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Despite their high potential for drug-drug interactions (DDI), clinical DDI studies of antiretroviral drugs (ARVs) are often lacking, because the full range of potential interactions cannot feasibly or pragmatically be studied, with some high-risk DDI studies also being ethically difficult to undertake. Thus, a robust method to screen and to predict the likelihood of DDIs is required. We developed a method to predict DDIs based on two parameters: the degree of metabolism by specific enzymes, such as CYP3A, and the strength of an inhibitor or inducer. These parameters were derived from existing studies utilizing paradigm substrates, inducers, and inhibitors of CYP3A to assess the predictive performance of this method by verifying predicted magnitudes of changes in drug exposure against clinical DDI studies involving ARVs. The derived parameters were consistent with the FDA classification of sensitive CYP3A substrates and the strength of CYP3A inhibitors and inducers. Characterized DDI magnitudes (; n; = 68) between ARVs and comedications were successfully quantified, meaning 53%, 85%, and 98% of the predictions were within 1.25-fold (0.80 to 1.25), 1.5-fold (0.66 to 1.48), and 2-fold (0.66 to 1.94) of the observed clinical data. In addition, the method identifies CYP3A substrates likely to be highly or, conversely, minimally impacted by CYP3A inhibitors or inducers, thus categorizing the magnitude of DDIs. The developed effective and robust method has the potential to support a more rational identification of dose adjustment to overcome DDIs, being particularly relevant in an HIV setting, given the treatment's complexity, high DDI risk, and limited guidance on the management of DDIs.

Objective: The prevalence of overweight and obesity is increasing among HIV-infected patients. Whether standard antiretroviral drug dosage is adequate in heavy individuals remains unresolved. We assessed the virological and immunological responses to initial efavirenz (EFV)-containing regimens in heavy compared to normal-weight HIV-infected patients. Design: Observational European cohort collaboration study. Methods: Eligible patients were antiretroviral-naive with documented weight prior to EFV start and follow-up viral loads after treatment initiation. Cox regression analyses evaluated the association between weight and time to first undetectable viral load ( 50 copies/ml) after initial suppression over 5 years of follow-up. Recovery of CD4(+) cell count was evaluated 6 and 12 months after EFV initiation. Analyses were stratified by weight (kg) group (I - 55, 80, 85, 90, 95). Results: The study included 19 968 patients, of whom 9.1, 68.3, 9.1, 5.8, 3.5, and 4.3% were in weight groups I-VI, respectively. Overall, 81.1% patients attained virological suppression, of whom 34.1% subsequently experienced viral load rebound. After multiple adjustments, no statistical difference was observed in time to undetectable viral load and virological rebound for heavier individuals compared to their normal weight counterparts. Although heaviest individuals had significantly higher CD4(+) cell count at baseline, CD4(+) cell recovery at 6 and 12 months after EFV initiation was comparable to normal-weight individuals. Conclusion: Virological and immunological responses to initial EFV-containing regimens were not impaired in heavy individuals, suggesting that the standard 600 mg EFV dosage is appropriate across a wide weight range. (C) 2015 Wolters Kluwer Health, Inc. All rights reserved

• Nevirapine pharmacokinetics are affected by several factors including CYP2B6 single nucleotide polymorphisms (SNPs). These genetic profiles are more common in African patients and they affect the drug clearance being associated with higher trough concentrations. Pharmacokinetic/pharmacogenetic (PK/PG) studies are difficult to perform in remote areas where refrigeration is not available, although dried plasma and dried blood methods have been validated.