Pharmacogenetic and Pharmacoecological Determinants of Therapeutic Response to Non-nucleoside Reverse Transcriptase Inhibitors Among Hiv Patients in Kenya

Abstract

Despite the efficacy of antiretroviral therapy (ART) and the advancement in the prognosis of persons living with HIV/AIDS, a considerable proportion of persons on ART do not realize or retain adequate virologic suppression. Currently, 68% of adults and 73% of children living with HIV in Kenya are receiving ART treatment following the implementation of the 2015 World Health Organization (WHO) test and treat guidelines. During this study, the first-line ART treatment recommendations in Kenya typically comprised of two nucleoside reverse transcriptase inhibitors (NRTIs) and one non-nucleoside reverse transcriptase inhibitor (NNRTI): either nevirapine (NVP) or efavirenz (EFV). High interpersonal variability in the pharmacokinetics of ARV drugs have been reported, which may jeopardize ART treatment gains if not appropriately managed. Therapeutic drug monitoring (TDM) of antiretrovirals (ARVs) purposes to identify elevated or sub-therapeutic ARV concentrations which allows for prompt dosage adjustments preventing patients’ exposure to toxic or subtherapeutic concentrations. Optimal ART outcomes inevitably require an understanding of the individual variation in response to ART. This study determined the pharmacogenetic and pharmacoecological determinant of sub-therapeutic responses to NVP and EFV among HIV patients in Nairobi Kenya. In this non-comparative cross-sectional study, a total of 599 HIV patients were recruited who provided 5ml blood samples 8 hours post ART medication as well as participated in a face to face structured interview. The CD4 cell, viral load, full blood hematology and blood chemistry measurements were determined according to manufactures’ instruction. HIV drug resistant mutations were identified using an in-house sequencing method while cytochrome P450 (CYP26B) and constitutive androstane receptor (CAR) single nucleotide polymorphisms (SNPs) were identified using Real Time Polymerase chain reaction (RT-PCR). The NVP and EFV plasma levels were measured using ultra-high-performance liquid chromatography with a tandem quadruple mass spectrometer (LC/MS/MS). All data were subjected to descriptive statistical analysis. The NVP and EFV plasma levels were first tested for normality and presented as the median and interquartile range (IQR). Inferential data analysis of drug plasma levels were evaluated using the student’s t-test and one-way ANOVA. Categorical variables were summarized as frequencies and percentages while the Pearson’s chi square and fisher-exact tests used for inferential analysis of categorical variables. Host factors relate to NVP and EFV plasma level either directly or indirectly by affecting ART adherence. The relationship between pharmacoecological factors with ART adherence was first evaluated using fisher-exact or chisquare and only significant variables were evaluated for association with NVP or EFV plasma level using quantile linear regression. Data analysis was done using STATA v 13 software (StataCorp LP, Texas, USA). The allele, genotype and haplotype frequency and deviation from Hardy-Weinberg of CYP26B and CAR single nucleotide polymorphism (SNP) was analyzed using an online SNPStats software (https://www.snpstats.net/start.htm). The level of significant was set at p<0.05. All the 599 patients enrolled (100% responses rate) had data for objective one, while 566 (94.5% responses rate) had data for objectives two, three and four. The median age of the 599 patients was 41 years [IQR 35-47 years] with the majority (60.3%) being female and 56.1% were receiving EFV based regimen. The CD4 cell count (mean ± SD) significantly increased at the12-month post ART initiation (301.7 ± 199.4 cell/ml to 329.4 ± 305.8 cell/ml; P<0.05). Hepatotoxicity and renal abnormalities occurred more frequently at month 12 compared to baseline; ALT (2.5% versus 10.5%), AST (5.3% vs 23.4%) and creatinine (63.4 vs 68.84%). Fewer patients