Thesis (Ph. D.)--University of Queensland, 2003. / Includes bibliographical references.

Tenofovir (TFV) and emtricitabine (FTC) are nucleoside reverse transcriptase inhibitors, often used in preexposure prophylaxis (PrEP) trials: where antiretroviral drugs are administered to high-risk, HIV-negative individuals to prevent HIV infection. Both drugs are safe when taken either daily or intermittently, which is ideal for PrEP regimens where adherence may not be high. The minimum number of doses estimated to confer high PrEP efficacy for a TFV/FTC regimen is four or more doses per week, resulting in a 95% lower risk of HIV acquisition. However, this is highly dependent on various host factors, of which adherence plays the largest role. The aim of the project was to develop a novel sensitive, specific, and robust direct method for the measurement of adherence, utilising tenofovir-diphosphate (TFV-DP) in dry blood spots (DBS) through LC-MS/MS analysis, to replace the current costly and laborious indirect method currently used to elucidate adherence of patients. This indirect method faces challenges, due to the polar nature of TFV and its metabolites, leading to separation and retention issues. The existing method applied a technique which separated the parent drug from the metabolite and then back-converted all metabolites to the parent drug before analysing the samples on LC-MS/MS. The developed alternative method aimed to reduce the time taken for each assay and the associated cost of consumables. TFV-DP is a highly polar compound and traditional reverse-phase chromatography has poor retention and separation capabilities when used to retain polar compounds, therefore alternative strategies were implemented. In this developed direct method, an anion exchange column was used along with a pH gradient, with the aim of improving separation and chromatography of TFV, TFV-DP, and tenofovir-monophosphate (TFV-MP). The method was optimised and validated using current U.S. Food and Drug Administration (FDA) and European Medical Agency (EMA) guidelines. The use of the anion exchange column resulted in a marked increase in retention time and allowed baseline separation of TFV, TFV-DP, and TFV-MP. Determination of TFV-DP from DBS was performed using three 3 mm DBS punches per sample, which underwent an extraction procedure followed by high-performance liquid chromatography with tandem mass spectrometry detection on an AB Sciex Qtrap 5500 mass spectrometer. The transitions of the protonated precursor ions were monitored at m/z 448.0 and 452.9 to the product ions m/z 350.0 and 354.9 for TFV-DP and the deuterated TFVDP internal standard, respectively. The method was validated over a range of 50–6400 fmol/punch for TFV-DP. The developed direct method had a lower limit of quantification (LLOQ) of 50 fmol/punch, which was higher than that of the indirect method; therefore, it had less sensitivity. The reduced sensitivity was acceptable, since the methods were meant for the measurement of adherence. The direct method had an ULOQ of 6400 fmol/punch, which was similar to that of the indirect method. The direct method also required significantly less on-bench sample processing and, therefore, was less time consuming and costly. To determine the suitability and accuracy of the direct method in comparison to the indirect method a comparative analysis was completed by analysing the same samples using both the indirect and direct method. The developed method met all the validation requirements and a strong correlation was observed between the results of the indirect and direct methods during the comparative analysis.

Includes bibliographical references.

Two 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.