• Dried plasma spots are useful tools for studying nevirapine PK with a good association to plasma concentrations and they can be used in rural areas since a cold chain is not necessary. Dried blood spots can be used to store and analyze patients' DNA for PG polymorphisms. Nevirapine trough concentrations in Burundese patients, not studied so far, are above the target concentration (3000 ng ml(-1) ) in 84% of patients. CYP2B6 (both at position 516 and 983) but not ABCB1 (3435 and 1236) SNPs as well as age correlate with higher nevirapine exposure.The pharmacokinetics (PK) and pharmacogenetics (PG) of nevirapine have been studied in rich and limited-resource countries. CYP2B6 single nucleotide polymorphisms (SNPs) have been associated with decreased drug clearance. We evaluated the PG determinants of nevirapine trough concentrations in a rural cohort in Burundi using easy to store and transport dried sample spot devices.A cross-sectional analysis in HIV-positive nevirapine-treated patients in Kiremba, north of Burundi, was performed in 2009. After blood withdrawal whole blood was stored on dried blood spots and plasma (after centrifugation) was placed on dried plasma spot devices and stored at room temperature. Nevirapine plasma and dried sample spot concentrations were compared to test the clinical usefulness of this method. SNPs in CYP2B6 and ABCB1 (using a real time PCR technique) were analyzed and associated with nevirapine plasma trough concentrations.Nevirapine concentrations measured on dried plasma spot devices were highly related to plasma concentrations in 60 patients, although a negative bias was observed (-18%). Nevirapine trough concentrations were above the target concentration (3000 ng ml(-1) ) in 84% of patients and they were associated with CYP2B6 SNPs (both at position 516 and 983). No effect of ABCB1 SNPs was noted.Dried plasma spot devices are accurate tools for measuring nevirapine concentrations in rural settings where refrigeration is not available, despite a moderate underestimation bias. They allowed the evaluation of nevirapine concentrations in a cohort of HIV-infected people in rural Burundi, confirming very good exposure and correlation with PG polymorphisms in the CYP2B6 encoding gene.

Antiretroviral therapy (ART) has revolutionised outcomes of HIV infection among people living with HIV (PLWH). But despite these great strides, ongoing concerns of drug-drug interactions, widespread use of herbal medication while using ART, safety in pregnancy and breastfeeding are challenges of HIV treatment in PLWH. Potent new ART are not widely used in most low and middle income countries (LMIC) due to insufficient safety data and fear of adverse events. This Thesis aimed at evaluating the pharmacokinetic (PK) safety of DTG in pregnant and breastfeeding women, drug drug interaction of DTG and Artemisinin Combination Therapy (ACT), and evaluate the factors that affect ART in PLWH such as use of herbal medication among PLWH. Therefore the thesis evaluated the following: Chapter 2 reviewed the clinical PK and pharmacogenetic, drug-drug interactions and safety of DTG in diverse populations from different studies, and concluded that DTG has a variable PK influenced by factors such as food, research conditions and population variability. Chapter 4 evaluated the PK of DTG in 3rd trimester of pregnancy and postpartum women and found no clinically significant difference in DTG PK between pregnancy and postpartum period in women. Safety and efficiency of HIV control was also evaluated and concluded that DTG efficiently control viral load within a short period of time even when women present late in pregnancy. xx Chapter 5 evaluated the drug-drug interaction of DTG and ACT, and concluded that DTG can be co-administered with ACT in treatment of malaria among PLWH on DTG. Chapter 6 evaluated the impact of pharmacogenetics and pregnancy on tenofovir and emtricitabine Pharmacokinetics. An estimated 1-2-fold increase in FTC blood concentration was observed in pregnant and postpartum women with ABCC2 12:g.154962860T>C T allele allele compared to women with CT and CC allele. Sample size was small and was recognise as a limitation. Therefore require verification with larger clinical studies and result should be interpreted with caution. Chapter 7 evaluated the widespread use of herbal medicines amongst PLWH and contamination of herbal medicines with ART in Nigeria as a recognised challenge of ART in sub-Saharan Africa. In conclusion, approximately 2-4% of maternal plasma DTG concentration was excreted in BM, and DTG PK changes were not clinically significant in both pregnancy and postpartum period. DTG Ctrough decreased by 37%, when DTG was administered with AL, 42% when administered with AS-AQ and 24% decrease in AUC0-24 when administered with AS-AQ, but were all above the protein adjusted IC90 for the Ctrough and does not warrant dose adjustment. An estimated 41.8% use of herbal medication was recorded amongst 742 PLWH attending HIV clinics. Herbal use preceded HIV therapy in 38.4% while 14.5% were yet to commence ART. A total of 3 (2%) out of 138 herbal samples evaluated, were contaminated with detectable levels of tenofovir and emtricitabine which is a concern, though implication is unknown.

