiside, artemisox, and artemisone in a murine model. Following oral administration, the composite Cmax value of artemiside plus its metabolites artemisox and artemisone formed in vivo is some 2.6-fold higher than that attained following administration of artemisone alone. Given that efficacy of short half-life rapidly-acting antimalarial drugs such as the artemisinins is associated with Cmax, it is apparent that artemiside will be more active than artemisone in vivo, due to additive effects of the metabolites. As is evident from earlier data, artemiside indeed possesses appreciably greater efficacy in vivo against murine malaria. Overall, the higher exposure levels of active drug following administration of artemiside coupled with its synthetic accessibility indicate it is much the preferred drug for incorporation into rational new artemisinin combination therapies.
- ItemOpen AccessCorrection to: Pharmacokinetic profile of amodiaquine and its active metabolite desethylamodiaquine in Ghanaian patients with uncomplicated falciparum malaria(2021-03-19) Anyorigiya, Thomas A; Castel, Sandra; Mauf, Katya; Atuguba, Frank; Ogutu, Bernhards; Oduro, Abraham; Dosoo, David; Asante, Kwaku-Poku; Owusu-Agyei, Seth; Dodoo, Alexander; Hodgson, Abraham; Binka, Fred; Workman, Lesley J; Allen, Elizabeth N; Denti, Paolo; Wiesner, Lubbe; Barnes, Karen IAn amendment to this paper has been published and can be accessed via the original article.
- ItemOpen AccessDetermination of biomarkers for toxicity and antiretroviral adherence in hair in South African patients(2018) Johnston, Jenna; Wiesner, Lubbe; Smith, PeterBackground: Substance abuse is one of the many factors associated with poor levels of antiretroviral adherence and is also prevalent among HIV-infected individuals. Ethyl glucuronide, a minor metabolite of alcohol, is a stable biomarker in hair that can be used to detect and monitor alcohol consumption over long time periods. Drugs of abuse are also detected in hair. Hair provides a longer window of drug detection compared to blood and urine. Recently, hair has also been studied as an alternative matrix for adherence monitoring and concentrations of antiretrovirals in hair have been shown to be closely correlated with virologic outcomes. This study investigated the impact of substance abuse on adherence among HIV-infected patients attending an antiretroviral therapy clinic in Cape Town by measuring drug concentrations in hair. Efavirenz levels in hair were also measured to investigate the usefulness of using hair analysis as a method of adherence monitoring within the South African context. Method: This study describes the development and validation of three liquid chromatography tandem mass spectrometry methods of hair analysis. The first method developed was for the quantification of ethyl glucuronide in 20 mg samples of hair. This method was validated over the calibration range 7.5 - 480 pg/mg. Secondly, a qualitative method was developed to screen hair samples for amphetamine, methamphetamine, cocaine, benzoylecgonine, cocaethylene and methaqualone. The final method developed was for the quantification of efavirenz in 0.2 mg samples of hair. This method was validated over the calibration range 0.625 - 40 ng/mg. The validated methods were applied to 257 samples of hair collected from 135 HIV-infected patients during visits to the clinic at weeks 16, 32 and 48. The results generated from the analysis of the hair samples were analysed in the context of additional adherence measurements collected for a related randomized controlled study. Results: Analysis of the hair samples for ethyl glucuronide demonstrated that 27% of the samples analysed contained levels above 30 pg/mg which is the cutoff value suggested by the Society of Hair Testing to identify heavy drinkers. The results also show limitations to using the CAGE alcohol abuse screening tool which had a poor sensitivity of only 28.8%. Eight (5.9%) out of the 135 participants were identified to be chronic drug users, and of these five (62.5%) were identified to be heavy drinkers as well. The most commonly abused drug identified in the screen was methaqualone. The median efavirenz levels at weeks 16, 32 and 48 were 5.52 ng/mg (IQR: 3.60 - 9.77), 5.75 ng/mg (IQR: 3.21 - 8.18) and 4.89 ng/mg (IQR: 3.10 - 7.94) respectively. Participants with the poor CYP2B6 metaboliser genotype had significantly higher median efavirenz hair concentrations compared to participants with intermediate and extensive genotypes (P < 0.0001). Efavirenz levels in hair and plasma samples were strongly correlated throughout the study (Spearman correlation coefficients: 0.672 - 0.741, all P values < 0.0001). Substance abuse had no impact on adherence measured by an electronic adherence monitoring device. No significant correlation was observed between adherence and levels of efavirenz in hair. Conclusions: Methods of hair analysis were developed and successfully applied to hair samples in the context of better understanding the impact of substance abuse on adherence. The results from the analysis of the hair samples provided insight into the prevalence of substance abuse among HIV-infected patients. The strong correlation observed between levels of efavirenz in hair and plasma suggest that, in this subset of HIV-infected patients, a single plasma concentration was as good an adherence measure as a hair concentration. The hair analysis methods developed and validated in this study are novel in South Africa and demonstrate the potential of this matrix to be used in various contexts within the country.
