Highly active antiretroviral therapy (HAART) has improved the life expectancy of HIV–1–infected patients dramatically since it was launched in 1996, but there are still many challenges in the provision of HAART, especially to children in resource limited countries. Efavirenz (EFV), a non–nucleoside reverse transcriptase inhibitor (NNRTI) forms part of the recommended national first line antiretroviral treatment regimen for children older than 3 years and weighing more than 10 kg in South Africa. Limited pharmacokinetic information on EFV plasma concentrations in sub–Saharan HIV–1– infected children is available. EFV is primarily metabolised by hepatic CYP2B6 isoenzymes. The CYP2B6 gene is characterised by extensive inter–individual variability in hepatic expression and activity. The single nucleotide change, 516G>T, on the CYP2B6 gene has consistently been associated with elevated EFV plasma levels in different ethnic populations and these are more frequently observed in populations of African descent. The recommended therapeutic range of EFV plasma levels is 1–4 ug/ml and the Cmin should be above 1 ug/ml. In this prospective study (PK/PD.EFV.07) cohort, 60 black children, both genders, with no prior exposure to antiretroviral therapy and eligible for antiretroviral therapy (ART) were enrolled and followed up at 1, 3, 6, 12, 18 and 24 months post HAART initiation. They all attended the outpatient clinic at Harriet Shezi Children’s Clinic, Chris Hani Baragwanath Hospital, Soweto, South Africa. The required ethics approval was obtained to conduct this study. The objectives of this investigation were to: develop and validate a suitable LCMS/ MS method to accurately determine plasma EFV levels from this study population, determine the prevalence and effect of CYP2B6 516G>T polymorphism on EFV plasma levels, determine the population pharmacokinetic clearance (CL/F) value of EFV, identify covariates that influence the clearance of EFV in HIV–1– infected children, and investigate specific pharmacodynamic effects and therapeutic outcomes of this EFV–based regimen within this paediatric population over the 24 months post–HAART initiation. The main findings of the measured mid–dose EFV plasma concentrations showed that sub–therapeutic concentrations (<1 ug/ml) accounted for 18% (116/649), within therapeutic range (1–4 ug/ml) represented 52.5% (341/649), and concentrations above the therapeutic range (>4 ug/ml) represented 29.5% (192/649). A significant number of the samples (47.5%) were outside the accepted therapeutic range during this 24 month follow–up period. Possible reasons contributing to this include genetic variation in drug metabolism and non–adherence. Genotype results on all 60 study participants were: 23% 516 T/T homozygotes, 42% 516 G/G homozygotes and 35% 516 G/T heterozygotes. The 516 T–allelic variant frequency was relatively high at 41%. This also supports and explains why such a large number (29.5%) of the mid–dose interval plasma samples were above (>4 ug/ml) the accepted therapeutic range. Repeated measures ANOVA confirmed that CYP2B6 516 G/G, G/T and T/T genotypes were consistently predictive of the log EFV concentrations at all times (P = 0.0001). The total median (IQR) EFV plasma concentrations over the 24 months post–HAART when pooled, were 6.36 (3.47 - 7.28) for T/T, 2.55 (1.62 - 3.59) for G/T, and 1.41 (1.02 - 1.74) ug/ml for G/G groups respectively (P<0.00001). Multiple comparisons by groups revealed that the EFV plasma concentrations between the T/T and G/G (P=0.000002) and between G/T and G/G (P=0.009) were statistically significant. However, the differences between the EFV plasma concentrations of the T/T and G/T groups were not significantly different (P=0.074). This supports previous results that the presence of the 516 T–allelic variant is responsible for the higher EFV plasma concentrations within individuals presenting with this single nucleotide mutation on the CYP2B6 gene. This EFV–based treatment was well tolerated even at plasma concentrations above the therapeutic range (>4 ug/ml) and most side effects subsided spontaneously. 89% of the participants were virally suppressed at 24 months post–HAART. The efficacy of this EFV–based treatment did not affect the three genotype groups differently and they showed similar improvement in their immunological (CD4–cell count and CD4%) markers and reduction in viral load over the 24 months post– HAART initiation. We found no association of the CYP2B6 516G>T polymorphism and side effects reported after 1 month of treatment within this study population. The final population pharmacokinetic (PK) estimates for EFV clearance (CL/F) were, 2.46, 4.60, and 7.33 l/h for the T/T, G/T, and G/G respective genotype groups. The volume of distribution (V/F) estimate was 89.52 l. The importance of interoccasion variability (IOV) in a PK model for a longitudinal study was again highlighted by this investigation. To our knowledge, this is the first study in black South African HIV–1–infected children with measured sequential EFV plasma concentrations which also investigated the influence of the CYP2B6 516G>T polymorphism on EFV plasma concentrations and the population clearance (CL/F) value of EFV in a longitudinal study over a period of 24 months post–HAART initiation. / Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2011.