Includes abstract. / Includes bibliographical references. / The pharmacokinetics of rifampicin, isoniazid, pyrazinamide and ethambutol in 78 patients with tuberculosis were described using non-linear mixed effects modeling. Pharmacodynamic data was comprised of weekly sputum liquid culture (using mycobacterial growth indicator tubes) time to detection results from 144 patients during the first 2 months of treatment. The effect of drug exposure on patient outcomes was investigated. To determine the adequacy of ofloxacin drug exposure, the probability of attaining the required area-under-the-curve to minimum inhibitory concentration ratio (AUC/MIC) of ofloxacin was determined in 65 patients on treatment for multidrug resistant tuberculosis. To improve efficiency in the clinical development of new drug regimens, clinical trial simulation was used to determine the optimal study design for a study investigating the efficacy of a new antitubercular drug regimen. The SLCO1B1 rs4149032 polymorphism existed at a high frequency of 0.70 in South Africans and resulted in a 28% decrease in bioavailability of rifampicin. The rifampicin peak concentration was a significant predictor of the 2 month treatment outcomes. A semimechanistic time to event model was developed to analyze days to positivity (time to detection) data. The model was comprised of a biexponential decay model describing bacillary decline in sputum from patients, followed by a logistic model with a lag time for growth of the mycobacteria in liquid culture. For the current 800 mg daily dose of ofloxacin, the probability of attaining an AUC/MIC target ratio of at least 100 was only 0.45. Based on clinical trial simulation, the optimum parallel study design was comprised of 125 study participants in each of 2 arms to achieve a study power of at least 80%. Increasing the study length beyond 42 days reduced study power perhaps due to increased amounts of censored data. Higher doses of rifampicin are required in the majority of South African patients with tuberculosis. A novel pharmacodynamic model of tuberculosis treatment is presented, which can be used for investigation of covariates such as drug exposure. Ofloxacin should be replaced with a more potent fluoroquinolone for treatment of multidrug resistant tuberculosis. Clinical trials should not be unduly long otherwise this may compromise study power.

The 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.

Includes bibliographical references. / Globally Sub-Saharan Africa carries the biggest burden of patients infected with human immunodeficiency virus (HIV). Tuberculosis is the most common opportunistic infection in patients infected with HIV. Although antiretroviral therapy (ART) has decreased the burden of tuberculosis in HIV-infected patients, the incidence of tuberculosis remains higher than in the general population. HIV-tuberculosis co-infection requires dual treatment with ART and tuberculosis treatment, exposing patients to multiple drug-drug interactions. As ART programs mature, more patients will be changed from first-line to second-line ART. In South Africa, the adult second-line ART consists of the protease inhibitor (PI) lopinavir/ritonavir (LPV/r) and 2 nucleoside reverse transcriptase inhibitors (NRTls). This review will focus on the data of the drug-drug interactions between the PIs and rifampicin, with an emphasis on LPV/r.

Aims: There are limited therapeutic options for drug-induced liver injury (DILI). N-acetylcysteine (NAC ) is known to be of benefit in management of DILI due to paracetamol overdose and may also be useful in the management of non-paracetamol DILI. Our objective was to systematically review evidence for the use of NAC as a therapeutic option for non-paracetamol DILI. Methods: We conducted a systematic review of the benefit and harm of NAC in non-paracetamol DILI. We searched for randomized controlled trials (RCTs) and prospective cohort studies. We searched several bibliographic databases (including PubMed, Scopus, CINAHL, CENTRAL), grey literature sources, conference proceedings and ongoing trials. Our pre-specified primary outcomes were all cause and DILI related mortality, time to normalisation of liver biochemistry and adverse events. Secondary outcomes were proportion receiving liver transplant, time to transplantation, transplant-free survival and hospitalization duration. Two reviewers independently assessed studies for inclusion and quality and extracted data. Results: We identified one RCT of NAC versus placebo in patients with non-paracetamol acute liver failure. There was no difference in the primary outcomes of overall survival at 3-weeks between NAC [70%, 95% Confidence Interval (CI)= 60% to 81%, n=81 ] and placebo (66%, 95% C I= 56% to 77%, n=92 ). NAC significantly improved the secondary outcomes of transplant-free survival compared with placebo : 40% NAC ( 95% CI= 28% to 51%) versus 27% placebo ( 95% CI= 18% to 37%). A subgroup analysis according to aetiology found improved transplant-free survival in patients with non-paracetamol DILI; NAC (58%, n=19 ) versus placebo (27%, n=26 ); odds ratio (OR) 0.27 (95% CI= 0.076 to 0.942 ). O verall survival was similar NAC ( 79% ) v ersus placebo ( 65% ); OR 0.5 0 ( 95% CI= 0.13 to 1.98 ). Conclusion : Current available evidence is limited and does not allow for any firm conclusions to be made regarding the role of NAC in non-paracetamol DILI. We therefore highlight the need for further research in this area.