- ItemOpen AccessDetermination of Rifapentine and 25-O-desacetyl Rifapentine from 100 µl human breastmilk by LC-MS/MS using protein precipitation and solid phase extraction(2019) Mkhize, Buyisile; Wiesner, Lubbe; Kellermann, Tracy; Norman, JenniferThere is currently no information available on the transfer of the second-line anti-TB drug, rifapentine and its metabolite, into breastmilk. The subsequent implications to the breastfed infant, as well as consequences of long-term exposure to potentially sub-therapeutic drug levels with regards to the development of drug resistant bacteria is therefore not known. A liquid chromatography method with detection by mass spectrometry (LC-MS/MS) is described for the quantification of rifapentine and its metabolite, 25-O-desacetyl rifapentine in human breastmilk, using rifampicin-d3 as an internal standard. An AB Sciex 4000 mass spectrometer at unit resolution in the multiple reaction monitoring (MRM) mode was used to monitor the transition of the protonated precursor ions m/z 877.5, m/z 835.4 and m/z 827.4 to the product ions m/z 151.1, m/z 453.2 and m/z 151.200 for rifapentine, 25-Odesacetyl rifapentine and rifampicin-d3, respectively. Ions were produced using Electro spray ionisation (ESI) in the positive ionisation mode. An Agilent Poroshell 120 EC-C18 (4.6 x 50 mm, 2.7 μm) column was used for chromatographic separation using an isocratic method of acetonitrile containing 0.1% formic acid and water containing 10% methanol and 0.1% formic acid (55:45, v/v), at a flow rate of 450 µl per minute. The retention times for rifapentine, 25- O-desacetyl rifapentine and rifampicin-d3 were ≈2.67, ≈1.88 and ≈1.96 minutes, respectively. The method was developed and validated according to FDA guidelines. The extraction method consisted of a combination of protein precipitation and C18 solid phase extraction. Rifapentine and 25-O-desacetyl rifapentine showed no significant carry over on the Agilent autosampler. The method was reproducible when analysed with human breastmilk from six different sources from Western Cape Maternity Breastmilk Bank. Rifapentine mean extraction yield was 84.2% (%CV = 1.7) and that of 25-O-desacetyl rifapentine was 71.1% (%CV = 10.8). Rifapentine had a mean process efficiency of 80.4% (%CV = 4.7) and that of 25-O-desacetyl rifapentine was 95.7% (%CV = 5.7). Intra- and inter day validations over 3 days were performed. The calibration curves fit a quadratic regression with 1/x weighting over a concentration range of 2 - 2000 ng/ml for both rifapentine and 25-Odesacetyl rifapentine based on the analyte/internal standard peak area ratios, the accuracy ranged from 92.9% to 105.5% for both rifapentine and 25-O-desacetyl rifapentine standards. The Quality Controls accuracy ranged from 97.4% to 106.0% for both rifapentine and 25-Odesacetyl rifapentine. Stock solutions were shown to be stable for 69 days at -80°C. v Rifapentine and 25-O-desacetyl rifapentine were stable in human breastmilk for up to 72 hours at approximately -80°C and -20°C, on benchtop for ≈4.5 hours on ice and after three freeze-thaw cycles. Rifapentine and 25-O-desacetyl rifapentine were shown to be stable on the autosampler over a period of approximately 48 hours after which the entire batch could be reinjected. Autosampler stability revealed a decrease in peak area ratios, indicating that a partial batch cannot be reinjected after 48 hours in case of instrument failure. This method will be utilized in the analysis of patient samples from a clinical study in South Africa in breastfeeding women with tuberculosis.
- ItemOpen AccessDetermination of total, unbound, and intracellular concentrations of the antiretroviral drugs Efavirenz, Lopinavir, and Ritonavir(2022) Kriegler Foster, Katie; Wiesner, Lubbe; Kellermann, TracyEfavirenz, lopinavir, and ritonavir are antiretroviral drugs used for the treatment of HIV in South Africa. Plasma concentrations of these drugs are routinely monitored to ensure efficacy, minimise adverse effects, and adjust dosing. However, variability exists in patient treatment response and tolerability, which cannot always be explained by the therapeutic drug monitoring results. This may be due to variability in the amount of drug reaching the target site within the HIV-infected cells. Therefore, intracellular drug concentrations could provide a more accurate depiction of drug exposure. An alternative to intracellular drug concentrations could be the quantitation of drug not bound to plasma proteins as this is the portion able to diffuse into tissues and cells to exert a therapeutic effect. A method is described for the quantification of intracellular efavirenz, lopinavir, and ritonavir from one million human peripheral blood mononuclear cells. In addition, the quantification of unbound efavirenz, lopinavir, and ritonavir from human plasma using ultracentrifugation is demonstrated, including a novel surrogate matrix. The two methods were validated according to the United States Food and Drug Administration and European Medicines Agency guidelines and proven to be accurate, precise, and reproducible. Both methods were submitted to the United States National Institute of Allergy and Infectious Diseases' Clinical Pharmacology Quality Assurance group for review and have been approved for use on clinical samples. A proof-of-concept correlation study of intracellular, unbound, and total drug concentrations is described using blood samples from six HIV-positive patients. A further patient unresponsive to lopinavir treatment, despite total plasma concentrations within the normal therapeutic range, was also evaluated. Paired plasma and cell samples indicated that the drug reached the target site within the cells, eliminating a possible cause of treatment failure. These findings show the utility and validity of these methods in a clinical setting to provide an overall view of treatment response and support their novel application in individualised patient care in South Africa.