Highly active antiretroviral therapy (HAART) has improved the life expectancy of HIV–1–infected patients dramatically since it was launched in 1996, but there are still many challenges in the provision of HAART, especially to children in resource limited countries. Efavirenz (EFV), a non–nucleoside reverse transcriptase inhibitor (NNRTI) forms part of the recommended national first line antiretroviral treatment regimen for children older than 3 years and weighing more than 10 kg in South Africa. Limited pharmacokinetic information on EFV plasma concentrations in sub–Saharan HIV–1– infected children is available. EFV is primarily metabolised by hepatic CYP2B6 isoenzymes. The CYP2B6 gene is characterised by extensive inter–individual variability in hepatic expression and activity. The single nucleotide change, 516G>T, on the CYP2B6 gene has consistently been associated with elevated EFV plasma levels in different ethnic populations and these are more frequently observed in populations of African descent. The recommended therapeutic range of EFV plasma levels is 1–4 ug/ml and the Cmin should be above 1 ug/ml. In this prospective study (PK/PD.EFV.07) cohort, 60 black children, both genders, with no prior exposure to antiretroviral therapy and eligible for antiretroviral therapy (ART) were enrolled and followed up at 1, 3, 6, 12, 18 and 24 months post HAART initiation. They all attended the outpatient clinic at Harriet Shezi Children’s Clinic, Chris Hani Baragwanath Hospital, Soweto, South Africa. The required ethics approval was obtained to conduct this study. The objectives of this investigation were to: develop and validate a suitable LCMS/ MS method to accurately determine plasma EFV levels from this study population, determine the prevalence and effect of CYP2B6 516G>T polymorphism on EFV plasma levels, determine the population pharmacokinetic clearance (CL/F) value of EFV, identify covariates that influence the clearance of EFV in HIV–1– infected children, and investigate specific pharmacodynamic effects and therapeutic outcomes of this EFV–based regimen within this paediatric population over the 24 months post–HAART initiation. The main findings of the measured mid–dose EFV plasma concentrations showed that sub–therapeutic concentrations (<1 ug/ml) accounted for 18% (116/649), within therapeutic range (1–4 ug/ml) represented 52.5% (341/649), and concentrations above the therapeutic range (>4 ug/ml) represented 29.5% (192/649). A significant number of the samples (47.5%) were outside the accepted therapeutic range during this 24 month follow–up period. Possible reasons contributing to this include genetic variation in drug metabolism and non–adherence. Genotype results on all 60 study participants were: 23% 516 T/T homozygotes, 42% 516 G/G homozygotes and 35% 516 G/T heterozygotes. The 516 T–allelic variant frequency was relatively high at 41%. This also supports and explains why such a large number (29.5%) of the mid–dose interval plasma samples were above (>4 ug/ml) the accepted therapeutic range. Repeated measures ANOVA confirmed that CYP2B6 516 G/G, G/T and T/T genotypes were consistently predictive of the log EFV concentrations at all times (P = 0.0001). The total median (IQR) EFV plasma concentrations over the 24 months post–HAART when pooled, were 6.36 (3.47 - 7.28) for T/T, 2.55 (1.62 - 3.59) for G/T, and 1.41 (1.02 - 1.74) ug/ml for G/G groups respectively (P<0.00001). Multiple comparisons by groups revealed that the EFV plasma concentrations between the T/T and G/G (P=0.000002) and between G/T and G/G (P=0.009) were statistically significant. However, the differences between the EFV plasma concentrations of the T/T and G/T groups were not significantly different (P=0.074). This supports previous results that the presence of the 516 T–allelic variant is responsible for the higher EFV plasma concentrations within individuals presenting with this single nucleotide mutation on the CYP2B6 gene. This EFV–based treatment was well tolerated even at plasma concentrations above the therapeutic range (>4 ug/ml) and most side effects subsided spontaneously. 