The success of our first-line antimalarial treatments is threatened by increased drug resistance in Plasmodium parasites. This makes the development of novel drugs critical to combat malaria. Historically, natural products have been an excellent source of novel antimalarial compounds and thus are an ideal place to search for potential drugs. Filamentous members of the bacterial phylum, Actinobacteria, are well-known antibiotic producers, but their antimalarial potential has not been well investigated. This makes these actinobacteria a potentially valuable source of novel antimalarial compounds. To evaluate the antimalarial potential of the filamentous actinobacteria, uncharacterized environmental actinobacterial strains from the Meyers laboratory culture collection, as well as the type strains of new actinobacterial species identified and characterized in the Meyers laboratory, were screened for antiplasmodial activity against drug-sensitive Plasmodium falciparum, NF54. Liquid cultures were extracted using the mid-polar solvent, ethyl acetate, with the aim of discovering drug-like molecules that can be administered orally. Thirty-one strains of actinobacteria belonging to eight genera (Actinomadura, Amycolatopsis, Gordonia, Kribbella, Micromonospora, Nocardia, Nonomuraea, and Streptomyces) were screened revealing fourteen active strains. Eight strains were identified for further study as the displayed antiplasmodial efficacy matching predefined criteria. Of these eight candidates, Streptomyces strain PR3 was selected, as it showed excellent antiplasmodial efficacy, no cytotoxicity against Chinese Hamster Ovary (CHO) or liver HepG2 cell lines, no haemotoxicity, and was easy to culture. Bioassay-guided fractionation of the crude extracts of strain PR3, supported by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) analysis, was conducted to isolate and identify the compounds responsible for the antiplasmodial activity. During purification by solid phase extraction (SPE), a novel class of compounds was isolated. The structure of these compounds was elucidated by HRMS and NMR analysis and determined to be a series of crown ethers with a methylated backbone. These methylated crown ethers (MCE) were not produced by strain PR3, but by the cyclization of polypropylene glycol (PPG) oligomers from Amberlite® XAD-16N 20–60 mesh resin under aqueous conditions. The MCEs displayed weak antiplasmodial activity against P. falciparum NF54, without cytotoxicity against the Chinese Hamster Ovary, HepG2 cell lines, nor human erythrocytes. To the author's knowledge, the MCEs are novel compounds, and this is the first time the cyclization of PPG oligomers into crown ethers has been reported. As the MCEs were not responsible for strain PR3's potent antiplasmodial activity, further study was conducted. Using the Global Natural Product Social molecular networking (GNPS) workflow, genome mining, and NMR analysis, it was revealed that the cyclodepsipeptides, valinomycin, montanastatin, and nine other novel analogues were responsible for the high antiplasmodial activity detected. A review of the literature revealed that the structure of four of these analogues had been predicted, based on MS/MS and the biosynthesis of valinomycin. Using the same described biosynthetic logic and MS/MS analysis, two new cyclodepsipeptides, compounds 1054 and 1068, were elucidated. Unfortunately, chromatographic systems developed were unable to purify the cyclodepsipeptides, and individual evaluation of their antiplasmodial efficacy and host selectivity was not possible. The fraction containing the cyclodepsipeptides exhibited strong antiplasmodial activity against the drug-sensitive, NF54 and multidrug-resistant K1, strains of P. falciparum. No cytotoxicity was displayed against the CHO cell line and no haemotoxicity was seen against human erythrocytes. Moderate toxicity was exhibited against the liver HepG2 cell line; however, the selectivity index of the cyclodepsipeptides suggested that they are selectively targeting the Plasmodium parasites. Overall, these results are positive, and further study of the individual cyclodepsipeptides is warranted. During the investigation, discrepancies were noticed between different fractions in terms of antiplasmodial activity. These fractions contained both the MCEs and, cyclodepsipeptides along with a range of impurities, yet they displayed potent antiplasmodial activity. Further study suggested that combination of the MCEs and cyclodepsipeptides elicits a synergistic response and improves antiplasmodial efficacy. This was determined independently using two models, the fixed-ratio isobologram method and the CompuSyn programme based on the massaction law principle. The workflow developed during this investigation demonstrates how new technologies can be used to dereplicate and elucidate bioactive natural products. This workflow can be utilized to continue this research and identify new natural products that can combat malaria