- ItemOpen AccessThe determination of β-endosulfan and endosulfan sulfate in human serum with dialkylphosphate metabolites as urinary markers using LC-MS/MS electrospray ionization(2017) Bergh, Werner; Wiesner, Lubbe; Van der Merwe, Marthinus JohannesTwo separate bioanalytical methods were developed, validated and applied to determine agricultural exposure to organochlorine and organophosphorus pesticides using different biological matrices as reference sources. The method that was validated for the quantification of the organochlorine compounds was used to simultaneously determine β-endosulfan [6, 7, 8, 9, 10, 10- hexacloro-1, 5, 5a, 6, 9, 9a- hexahydro- 6, 9-methano-2, 4, 3-benzodioxathiepin-3- oxide] and one of its main metabolites, endosulfan sulfate, in human serum. In a second bioanalytical method, urinary dialkylphosphate metabolites have been assessed as markers to estimate the exposure to organophosphorus pesticides, focusing on three of the six organophosphorus urinary metabolites, namely dimethyl phosphate, dimethyl thiophosphate and diethyl phosphate. For both the bioanalytical methods, liquid-liquid extraction was used for sample preparation and high performance liquid chromatography with tandem mass spectrometry as detection method due to its high sensitivity and selectivity. Chromatographic separation for both bioanalytical methods was achieved by performing reverse phase chromatography on C18 analytical columns. Isocratic elution with a mobile phase composed of acetonitrile, methanol and water was employed for the analysis of the organochlorine compounds while the organophosphorus compounds were eluted using gradient elution with a mobile phase consisting of acetonitrile and 20 mM ammonium acetate. A triple quadrupole mass spectrometer equipped with an electrospray ionization source operating in the negative ionization mode was used for mass detection of all the analytes, employing multiple reaction monitoring as scan mode. Calibration standards and quality control samples for both analyses were prepared in the biological matrix in which the samples for each determination were collected, i.e. serum for the determination of the organochlorine compounds and stripped urine for the organophosphorus compounds. Deuterated internal standards were used in the bioanalytical method for the determination of the organophosphorus compounds whereas the organochlorine compounds were determined without the use of an internal standard due to unavailability of suitable internal standards. The calibration ranges for the determination of β-endosulfan and endosulfan sulfate were 0.8 ng/ml to 200 ng/ml and 0.117 ng/ml to 30 ng/ml, respectively, and 1.0 ng/ml to 30 ng/ml for the dialkylphosphate metabolites of the organophosphorus compounds. These sensitive and robust quantitation methods were successfully applied to quantify 219 serum and 187 urine samples that were collected from agricultural workers with the purpose to determine whether they were exposed to any of the investigated organochlorine or organophosphorus compounds. No traces of β-endosulfan and endosulfan sulfate were found in any of the serum samples that were analyzed, however, significant amounts of the three organophosphorus compounds dimethyl phosphate, dimethyl thiophosphate and diethyl phosphate were present in the urine samples.
- ItemOpen AccessDevelopment and validation of liquid chromatography mass spectrometry (L/C/MS/MS) assay for the determination of plasma 4betahydroxycholestrol and cholesterol in HIV infected children in Africa(2015) Ngwalero, Precious; Wiesner, Lubbe; McIlleron, Helen4β-hydroxycholesterol (4β-OHC) is a metabolite of cholesterol formed by Cytochrome (CYP) 3A4/5/7 enzymes. It has recently been proposed as an endogenous biomarker forCYP3A4/5/7 activity. This may be useful in prediction of drug-drug interactions and other metabolic processes affected by regulators of CYP3A activity. The aim of this study was to develop and validate an LC/MS/MS assay for the determination of 4β-OHC in human plasma and use 4β-OHC as a biomarker of CYP3A4/5/7 metabolism in HIV-infected children with and without treatment in Africa. Determination of 4β-OHC from plasma was performed by saponification and derivatisation reaction processes followed by high performance liquid chromatography with MS/MS detection on an AB Sciex Qtrap 5500 mass spectrometer. Since 4β-OHC is an endogenous metabolite in human plasma, a stable isotope labelled (SIL) analogue, 4β-OHC-D7, was used as a surrogate analyte for the preparation of calibration standards and quality controls. A second SIL analogue, 4β-OHC-D4 was used as the internal standard. The transitions of the protonated derivatised products were monitored atm/z 613, 620 and 617 to the product ions m/z 490, 497 and 494 for 4β-OHC, 4β-OHC-D7and 4β-OHC-D4 respectively. The calibration curve fitted a quadratic (weighted by1/concentration2) regression over the range 2-500 ng/ml. Validation accuracy and precision statistics summary for three consecutive runs were between 98.9% and 103%, and 3.5%and 12% respectively of all quality controls. The assay's recovery, selectivity and analyte stability were established. The validated assay was successfully applied on clinical samples, where 4β-OHC was used as a biomarker to investigate the levels of CYP3A induction in HIV-infected children with and without treatment containing non-nucleoside reverse transcriptase inhibitors (NNRI).It was found that plasma 4β-OHC concentrations at baseline were significantly lower in children belonging to the naïve group compared to nevirapine (NVP) and efavirenz (EFV)groups. When NVP and EFV groups were compared at non-baseline treatment weeks, the median 4β-OHC concentrations were significantly higher in EFV group than the NVP group. Regarding the effect of time on treatment, a significant increase in 4β-OHC concentrations was observed from baseline to each of the non-baseline weeks in naïve group. Conversely, in the NVP group, there was a significant decrease in 4β-OHC concentrations from baseline to each of the non-baseline weeks. Time did not show any significant effect on 4β-OHC concentrations in EFV group. Furthermore, at baseline, age, sex and weight did not affect 4β-OHC concentrations in all the three groups. This study has provided a method that would be utilised to determine plasma 4β-OHC concentrations using relatively small volumes - typical of samples taken from children. The results of this study suggest that children on antiretroviral therapy (ART) are at risk of effects of CYP3A induction, as indicated by the increase of 4β-OHC concentrations in the NVP and EFV groups. Additionally, prolonged use of the ART may activate some nuclear receptors that regulate CYP3A enzyme activity thereby negatively affecting, for example, the regulation of lipid and glucose metabolism. The developed method may therefore be useful in predicting drug-drug interactions in the context of multiple therapy and may also be used in predicting other metabolic processes affected by regulators of CYP3A activity. Further prospective studies with larger sample sizes are required to confirm and build on the evidence shown in this study.