89% of the participants were virally suppressed at 24 months post–HAART. The efficacy of this EFV–based treatment did not affect the three genotype groups differently and they showed similar improvement in their immunological (CD4–cell count and CD4%) markers and reduction in viral load over the 24 months post– HAART initiation. We found no association of the CYP2B6 516G>T polymorphism and side effects reported after 1 month of treatment within this study population. The final population pharmacokinetic (PK) estimates for EFV clearance (CL/F) were, 2.46, 4.60, and 7.33 l/h for the T/T, G/T, and G/G respective genotype groups. The volume of distribution (V/F) estimate was 89.52 l. The importance of interoccasion variability (IOV) in a PK model for a longitudinal study was again highlighted by this investigation. To our knowledge, this is the first study in black South African HIV–1–infected children with measured sequential EFV plasma concentrations which also investigated the influence of the CYP2B6 516G>T polymorphism on EFV plasma concentrations and the population clearance (CL/F) value of EFV in a longitudinal study over a period of 24 months post–HAART initiation. / Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2011.

Malaria is one the world’s most devastating diseases. Several classes of drugs are used to treat malaria. Artemisinin combination therapy is the first line treatment of uncomplicated malaria. The artemisinin derivative, artemisone in conjunction with the Pheroid® drug delivery system, is the focus of this thesis. The impact of the Pheroid® on the bioavailability of artemisone was evaluated in vervet monkeys. The resulting artemisone plasma levels were much lower (Cmax of 47 and 114 ng/mL for reference and Pheroid® test formulations respectively) than expected for the dosages administered (60 mg/kg). The Pheroid® improved the pharmacokinetic profile of artemisone in a clinically significant manner. The metabolism of artemisone was assessed in vitro by using human and monkey liver and intestinal microsomes, and recombinant CYP3A4 enzymes. The Pheroid® inhibits the microsomal metabolism of artemisone. In addition, there is a species difference in artemisone metabolism between man and monkey since the in vitro intrinsic clearance of the reference formulation with monkey liver microsomes is ~8 fold higher in the monkey liver microsomes compared to the human liver microsomes and the estimated in vivo hepatic clearance for the monkey is almost twofold higher than in humans. Artemisone has potent antimalarial activity. Its in vitro efficacy was approximately twofold higher than that of either artesunate or dihydroartemisinin when evaluated against P. falciparum W2, D6, 7G8, TM90-C2B, TM91-C235 and TM93-C1088 parasite strains. The Pheroid® drug delivery system did not improve or inhibit the in vitro efficacy of artemisone or DHA. Artemisone (reference and Pheroid® test formulations) and metabolite M1 abruptly arrested the growth of P. falciparum W2 parasites and induced the formation of dormant ring stages in a manner similar to that of DHA. Interaction of artemisone with the p-glycoprotein (p-gp) efflux transporter was investigated. Artemisone stimulates ATPase activity in a concentration-dependent manner, whereas the Pheroid® inhibited this p-gp ATPase activity. P-gp ATPase activity stimulation was fourfold greater in human than cynomolgus monkey MDR1 expressed insect cell membranes. Artemisone alone and artemisone entrapped in Pheroid® vesicles showed moderate apical to basolateral and high basolateral to apical permeability (Papp) across Caco-2 cells. The Papp efflux ratio of artemisone and artemisone entrapped in Pheroid® vesicles were both >5, and decreased to ~1 when the p-gp inhibitor, verapamil, was added. Therefore, artemisone is a substrate for mammalian p-gp. The cytotoxic properties of Pheroid® on Caco-2 cells were assessed and the pro-Pheroid® seems to be non-toxic at concentrations of 1.25%. Vervet monkey plasma caused antibody-mediated growth inhibition of P. falciparum. Heat inactivated or protein A treatment proved useful in the elimination of the growth-inhibitory activity of the drug-free plasma. Plasma samples containing artemisone could not be analysed by the ex-vivo bioassay method. The dual labelling ROS assay did not prove to be useful in the evaluation of ROS production by artemisone and the Pheroid® delivery system. In conclusion, entrapment of artemisone in the Pheroid® delivery system improves the pharmacokinetic properties of artemisone, but does not improve or inhibit its antimalarial efficacy in vitro. The Pheroid® inhibited both the microsomal metabolism of artemisone and P-gp ATPase activity and was shown to be non-toxic at clinically usable concentrations. / PhD (Pharmaceutics), North-West University, Potchefstroom Campus, 2014