Background: Tuberculosis (TB) is the most common opportunistic infection in children with human immunodeficiency virus (HIV) infection in developing countries, and co-treatment for HIV infection and TB is frequently indicated. Efavirenz and lopinavir/ritonavir (ratio 1:1) as part of antiretroviral therapy are used in combination with rifampicin-based antitubercular treatment in South African TB/HIV co-infected children. Adult studies show that concomitant rifampicin significantly reduces efavirenz and lopinavir plasma concentrations. However, the pharmacokinetics (PK) of efavirenz and lopinavir/ritonavir are poorly characterized in children, especially African children and no study has evaluated the effect of rifampicin-based antitubercular treatment on efavirenz and lopinavir/ritonavir plasma concentrations in children. Although therapeutic drug monitoring (TDM) is recommended in selected patients (including young children and patients receiving concomitant antitubercular treatment), TDM is seldom available in resource-constrained countries. There is an urgent need to develop a field friendly method which requires small volumes of blood, and inexpensive processing and storage conditions. Furthermore, because HIV replicates in the cells, efavirenz and lopinavir need to penetrate into these infected cells to inhibit viral replication. Therefore, directly measurement of intracellular concentrations of these drugs in HIV-infected children could provide better understanding of drug exposure at the action site. It is also important to evaluate the effects of frequently co-administered drugs on intracellular accumulation of efavirenz and lopinavir. Objectives: 1) To evaluate efavirenz and lopinavir/ritonavir plasma concentrations and determine the effects of rifampicin on efavirenz and lopinavir/ritonavir PK in HIV-infected African children with and without rifampicin-based antitubercular treatment. 2) To develop and validate the dried blood spot (DBS) method as an alternative to conventional plasma methods of drug concentration measurement in TDM. 3) To evaluate in vivo intracellular concentrations of efavirenz and lopinavir/ritonavir in HIV-infected children with and without concomitant antitubercular treatment. 4) To determine the in vitro modulation effects on the intracellular accumulation of efavirenz IV and lopinavir in human peripheral blood mononuclear cells (PBMCs) by drug efflux protein inhibitors, as well as frequently co-administered rifampicin and ritonavir (at low dose; as pharmacoenhancer). Methods: 1) Plasma efavirenz and lopinavir/ritonavir concentrations were measured by validated liquid chromatography/tandem mass spectrometry (LC/MS/MS) method in TB/HIV co-infected children during and after rifampicin-based antitubercular treatment as well as in a group of controls (HIV-infected children without TB). Children in the efavirenz study (n= 30) were receiving standard doses of efavirenz as part of antiretroviral treatment. Trough concentrations (Cmin) of efavirenz were estimated by extrapolation of the log-linear concentration-time line to 24 hours after the previous dose. Children in the lopinavir/ritonavir study were receiving additional ritonavir (lopinavir: ritonavir ratio 1:1) during antitubercular treatment (n= 15), and standard doses of lopinavir/ritonavir (LPV/r; ratio 4:1) after antitubercular treatment, and in controls (n= 15). The PK of lopinavir and ritonavir were characterized from concentration-time curves using WinNonlin version 4.1 by non-compartmental analysis. 2) Aliquots of 50 μ L of whole blood from the efavirenz and lopinavir/ritonavir studies were dried onto filter paper. The drug concentrations were analyzed using validated LC/MS/MS method. The effects of high temperature and direct sunlight on the stabilities of these antiretroviral drugs in DBS samples were tested. 