- ItemOpen AccessDevelopment and validation of selective and sensitive LC-MS/MS Methods for determination of para-aminosalicyclic acid and cycloserine/terizidone applicable to clinical studies for the treatment of tuberculosis(2018) Smit, Michiel Johannes; Wiesner, Lubbe; Castel, SandraA method was validated for the quantification of para-aminosalicylic acid (PAS) in human plasma. The technique consisted of a protein precipitation extraction, followed by high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) detection. Rilmenidine was used as the internal standard (ISTD). Analyte mean extraction yields determined were ~100.3% (CV % = 3.3). The extraction procedure was followed by liquid chromatographic separation using a Phenomenex Synergi Hydro-RP (150 x 2.0 mm, 4µm) analytical column. An isocratic mobile phase containing methanol, water and formic acid (40:59.8:0.2, v/v/v) was used at a flow-rate of 300 µl per minute. The retention times for PAS and rilmenidine were, ~2.4 and ~1.6 minutes, respectively. An AB Sciex API 3000 mass spectrometer at unit resolution in the multiple reaction monitoring (MRM) mode was used to monitor the transition of the protonated precursor ions m/z 154.1 and m/z 181.2 to the product ions m/z 80.2 and m/z 95.2 for PAS and the ISTD, respectively. Electro Spray Ionisation (ESI) was used for ion production. Accuracy and precision were assessed over three consecutive, independent runs. The calibration curve fits a quadratic (weighted by 1/x concentration) regression for PAS over the range 0.391 – 100 µg/ml, based on peak area ratios. A 1:1 and 1:4 dilution of the QC Dilution sample showed that concentrations of up to 160 µg/ml of PAS in plasma could be analysed reliably when diluted into the calibration range. Endogenous matrix components were found to have an insignificant effect on the reproducibility of the method, when human plasma originating from eight different sources were analysed. PAS was found to be stable in human plasma for 21 months kept at ~-80°C, for up to 21 hours at room temperature and when subjected to 3 freeze-thaw cycles. Stock solutions of PAS in methanol were stable for 2 days when stored at ~80°C and for 24 hours when stored at room temperature, ~4°C and ~-20°C. Plasma extracts of the analyte/ISTD ratio were shown to be stable on instrument over a period of ~55 hours. Reinjection reproducibility experiments indicated that an assay batch may be re-injected within 58 hours. Quantification of PAS in plasma was not significantly affected by the presence of haemolysed blood (2%) in plasma and when Lithium Heparin was used as anti-coagulant instead of K3EDTA. The best marker for terizidone pharmacokinetics is the analysis of cycloserine, a small polar drug with limited potential for absorbing UV that makes it difficult to analyse. A method was validated for the quantification of cycloserine in human plasma, and consisted of a protein precipitation extraction and derivatization, followed by high performance liquid chromatography with MS/MS detection. No ISTD was used as no suitable match could be found. The mean extraction yield determined was ~77% (CV% = 10.7). The extraction procedure was followed by liquid chromatographic separation using a Gemini NX C18 (50 x 2.0 mm, 5µ) analytical column. An isocratic mobile phase containing acetonitrile, water and formic acid (30:69.9:0.1, v/v/v) was used at a flow-rate of 300 µl per minute. The retention time for cycloserine was ~ 1.5 minutes. An AB Sciex API 3000 mass spectrometer at unit resolution in the MRM mode was used to monitor the transition of the protonated precursor ion m/z 335.9 to the product ion m/z 157.2 for cycloserine. ESI was used for ion production. Accuracy and precision were assessed over three consecutive, independent runs. The calibration curve fits a quadratic (weighted by 1/x concentration) regression for cycloserine over the range 0.313 – 40.0 µg/ml, based on peak areas. A 1:4 dilution of the QC Dilution sample showed that concentrations of up to 64.0 µg/ml of cycloserine in plasma could be analysed reliably when diluted into the calibration range and no carry over peaks were observed. Endogenous matrix components were found to have no effect on the reproducibility of the method when human plasma originating from six different sources was analysed. Cycloserine was found to be stable in human plasma for up to 18 hours at room temperature, and when subjected to 3 freeze-thaw cycles. Stock solutions of cycloserine in water and methanol were stable for 10 days when stored at ~ -80°C and for 18 hours when stored at room temperature, ~ 4°C and ~ -20°C. Long term stability in plasma has been proven for 17 months at -80°C. Plasma extracts of the analyte were shown to be stable on instrument over a period of ~ 29 hours. Reinjection reproducibility experiments indicate that an assay batch may be re-injected within 29 hours. Cycloserine is stable in whole blood (on ice) for up to 30 minutes. Both validated methods presented performed well on clinical samples generated from a multi drug resistant TB (MDR-TB) research study in children dosed with PAS and terizidone.
