This is a study of the role of oxygen free radicals in isoniazid induced toxicity and its implications on the safety of isoniazid containing regimens in elderly patients. During isoniazid metabolism, toxic metabolites are produced which, together with isoniazid, may lead to toxicity by mechanism (s) yet unknown. Understanding the mechanism of toxicity is important for effective management and prevention of isoniazid induced toxicity. Elderly patients take priority for this consideration because isoniazid is more toxic in these people. Consequently, "the therapeutic importance of isoniazid metabolism in elderly patients" was found a suitable title for this work. lsoniazid metabolites; acetylisoniazid, monoacetyl-hydrazine and diacetylhydrazine were synthesized and characterized by thin layer chromatography, high performance liquid chromatography (HPLC), ultra-violet absorption spectra and mass spectrometry. Thereafter, these compounds together with hydrazine were used in the subsequent experiments.

Although several dozen different compounds are able to transiently alter chloroquine resistance via chemosensitisation, the phenomenon has never evolved beyond laboratory practice as a result of in vivo difficulties. Chemosensitising compounds either need to be administered at doses which are toxic to the host in order to reverse resistance, or the drug is so highly bound to serum proteins that there is an insufficient circulating quantity available to restore sensitivity. Nine chemosensitisers were evaluated in vitro against several resistant isolates of the malaria parasite in order to develop a cocktail treatment of three compounds which could reverse resistance additively or synergistically when used at low doses with chloroquine. This would bypass any toxicity issues which might arise from the use of a high dose of a single agent. Six of the chemosensitisers were selected for combination into six different cocktails which were tested in vitro. Each cocktail contained one antidepressant, one antihistamine and one antipsychotic. Low doses of each drug were able to alter resistance to a small extent singly and in combination; this was shown by determining the effect of drugs and cocktails on both chloroquine transport using radiolabelled chloroquine, and chloroquine efficacy using the lactate dehydrogenase assay for parasite viability. The reversal activity was shown to be additive in the cocktail treatments and not synergistic, and was highly dose-dependent. There was no direct correlation between the change in chloroquine transport and the extent of resistance reversal. The chemosensitisers' effect on chloroquine transport was evaluated in a mouse model of malaria and shown to be similar to that seen against cultured human parasites; following this, the cocktails were tested for efficacy in mice infected with chloroquine-resistant malaria. Five of the six cocktails were able to significantly alter parasite survival in the mice in conjunction with a low dose of chloroquine. Drug levels in the mice were quantified via mass spectrometry and liquid chromatography in order to correlate the efficacy data. One of the compounds in the failed treatment was shown to circulate at low levels in the animals and this is possibly why that treatment, although effective in vitro, did not yield a result in vivo.

BACKGROUND: Antiretroviral therapy (ART), notably efavirenz and lopinavir, have been associated with metabolic abnormalities known to increase cardiovascular risk. Efavirenz and lopinavir pharmacokinetics demonstrate considerable interindividual variability, which in part, may be explained by host genetic factors. Mitochondrial DNA (mtDNA) variation influences ART related metabolic complications. However, the associations between genetic polymorphisms and pharmacokinetics of antiretroviral drugs, and their associations with metabolic complications, are incompletely understood. We explored associations of mitochondrial DNA (mtDNA) haplogroups and ART related metabolic complications, characterized relationships between genetic polymorphisms and plasma efavirenz concentrations, and investigated associations between plasma efavirenz/lopinavir concentrations and lipid and glucose concentrations in HIVinfected Black South Africans. METHODS: We collected clinical and laboratory data from HIV infected patients on ART from Cape Town. We sequenced the mitochondrial genome and determined African mtDNA haplogroups. We genotyped 241 polymorphisms in genes potentially relevant to efavirenz metabolism and transport. We measured steady state efavirenz and lopinavir concentrations and used regression analyses to determine associations with metabolic parameters.

This pharmacokinetic sub-study was nested within the phase-III OFLOTUB study investigating the shortening of tuberculosis treatment. A total of 343 adults enrolled in Benin, Guinea, Senegal, and South Africa were randomized to receive rifampicin, isoniazid, pyrazinamide, and ethambutol in the standard 6-month control regimen or the 4-month test regimen where gatifloxacin replaced ethambutol. The pharmacokinetics of all drugs was described at first dose and steady-state using nonlinear mixed-effects modelling, and individual exposures were summarised as area under the concentration-time curve (AUC0-24) and peak concentration. Autoinduction of rifampicin metabolism was characterized with a semi-mechanistic enzyme turn-over model. Gatifloxacin dose was evaluated using Monte Carlo simulations. Lastly, Classification & Regression Tree (CART) analysis techniques were used to identify factors predictive of 2-month culture conversion or 24-month long-term composite outcome. Consistent with literature findings, approximately 73.0, 43.0, 0.3, 6.0 and 0.0% of patients failed to achieve previously reported target concentrations for rifampicin, isoniazid, pyrazinamide, ethambutol, and gatifloxacin, respectively.

RATIONALE: 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.

Background: 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.