Malaria is one the world’s most devastating diseases. Several classes of drugs are used to treat malaria. Artemisinin combination therapy is the first line treatment of uncomplicated malaria. The artemisinin derivative, artemisone in conjunction with the Pheroid® drug delivery system, is the focus of this thesis. The impact of the Pheroid® on the bioavailability of artemisone was evaluated in vervet monkeys. The resulting artemisone plasma levels were much lower (Cmax of 47 and 114 ng/mL for reference and Pheroid® test formulations respectively) than expected for the dosages administered (60 mg/kg). The Pheroid® improved the pharmacokinetic profile of artemisone in a clinically significant manner. The metabolism of artemisone was assessed in vitro by using human and monkey liver and intestinal microsomes, and recombinant CYP3A4 enzymes. The Pheroid® inhibits the microsomal metabolism of artemisone. In addition, there is a species difference in artemisone metabolism between man and monkey since the in vitro intrinsic clearance of the reference formulation with monkey liver microsomes is ~8 fold higher in the monkey liver microsomes compared to the human liver microsomes and the estimated in vivo hepatic clearance for the monkey is almost twofold higher than in humans. Artemisone has potent antimalarial activity. Its in vitro efficacy was approximately twofold higher than that of either artesunate or dihydroartemisinin when evaluated against P. falciparum W2, D6, 7G8, TM90-C2B, TM91-C235 and TM93-C1088 parasite strains. The Pheroid® drug delivery system did not improve or inhibit the in vitro efficacy of artemisone or DHA. Artemisone (reference and Pheroid® test formulations) and metabolite M1 abruptly arrested the growth of P. falciparum W2 parasites and induced the formation of dormant ring stages in a manner similar to that of DHA. Interaction of artemisone with the p-glycoprotein (p-gp) efflux transporter was investigated. Artemisone stimulates ATPase activity in a concentration-dependent manner, whereas the Pheroid® inhibited this p-gp ATPase activity. P-gp ATPase activity stimulation was fourfold greater in human than cynomolgus monkey MDR1 expressed insect cell membranes. Artemisone alone and artemisone entrapped in Pheroid® vesicles showed moderate apical to basolateral and high basolateral to apical permeability (Papp) across Caco-2 cells. The Papp efflux ratio of artemisone and artemisone entrapped in Pheroid® vesicles were both >5, and decreased to ~1 when the p-gp inhibitor, verapamil, was added. Therefore, artemisone is a substrate for mammalian p-gp. The cytotoxic properties of Pheroid® on Caco-2 cells were assessed and the pro-Pheroid® seems to be non-toxic at concentrations of 1.25%. Vervet monkey plasma caused antibody-mediated growth inhibition of P. falciparum. Heat inactivated or protein A treatment proved useful in the elimination of the growth-inhibitory activity of the drug-free plasma. Plasma samples containing artemisone could not be analysed by the ex-vivo bioassay method. The dual labelling ROS assay did not prove to be useful in the evaluation of ROS production by artemisone and the Pheroid® delivery system. In conclusion, entrapment of artemisone in the Pheroid® delivery system improves the pharmacokinetic properties of artemisone, but does not improve or inhibit its antimalarial efficacy in vitro. The Pheroid® inhibited both the microsomal metabolism of artemisone and P-gp ATPase activity and was shown to be non-toxic at clinically usable concentrations. / PhD (Pharmaceutics), North-West University, Potchefstroom Campus, 2014