- ItemOpen AccessDiscovery of Novel Cyclic Ethers with Synergistic Antiplasmodial Activity in Combination with Valinomycin(Multidisciplinary Digital Publishing Institute, 2021-12-10) Watson, Daniel J.; Meyers, Paul R.; Acquah, Kojo Sekyi; Dziwornu, Godwin A.; Barnett, Christopher Bevan; Wiesner, LubbeWith drug resistance threatening our first line antimalarial treatments, novel chemotherapeutics need to be developed. Ionophores have garnered interest as novel antimalarials due to their theorized ability to target unique systems found in the Plasmodium-infected erythrocyte. In this study, during the bioassay-guided fractionation of the crude extract of Streptomyces strain PR3, a group of cyclodepsipeptides, including valinomycin, and a novel class of cyclic ethers were identified and elucidated. Further study revealed that the ethers were cyclic polypropylene glycol (cPPG) oligomers that had leached into the bacterial culture from an extraction resin. Molecular dynamics analysis suggests that these ethers are able to bind cations such as K+, NH4+ and Na+. Combination studies using the fixed ratio isobologram method revealed that the cPPGs synergistically improved the antiplasmodial activity of valinomycin and reduced its cytotoxicity in vitro. The IC50 of valinomycin against P. falciparum NF54 improved by 4–5-fold when valinomycin was combined with the cPPGs. Precisely, it was improved from 3.75 ± 0.77 ng/mL to 0.90 ± 0.2 ng/mL and 0.75 ± 0.08 ng/mL when dosed in the fixed ratios of 3:2 and 2:3 of valinomycin to cPPGs, respectively. Each fixed ratio combination displayed cytotoxicity (IC50) against the Chinese Hamster Ovary cell line of 57–65 µg/mL, which was lower than that of valinomycin (12.4 µg/mL). These results indicate that combinations with these novel ethers may be useful in repurposing valinomycin into a suitable and effective antimalarial.
- ItemOpen AccessDrug discovery at the site of pulmonary tuberculosis(2018) Tanner, Lloyd; Wiesner, Lubbe; Warner, Digby; Haynes, RichardThe protracted duration of standard tuberculosis (TB) therapy suggests the inadequacy of current first line TB drugs to eliminate the causative agent Mycobacterium tuberculosis (Mtb). Among multiple potential causes, this may be due to poor distribution of TB drugs into the pulmonary lesions in which the bacilli reside. In attempting to explore this possibility, the study described here aimed to assess selected novel compounds (phenoxazine, artemisinin, and decoquinate derivatives) proposed to induce redox-cycling in efficacious combinations, as well as their distribution to TB-relevant lesions, for potential clinical use against TB. To this end, specific in vitro absorption, distribution, metabolism, and excretion (ADME) assays were performed to predict the abilities of the compounds to penetrate different TB microenvironments. Penetration into murine blood and organs was assessed via pharmacokinetic (PK) profiling. Complementary analyses involving murine epithelial lining fluid allowed for more detailed analyses of the potential of these novel compounds to penetrate the deeper recesses of the lung. In order to gain a greater understanding of the potential efficacy of the compounds in an intracellular environment, THP-1 macrophage-like cells were infected with Mtb, treated with anti-TB agents, and sampled at different time-points. Samples were analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays and drug concentrations were determined and related to efficacy measurements using colony forming unit counts. Promising combinations of novel drugs were identified in a two dimensional synergy assay; these combinations showed synergistic activity in the infected macrophage model. The compounds showed marked differences in their abilities to accumulate within infected macrophages. Differential uptake was also indicated by the results of the PK studies involving murine blood and organ uptake, and the in vitro ADME assays. These results enable PK modelling on the putative drug target to be carried out, allowing for the determination of more accurate dosing. In addition, results indicate the need for further studies, including investigations of the impact of macrophage structural organisation (three-dimensional model) on compound efficacy and in vivo studies in a relevant mouse model of TB disease. The identification of a potentially efficacious two drug combination which might penetrate to the site of pulmonary TB supports the utility of this approach in the preclinical drug discovery and development pipeline.
- ItemOpen AccessEffect of Moringa oleifera Lam. leaf powder on the pharmacokinetics of nevirapine in HIV-infected adults: a one sequence cross-over study(BioMed Central, 2017-03-14) Monera-Penduka, Tsitsi G; Maponga, Charles C; Wolfe, Alan R; Wiesner, Lubbe; Morse, Gene D; Nhachi, Charles F BBackground: Moringa oleifera Lam., an herb commonly consumed by HIV-infected people on antiretroviral therapy, inhibits cytochrome P450 3A4, 1A2 and 2D6 activity in vitro; and may alter the pharmacokinetics (PK) of antiretroviral drugs metabolized via the same pathways. However, in vitro drug interaction activity may not translate to a clinically significant effect. Therefore, the effect of moringa leaf powder on the PK of nevirapine in HIV-infected people was investigated. Methods: Adult patients at steady-state dosing with nevirapine were admitted for 12-h intensive PK sampling follow ing a 21-day herbal medicine washout. Blood sampling was repeated after 14 days of nevirapine and moringa (1.85 g leaf powder/day) co-administration. Nevirapine plasma concentrations were determined by liquid chromatographytandem mass spectrometry. To assess the effect of moringa on nevirapine PK, the change in nevirapine area under the plasma concentration–time curve (AUC) was determined. The mean difference in pre- and post-moringa nevirapine, maximum concentration (Cmax) and concentration at 12 h (C12h) were also calculated. The PK parameters were com pared by assessing the post/pre geometric mean ratios (GMRs) and associated 90% confidence intervals (CIs). Results: Pharmacokinetics analyses were performed on the results from 11 participants for whom complete data were obtained. The post/pre GMRs and associated 90% CIs for nevirapine were 1.07 (1.00–1.14) for the AUC; 1.06 (0.98–1.16) for Cmax and 1.03 (0.92–1.16) for C12h. Conclusion: Co-administration of Moringa oleifera Lam. leaf powder at the traditional dose did not significantly alter the steady-state PK of nevirapine. Trial registration number NCT01410058 (ClinicalTrials.gov)