Two separate assays for prizidilol have been developed. One relies on separation by high performance liquid chromatography, and the other on separation by thin layer chromatography. Both of these methods have several advantages over the existing assay developed by JC Pearce for Smith Kline and French Ltd. (SK & F). Firstly, both methods utilize pepsin to digest plasma protein since it is known that precipitation with trichloroacetic acid of the protein leads to losses by binding at different rates of prizidilol and of the internal standard. The SK & F assay might therefore be less sensitive and precise than necessary. Secondly, use of the complexing reagent, quinolin-3-al, leads to greater sensitivity of detection than the reagent, anisaldehyde, previously employed. The minimum level of quantitation of the developed assays are approximately 30 μg/1 by HPLC and 15 μg/1 by TLC, as against 50 μg/1 by the earlier HPLC method originating from SK & F laboratories. Finally, the TLC method is much quicker to perform than the previous assay.

Includes bibliographical references.

Tuberculosis (TB) currently kills more people than any other infectious disease worldwide, the highest burden being in Africa and Asia (1). Therapy recommended for drug sensitive TB consists of a cocktail of isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA) and ethambutol (EMB), all given in a 2-month intensive phase, followed by only INH and RIF in a 4-month continuation phase. Clinical studies seeking to optimize dosing, gain more knowledge on the pharmacokinetics and pharmacodynamics of the drugs and compare current therapy to alternative regimens are required (2, 3). Therapeutic drug monitoring (TDM) is frequently carried out in cases responding poorly to therapy (3, 4). Both clinical studies and TDM require bioanalytical methods for quantifying drug concentrations in biological fluids. Several methods have been developed, mostly analysing individual drugs but a few analyse combinations. Ideally, quantification of all four drugs in one method is desirable as it is economical and allows high throughput. A method was developed and validated for the quantification of first line anti-tuberculosis drugs EMB, INH, PZA and RIF and the metabolites N-acetyl isoniazid (AcINH) and 25- desacetyl rifampicin (desRIF). Sample preparation consisted of protein precipitation, followed by high performance liquid chromatography (HPLC) with tandem mass spectrometry (MS/MS) detection. Deuterated internal standards for each analyte (AcINH-d4, desRIF-d3, EMB-d4, INH-d4, PZA-15N,d3 and RIF-d3) were used. Mean recoveries of the analytes from plasma were as follows: AcINH 106.5%, DesRIF 123.2%, EMB 105.3%, INH 110.1%, PZA 132.0% and RIF 127.7%. Sample preparation was followed by reverse phase liquid chromatography on an Agilent 1200 series HPLC system using an Agilent Poroshell 120 EC- C18 2.7µm, 4.6 X 50mm analytical column. Separation of all analytes was achieved using a mobile phase gradient consisting of an aqueous mobile phase A (0.05% formic acid in water) and an organic mobile phase B (0.05% formic acid in a mixture of methanol and acetonitrile, 1:1). A T-junction splitter was used to reduce the mobile phase flow to the ion source by about 30%. Retention times for AcINH, desRIF, EMB, INH, PZA and RIF were 2.45, 5.40, 1.75, 2.22, 4.30 and 5.68 minutes respectively. An AB Sciex API 4000 triple quadrupole mass spectrometer at unit mass resolution in the multiple reaction monitoring (MRM) mode was used for detection, monitoring the following transitions for the six analytes: AcINH 180 → 121, desRIF 784 → 752, EMB 205 → 116, INH 138 → 79, PZA 124 → 81 and RIF 823 → 792. An electrospray ionisation (ESI) source in the positive ion mode was used to couple the mass spectrometer to the LC system. Accuracy and precision were assessed over three consecutive and independent runs. The calibration curves fit quadratic regressions for all analytes, with weighting of 1/x (where x=concentration) for all except PZA which had a weighting of 1/x2 over the calibration range. Calibration ranges in µg/ml were as follows: AcINH 0.050 – 12.5, desRIF 0.040 – 10.0, EMB 0.020 – 5.00, INH 0.100 – 25.0, PZA 0.32. – 80.0 and RIF 0.120 – 30.0, based on peak area ratios. A 1:4 dilution of the QC Dilute sample showed that concentrations of up to 20.0 µg/ml for AcINH, 16.0 µg/ml for desRIF, 8.00 µg/ml for EMB, 40.0 µg/ml for INH, 128 µg/ml for PZA and 48.0 µg/ml for RIF in plasma could be analysed reliably when diluted into the calibration range. No significant carry-over was observed for all analytes. The method was shown to be reproducible when human plasma samples from six different sources were analysed and endogenous matrix components had no significant effect on the assay. All analytes were stable in plasma for at least four hours on ice, and when subjected to three freeze-thaw cycles. Reinjection reproducibility experiments showed that all analytes except PZA could be reliably analysed by re-injecting an entire batch after about 48 hours. Quantification of AcINH, INH and RIF was not significantly affected by 2% hemolysis of sample while desRIF, EMB and PZA were significantly affected. Data was analysed using Analyst ® version 1.6.2 software. With wide calibration ranges, the assay is suitable for both routine TDM and PK studies. Concurrent analysis of metabolites allows inferences to be made on the PK of the two main TB drugs. The total run time of 6.5 minutes per sample combined with the simple sample preparation procedure, the method is more economical on both time and resources than single analyte assays.

Includes abstract. Includes bibliographical references.