3) Intracellular concentrations of efavirenz, lopinavir and ritonavir were measured in trough concentrations of 11 TB/HIV co-infected children using a validated LC/MS/MS method. Six children were receiving double dose of LPV/r (4:1) with concomitant rifampicin; 5 children were receiving standard doses of efavirenz with rifampicin-based antitubercular treatment, 3 of them had intracellular concentrations measured again after completing rifampicin-based antitubercular treatment. 4) in vitro intracellular accumulation of efavirenz and lopinavir were measured in human PBMCs in the absence and presence of P-glycoprotein inhibitors (verapamil at 50 μ M, V furosemide at 50 μ M and cyclosporine A at 20 μ M) and frequently co-administered drugs at levels representing the average concentrations found in patients (ritonavir at 5 mg/L and rifampicin at 4 mg/L). The concentrations of efavirenz and lopinavir in PBMCs were determined by LC/MS/MS. Results and Conclusions: 1) The co-administration of rifampicin did not significantly reduce efavirenz estimated Cmin concentrations. A high proportion of children with and without concomitant antitubercular treatment had sub-therapeutic efavirenz concentrations despite being correctly dosed according to the manufacturer's instructions, raising concerns about the adequacy of current efavirenz dosing recommendations in children. The lopinavir key PK parameter, Cmin, was not significantly different in same group of children during and after rifampicin-based antitubercular treatment or compared to HIV-infected children without tuberculosis. The recommended minimum therapeutic concentration was achieved in 87% of children during antitubercular treatment and in 92% without concomitant antitubercular treatment. Therefore, in the context of limited options, LPV/r with additional ritonavir (ratio 1:1) is an acceptable approach to treat young children receiving concomitant rifampicin-based antitubercular treatment, although safety remains a concern and hepatic alanine transaminase levels should be monitored regularly. 2) Plasma and DBS concentrations of efavirenz, lopinavir and ritonavir were strongly correlated. The median (interquartile range, IQR) DBS/plasma concentration ratios for efavirenz, lopinavir and ritonavir were 0.93 (IQR 0.83, 1.08), 0.73 (IQR 0.61, 0.90) and 1.05 (IQR 0.74, 1.21), respectively. PK parameters of efavirenz and ritonavir were closely similar between DBS and plasma; whereas lopinavir pre-dose and Cmin (at 12 hours after lopinavir intake) concentrations were 16% lower in DBS samples. The 3 antiretroviral drugs in DBS samples were stable at 37 deg C for 7 days and with exposure to direct sunlight for 2 hours. DBS can be used as an alternative field-friendly method for efavirenz, lopinavir and ritonavir concentration monitoring. However, pre-dose and Cmin concentrations of lopinavir in DBS samples need to be increased by 16% when used to predict plasma concentrations. VI 3) In vivo median intracellular/plasma concentration ratios for efavirenz, lopinavir and ritonavir amongst 11 TB/HIV co-infected children during antitubercular treatment were 0.91 (IQR 0.54, 1.19), 0.22 (IQR 0.09, 0.31) and 4.17 (IQR 1.30, 7.33), respectively. Two children had efavirenz intracellular/plasma concentration ratios during vs. after antitubercular treatment: 1.00 vs. 0.61 and 0.27 vs. 0.79. 4) Furosemide significantly increased efavirenz and lopinavir accumulation in healthy human PBMC samples by 1.2- 1.5 fold. Whereas, neither verapamil nor cyclosporin A had significant effects on efavirenz or lopinavir intracellular accumulation. Despite being an inducer of P-glycoprotein, rifampicin increased the accumulation of both efavirenz and lopinavir to different extents in all 3 PBMC samples. The low-dose ritonavir (at the concentration found in HIV-infected patients) had no effect on intracellular accumulation of efavirenz and lopinavir at therapeutic concentrations.