- ItemOpen AccessEfficacy and pharmacokinetic evaluation of a novel anti-malarial compound (NP046) in a mouse model(BioMed Central Ltd, 2015) Abay, Efrem; van der Westuizen, Jan; Swart, Kenneth; Gibhard, Liezl; Lawrence, Nina; Dambuza, Ntokozo; Wilhelm, Anke; Pravin, Kendrekar; Wiesner, LubbeBACKGROUND: Even though malaria is a completely preventable and treatable disease, it remains a threat to human life and a burden to the global economy due to the emergence of multiple-drug resistant malaria parasites. According to the World Malaria Report 2013, in 2012 there were an estimated 207 million malaria cases and 627,000 deaths. Thus, the discovery and development of new, effective anti-malarial drugs are required. To achieve this goal, the Department of Chemistry at the University of the Free State has synthesized a number of novel amino-alkylated chalcones and analogues, which showed in vitro anti-malarial activity against both chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum strains. The lead compound (NP046) was selected for a comprehensive pharmacokinetic (PK) and in vivo efficacy evaluation in a mouse model. METHODS: In vivo efficacy: Water solutions of NP046 were administered orally at 50 and 10mg/kg using oral gavage and IV at 5 and 1mg/kg via the dorsal penile vein to Plasmodium berghei (ANKA strain) infected male C57BL/6 mice (n=5), once a day for four days. Blood samples were collected via tail bleeding in tubes containing phosphate buffer saline (PBS) on day five to determine the % parasitaemia by flow cytometry.In vivo PK: NP046 solutions in water were administered orally (50 and 10mg/kg) and IV (5mg/kg) to male C57BL/6 mice (n=5). Blood samples were collected via tail bleeding into heparinized tubes and analysed using a validated LC-MS/MS assay. Data obtained from the concentration-time profile was evaluated using Summit PK software to determine the PK parameters of NP046. RESULTS: NP046 inhibited parasite growth for the oral and IV groups. Better parasite growth inhibition was observed for the IV group. The PK evaluation of NP046 showed low oral bioavailability (3.2% and 6% at 50mg/kg and 10mg/kg dose, respectively and a moderate mean half-life ranging from 3.1 to 4.4hours. CONCLUSION: Even though the oral bioavailability of NP046 is low, its percentage parasite growth inhibition is promising, but in order to improve the oral bioavailability, structure-activity-relationship (SAR) optimization studies are currently being conducted.
- ItemOpen AccessEfficacy and safety of artemether–lumefantrine, artesunate–amodiaquine, and dihydroartemisinin–piperaquine for the treatment of uncomplicated Plasmodium falciparum malaria in three provinces in Angola, 2017(BioMed Central, 2018-04-03) Davlantes, Elizabeth; Dimbu, Pedro R; Ferreira, Carolina M; Florinda Joao, Maria; Pode, Dilunvuidi; Félix, Jacinto; Sanhangala, Edgar; Andrade, Benjamin N; dos Santos Souza, Samaly; Talundzic, Eldin; Udhayakumar, Venkatachalam; Owens, Chantelle; Mbounga, Eliane; Wiesner, Lubbe; Halsey, Eric S; Martins, José F; Fortes, Filomeno; Plucinski, Mateusz MBackground The Angolan government recommends three artemisinin-based combinations for the treatment of uncomplicated Plasmodium falciparum malaria: artemether–lumefantrine (AL), artesunate–amodiaquine (ASAQ), and dihydroartemisinin–piperaquine (DP). Due to the threat of emerging anti-malarial drug resistance, it is important to periodically monitor the efficacy of artemisinin-based combination therapy (ACT). This study evaluated these medications’ therapeutic efficacy in Benguela, Lunda Sul, and Zaire Provinces. Methods Enrollment occurred between March and July 2017. Study participants were children with P. falciparum monoinfection from each provincial capital. Participants received a 3-day course of a quality-assured artemisinin-based combination and were monitored for 28 (AL and ASAQ arms) or 42 days (DP arm). Each ACT was assessed in two provinces. The primary study endpoints were: (1) follow-up without complications and (2) failure to respond to treatment or development of recurrent P. falciparum infection. Parasites from each patient experiencing recurrent infection were genotyped to differentiate new infection from recrudescence of persistent parasitaemia. These parasites were also analysed for molecular markers associated with ACT resistance. Results Of 608 children enrolled in the study, 540 (89%) reached a primary study endpoint. Parasitaemia was cleared within 3 days of medication administration in all participants, and no early treatment failures were observed. After exclusion of reinfections, the corrected efficacy of AL was 96% (91–100%, 95% confidence interval) in Zaire and 97% (93–100%) in Lunda Sul. The corrected efficacy of ASAQ was 100% (97–100%) in Benguela and 93% (88–99%) in Zaire. The corrected efficacy of DP was 100% (96–100%) in Benguela and 100% in Lunda Sul. No mutations associated with artemisinin resistance were identified in the pfk13 gene in the 38 cases of recurrent P. falciparum infection. All 33 treatment failures in the AL and ASAQ arms carried pfmdr1 or pfcrt mutations associated with lumefantrine and amodiaquine resistance, respectively, on day of failure. Conclusions AL, ASAQ, and DP continue to be efficacious against P. falciparum malaria in these provinces of Angola. Rapid parasite clearance and the absence of genetic evidence of artemisinin resistance are consistent with full susceptibility to artemisinin derivatives. Periodic monitoring of in vivo drug efficacy remains a priority routine activity for Angola.
