Characterization of the steady state concentrations and pharmacogenetics of ritonavir boosted atazanavir in Kenyan HIV positive patients.

Abstract

Background Atazanavir is a protease inhibitor currently recommended for use as a second line agent in Human Immunodeficiency Virus (HIV) infected patients who have failed therapy on a first line regimen. The pharmacokinetics of atazanavir are highly variable and may be influenced by factors such as age, sex, weight and genetics. In Kenya, there is a paucity of local HIV patient data on atazanavir pharmacokinetics, the influence of cytochrome P450 subfamily 3A5 (CYP3A5) genetic polymorphisms on its disposition and the prevalence of hyperbilirubinemia in patients using the drug. Objectives We optimized and verified a high-performance liquid chromatographic method with ultra-violet detection (HPLC-UV) for the determination of plasma concentrations of atazanavir. We determined the prevalence of single nucleotide polymorphisms in CYP3A5 and uridine diphosphate glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1) enzymes. Thereafter, we examined whether polymorphisms in CYP3A5 enzymes were associated with atazanavir steadystate plasma concentrations. We determined the prevalence and risk factors of hyperbilirubinemia and investigated the effect of UGT1A1 polymorphisms on bilirubin levels and the risk of hyperbilirubinemia. Methods We conducted a cross sectional study enrolling 110 male and female HIV positive patients on ritonavir boosted atazanavir over two months from the Kenyatta National Hospital. Participants were 18 years and older and provided written informed consent. We abstracted data from patient files, conducted interviews and drew five blood samples: one at 30 minutes before the morning dose then 2 and 4 hours after the morning dose. The two additional samples were for genotyping and for determination of plasma bilirubin levels respectively. Atazanavir quantification was achieved by high performance liquid chromatography with ultraviolet detection at 261nm. Genetic analysis was performed by fast real time polymerase chain reaction for CYP3A5 and UGT1A1 single nucleotide polymorphisms. Statistical analysis was done in STATA version 13.1 and R i3.8.6 version 3.3.1 with the level of significance set at p value ≤ 0.05. Ethical approval was granted by the Kenyatta National Hospital and University of Nairobi Ethics and Research Review Committee (KNH-ERC/A/110). Results Optimization and verification of a HPLC-UV method for the quantification of plasma atazanavir levels was achieved. The coefficient of determination was > 0.99 and was linear between 100 -10000 ng/mL. The precision (% relative standard deviation) was 8,8% (SD 6.2) and the accuracy ranged from 91.3 - 120.8%. The limit of detection was 40 ng/mL whereas the limit of quantitation was 120ng/mL. The prevalence of single nucleotide polymorphisms was as follows: CYP3A5*3 TT wild type genotype - 24%, heterozygous TC genotype - 70%, CC homozygous genotype -6%; CYP3A5*6 CC wild type genotype - 2.7%, heterozygous CT genotype - 97.3%; CYP3A5*7 AA wild type genotype – 1.8%, heterozygous A/- genotype – 95.5%, -/- variant genotype - 2.7%; UGT1A1 CC genotype 16.4%, heterozygous CT 83.6%. A small proportion of participants had subtherapeutic plasma trough concentrations of atazanavir (13%) whereas 39% had supratherapeutic levels. CYP3A5*6 was found to influence the trough plasma concentrations of atazanavir (adjusted estimate 2151.1 ng/mL, 95% CI 275.5, 4026.8). The prevalence of grades III and IV hyperbilirubinemia among the participants was 12.7 and 1.8%. Hyperbilirubinemia of any grade was present in 57.3% of the participants Risk factors for hyperbilirubinemia were high atazanavir trough concentrations (aOR 1.001, 95% CI 1.001, 1.002), UGT1A1 heterozygosity (aOR 7.01, 95% CI 1.86, 34.01) and a positive history of alcohol use (aOR 0.18, 95% CI 0.03, 0.78). Conclusions CYP3A5 and UGT1A1 polymorphisms exist in this population. A significant proportion of participants had supratherapeutic trough plasma concentrations of atazanavir. CYP3A5 polymorphisms have an influence on the troughxvii concentrations of atazanavir. Hyperbilirubinemia is prevalent in patients on atazanavir and is influenced by UGT1A1 status, trough plasma concentrations of atazanavir pharmacokinetics and alcohol use.