The first-order degradation rate constant (kdeg) of drug metabolising enzymes (DMEs) is a known source of uncertainty in the prediction of time-dependent drug-drug interactions (DDIs) in physiologically-based pharmacokinetic (PBPK) modelling. There is a large disparity or paucity of published kdeg and related half-life (t1/2) values for DMEs. Physiologically-relevant kdeg values should ideally be derived in vivo to facilitate accurate DDI predictions. However, direct measurement of DME degradation in humans is not routinely possible and indirect measurements utilising changes in levels of specific endogenous substrates have only been described for a few DMEs. This thesis aims to develop an in vitro method of measuring DME protein degradation rates to improve the prediction accuracy of time-dependent DDIs. One in vitro approach of measuring protein degradation rates involves inhibiting de novo protein synthesis, followed by tracking residual protein or activity decline over time. Pharmacological protein synthesis inhibitor agents are commonly used for this purpose but may cause cytotoxicity. Four commonly used inhibitor agents were assessed for their capacity to inhibit protein synthesis without overt cytotoxicity. However, all selected compounds were too cytotoxic for subsequent use in kdeg studies. Small-interfering ribose nucleic acid (siRNA) can be added to in vitro systems to initiate gene-specific silencing by inhibiting messenger RNA (mRNA) translation. It was hypothesised that siRNA would inhibit de novo protein synthesis with less cytotoxicity owing to its specificity. CYP3A4 is the most widely studied cytochrome P450 (CYP) enzyme in terms of DDIs because of its well-recognised role in xenobiotic metabolism. Primary human hepatocytes were treated with CYP3A4-specific siRNA to suppress mRNA translation, followed by the tracking of enzyme activity and protein loss over time to derive kdeg. CYP3A4 kdeg was calculated at 0.019 (± 0.044) and 0.020 (± 0.0003) h-1 from protein and activity loss, respectively. These values were in good agreement with existing published values. The siRNA approach was subsequently applied to determine CYP2B6 kdeg. The CYP2B6 kdeg values derived from siRNA-treated hepatocytes were 0.081 (± 0.009) h-1 from protein loss and 0.058 (± 0.010) and 0.062 (± 0.006) h-1 from activity loss, which were assessed by different methods. The CYP2B6 kdeg values derived from untreated hepatocytes were similar to values in literature. This novel approach can now be used for other less well-characterised DMEs that are associated with time-dependent DDIs. Cellular protein abundance is a balance between synthesis and degradation. Dysregulation of the lysosomal or proteasomal protein degradation mechanisms affects steady-state protein levels and impacts on overall cellular functions. It was hypothesised that single nucleotide polymorphisms (SNPs) in the CYP3A4 protein degradation machinery could affect CYP3A4 protein abundance and downstream activity. Five SNPs were investigated for associations with plasma atazanair (ATV) concentrations, which was a surrogate measure for CYP3A4 activity. No associations were found and this was likely due to the lack of clear understanding of the specific mechanisms that commits CYP proteins for degradation. Further work in this field will identify targets that may be exploited in the future for more accurate measurements of DME kdeg. The data presented in this thesis enhances the understanding of methods used to study protein degradation and this can be applied to multiple fields of cellular research. Importantly, work herein has generated a novel approach to measuring kdeg of proteins that can be applied to other less-well characterised enzymes for better prediction of time-dependent DDIs.