- ItemOpen AccessEfficacy of artemether-lumefantrine in relation to drug exposure in children with and without severe acute malnutrition: an open comparative intervention study in Mali and Niger(BioMed Central, 2016-10-24) Denoeud-Ndam, Lise; Dicko, Alassane; Baudin, Elisabeth; Guindo, Ousmane; Grandesso, Francesco; Diawara, Halimatou; Sissoko, Sibiri; Sanogo, Koualy; Traoré, Seydou; Keita, Sekouba; Barry, Amadou; de Smet, Martin; Lasry, Estrella; Smit, Michiel; Wiesner, Lubbe; Barnes, Karen I; Djimde, Abdoulaye A; Guerin, Philippe J; Grais, Rebecca F; Doumbo, Ogobara K; Etard, Jean-FrançoisBackground: Severe acute malnutrition (SAM) affects almost all organs and has been associated with reduced intestinal absorption of medicines. However, very limited information is available on the pharmacokinetic properties of antimalarial drugs in this vulnerable population. We assessed artemether-lumefantrine (AL) clinical efficacy in children with SAM compared to those without. Methods: Children under 5 years of age with uncomplicated P. falciparum malaria were enrolled between November 2013 and January 2015 in Mali and Niger, one third with uncomplicated SAM and two thirds without. AL was administered under direct observation with a fat intake consisting of ready-to-use therapeutic food (RUTF – Plumpy’Nut®) in SAM children, twice daily during 3 days. Children were followed for 42 days, with PCR-corrected adequate clinical and parasitological response (ACPR) at day 28 as the primary outcome. Lumefantrine concentrations were assessed in a subset of participants at different time points, including systematic measurements on day 7. Results: A total of 399 children (360 in Mali and 39 in Niger) were enrolled. Children with SAM were younger than their non-SAM counterparts (mean 17 vs. 28 months, P < 0.0001). PCR-corrected ACPR was 100 % (95 % CI, 96.8–100 %) in SAM at both day 28 and 42, versus 98.8 % (96.4–99.7 %) at day 28 and 98.3 % (95.6–99.4 %) at day 42 in non-SAM (P = 0.236 and 0.168, respectively). Compared to younger children, children older than 21 months experienced more reinfections and SAM was associated with a greater risk of reinfection until day 28 (adjusted hazard ratio = 2.10 (1.04–4.22), P = 0.038). Day 7 lumefantrine concentrations were significantly lower in SAM than non-SAM (median 251 vs. 365 ng/mL, P = 0.049). Conclusions: This study shows comparable therapeutic efficacy of AL in children without SAM and in those with SAM when given in combination with RUTF, but a higher risk of reinfection in older children suffering from SAM. This could be associated with poorer exposure to the antimalarials as documented by a lower lumefantrine concentration on day 7. Trial registration: ClinicalTrials.gov: NCT01958905, registration date: October 7, 2013.
- ItemOpen AccessIsolation and characterisation of antiplasmodial compounds from Xerophyta species and the bioavailability, metabolic and efficacy evaluation of 9-0-acetylhydnocarpin in a mouse model(2008) Wiesner, Lubbe; Smith, Peter J; Campbell, William EIncludes abstract. Includes bibliographical references (leaves 237-265).
- ItemOpen AccessMelamine contamination in nutritional supplements - Is it an alarm bell for the general consumer, athletes, and 'Weekend Warriors'?(BioMed Central Ltd, 2015) Gabriels, Gary; Lambert, Mike; Smith, Pete; Wiesner, Lubbe; Hiss, DonavonBACKGROUND: Nutritional supplements are used or experimented with by consumers, notably these are; competitive and recreational athletes of all ages, and 'weekend warriors'. As a consequence the supplement industry has grown to meet the increasing demand. A Global Industry Analysts Inc. report indicates that the herbal supplement market has not declined during the worldwide recession, but in fact exhibited steady growth over the period 2008 to 2009. It is anticipated that the market will reach US$93.15 billion by the year 2015. These supplements may contain adulterated substances that may potentially have harmful short - and long-term health consequences to the consumer. "Scrap Melamine" is such an example, which has been implicated in the kidney failure and death of several cats, dogs and pigs. In China in 2008, reports described very severe health effects in infants and young children. At the time over 294 000 infants were screened and diagnosed with urinary tract stones and sand-like calculi associated with melamine in milk products, of which 50 000 infants were hospitalised, and at least six associated deaths, recorded. The extent that melamine contamination occurs in nutritional supplements is not known. Therefore, the aim of this study was to determine whether commercially available nutritional and traditional supplement products contain melamine, even though they are not declared by the manufacturer on the product label. METHODS: A total of 138 nutritional supplements products were obtained from (i) direct purchases from shops, pharmacies and outlets, (ii) directly from consumers, and (iii) from suppliers, manufacturers and distributors. The products were laboratory analysed for melamine, using Tandem Liquid Chromatography Mass Spectrometry. RESULTS: Forty-seven % of all the products (n=138) tested positive for melamine. Eight-two % of the South African produced products (n=27) tested positive and 58 % of the products imported into South Africa (n=50) tested positive. The median concentration estimate for melamine in the products tested were, 6.0 mug/g for the 138 supplements tested, 8.9 mug/g for South African produced products, and 6.9 mug/g for products imported into South Africa. CONCLUSION: The melamine (undeclared on product label) levels detected in the nutritional supplements products investigated were within the Tolerable Daily intake (TDI) limit guidelines of 200 mug/g as set by WHO and others. Melamine over exposure within the context of the nutritional supplements consumption in the products investigated should not be of concern to the consumer provided the recommended guidelines of daily product use are adhered to. Further investigation is warranted to determine, (i) the link of melamine as (part) substitute for the perceived total declared protein content on the product label, (ii) cyanuric and uric acid presence in the supplement products that could form chemical-complex formation with melamine and/or analogues that could cause adverse health effects.
- ItemOpen AccessPharmacokinetic profile of amodiaquine and its active metabolite desethylamodiaquine in Ghanaian patients with uncomplicated Falciparum malaria(2017) Anyorigiya, Thomas Atingane; Barnes, Karen I; Wiesner, LubbeRATIONALE: The accurate measurement of antimalarial drug concentrations in key target patient groups is essential to ensure optimal dosing for malaria treatment and to distinguish between inadequate drug exposure and antimalarial resistance. METHODS: A sensitive and selective liquid chromatography-tandem mass spectrophotometric method was developed and validated for the simultaneous determination of amodiaquine and its active metabolite, desethylamodiaquine in 20μl heparinised human capillary whole blood and capillary plasma. During validation no significant matrix effects were observed for the analytes or internal standards. This assay method was successfully used to describe the pharmacokinetics of amodiaquine and desethylamodiaquine in Ghanaian patients with uncomplicated falciparum malaria, who were followed up for 28 days in a therapeutic efficacy study of the fixed-dose combination therapy, artesunate-amodiaquine. RESULTS: The day 28 genotype-adjusted adequate clinical and parasitological response rate of artesunate-amodiaquine combination in 321 patients studied was above 97% in both the intention-to-treat and per protocol analyses in the two study sites. Amodiaquine and desethylamodiaquine pharmacokinetic parameters were defined for 225 patients (7 infants, 127 aged 1-4 years, 91 aged ≥5 years). Multivariate analysis of the effects of pre-defined covariates found that mg/kg dose, female gender, hyperparasitaemia and site of sample collection were independently associated with desethylamodiaquine pharmacokinetic parameters. Although the association between treatment outcome and desethylamodiaquine day 7 concentrations or area under the concentration-time curve could not be established due to high artesunate-amodiquine efficacy, median day 3 amodiaquine concentrations were associated with 13% reduction in the rate of parasite recurrence, p=0.021. Despite the significantly higher amodiaquine exposure (4988ng.h/ml, p<0.001) in infants compared to the older age groups, no significant safety concerns were identified. The ratio of the geometric mean of matched concentrations in capillary whole blood to capillary plasma in a subset of 163 field samples was approximately 2.04 [95% CI 1.90-2.19] for amodiaquine and 2.4 [95%CI 2.14-2.63] for desethylamodiaquine. CONCLUSION: Pharmacokinetic parameters of amodiaquine and desethylamodiaquine were determined in the largest single uncomplicated malaria pharmacokinetic study to date. Although efficacy was high across all age groups with currently recommended dosage regimens, factors were identified as potentially significant covariates that may require further investigation in pooled analyses.
- ItemOpen AccessPharmacokinetics of Isoniazid, Pyrazinamide, and Ethambutol in newly diagnosed pulmonary TB patients in Tanzania(Public Library of Science, 2015) Denti, Paolo; Jeremiah, Kidola; Chigutsa, Emmanuel; Faurholt-Jepsen, Daniel; PrayGod, George; Range, Nyagosya; Castel, Sandra; Wiesner, Lubbe; Hagen, Christian Munch; Christiansen, MichaelExposure to lower-than-therapeutic levels of anti-tuberculosis drugs is likely to cause selection of resistant strains of Mycobacterium tuberculosis and treatment failure. The first-line anti-tuberculosis (TB) regimen consists of rifampicin, isoniazid, pyrazinamide, and ethambutol, and correct management reduces risk of TB relapse and development of drug resistance. In this study we aimed to investigate the effect of standard of care plus nutritional supplementation versus standard care on the pharmacokinetics of isoniazid, pyrazinamide and ethambutol among sputum smear positive TB patients with and without HIV. In a clinical trial in 100 Tanzanian TB patients, with or without HIV infection, drug concentrations were determined at 1 week and 2 months post initiation of anti-TB medication. Data was analysed using population pharmacokinetic modelling. The effect of body size was described using allometric scaling, and the effects of nutritional supplementation, HIV, age, sex, CD4+ count, weight-adjusted dose, NAT2 genotype, and time on TB treatment were investigated. The kinetics of all drugs was well characterised using first-order elimination and transit compartment absorption, with isoniazid and ethambutol described by two-compartment disposition models, and pyrazinamide by a one-compartment model. Patients with a slow NAT2 genotype had higher isoniazid exposure and a lower estimate of oral clearance (15.5 L/h) than rapid/intermediate NAT2 genotype (26.1 L/h). Pyrazinamide clearance had an estimated typical value of 3.32 L/h, and it was found to increase with time on treatment, with a 16.3% increase after the first 2 months of anti-TB treatment. The typical clearance of ethambutol was estimated to be 40.7 L/h, and was found to decrease with age, at a rate of 1.41% per year. Neither HIV status nor nutritional supplementations were found to affect the pharmacokinetics of these drugs in our cohort of patients.