at month 12 had anemia (29.4% vs 56.4%), leucopenia (42.4% vs. 46.9%) and thrombocytopenia (6.5% vs. 84.1%) compared to baseline. The median [interquartile range – IQR] NVP plasma (n = 254) concentration was 6237.5 [4518 – 8964 ng/ml] and 2739.5 [1878 - 4891.5 ng/m] for EFV (n = 312). Majority 54.3% of patients had supra-therapeutic NVP plasma levels followed by 31.5% and 14.2% with sub-therapeutic and therapeutic plasma concentrations respectively (p < 0.001). Patients on EFV (63.8%, 31.7% and 4.5%) had therapeutic, supra-therapeutic and sub-therapeutic plasma concentrations respectively (p < 0.001). Thirteen CYP2B6 (329G>T, 341T>C, 444 G>T/C, 15582C>T, 516G>T, 548T>G, 637T>C, 785A>G, 18492C>T, 835G>C, 1459C>T and 21563C>T) and one CAR (540C>T) SNPs were detected among study patients. Hardy-Weinberg equilibrium could not be tested for CYP2B6 329G>T, 341T>C, 444 G>T/, 637T>C, 835G>C, and 548T>G SNPs due to lack of heterozygous and/or homozygous mutants. Linkage disequilibrium (LD) was observed among CYP2B6 15582C>T, 516G>T, 785A>G, 18492C>T, 983T>C, 21563C>T, 1459C>T and CAR 540C>T, resulting in 8 haplotypes among which CTGCTTCC and CGATTCCT had the highest and the lowest frequency. The most frequently occurring mutations with a prevalence of more than 30% were CYP2B6 516G>T, 785A>G and 21563C>T followed by CYP2B6 18492C>T and 15582C>T at a frequency of between 10 to 20%. The CAR (540C>T) and CYP2B6 983T>C occurred at a rate of 5 to 10% with CYP2B6 1459C>T, 329G>T, 341T>C, 444 G>T/C, 637T>C and 835G>C rarely occurred (>0.05%). The genotype and allele frequencies of SNPs were similar, regardless of the ART regimen. The following SNPs: CYP2B6 329 G>T; 15582C>T, 516G>T, 785A>G and 21563C>T, were associated with reduced NVP metabolism; while CYP2B6 18492C>T and 983T>C with increased NVP metabolism. The SNPs CYP2B6 15582C>T, 516G>T, 785A>G, and 21563C>T were associated with reduced metabolism of EFV and CYP2B6 18492C>T with increased EFV metabolism. The CYP2B6 and CAR inferred phenotypes associated with a reduced metabolism were found in 171/566 (30.2%) of all study patients; and 91/312 (29.2%) on EFV and 80/254 (31.5%) on NVP. Phenotypes associated with increased metabolism were found in 39.9% (n = 226/566) of the patients, 122/312 (39.1%) patients on EFV and 104/254 (40.9%) patients on NVP. On multivariate quantile regression analysis, factors that remained associated with high NVP plasma concentration included; feeling guilty for being HIV positive (adjusted β 954, 95% CI 192.7 to 2156.6; p =0.014) or feeling worthless for being HIV positive (adjusted β 852, 95% CI 64.3 to 1639.7; p =0.034), disclosing patient’s HIV status to neighbors (adjusted β 1731, 95% CI 376 to 3086; p = 0.012), regular uptake of porridge (adjusted β 1780, 95% CI 121.4 to 3438.6; p =0.036), missing taking current ARVs for the whole day or more (adjusted β 4287.9, 95% CI, 826.2 to 7749.6; p = 0.015), current white blood cell concentration (adjusted β 4012.4, 95% CI 1032.2 to 6992.6; p < 0.001). The SNPs associated with NVP plasma levels on multivariate analysis included; CYP2B6 329G>T (adjusted β 11343.2, 95% CI 5738.8 to 16947.6; p <0.001), CYP2B6 341T>C (adjusted β -8614.2, 95% CI -9621.7 to -7606.7; p < 0.001), CYP2B6 516G>T (adjusted β 3603.4, 95% CI 589.6 to 6617.2; p = 0.019), CYP2B6 18492 C >T (adjusted β -1301.4, 95% CI -2195.3 to -407.5; p = 0.004), CYP2B6 983T>C (adjusted β 2948, 95% CI 313.2 to 5582.8; p =0.028), CYP2B6 CAR 540 C>T (adjusted β -1543.2, 95% CI -2683.7 to -402.7; p =0.008) and number of SNPs per patients (adjusted β 1129.6, 95% CI 729.9 to 1529.3; p <0.001). On multivariate quantile regression analysis factors that remained significantly associated with high EFV plasma levels included; disclosing HIV positive status (adjusted β 363, 95% CI, 97.9 to 628.1; p = 0.007), obtaining ARV pill uptake information from other sources (adjusted β 18421.3, 95% CI, 16291.5 to 20551.2; p <0.001), stopping taking current ARVs due nausea (adjusted β 713, 95% CI 425.5 to 1000.5; p <0.001) and stopped taking current medication due to skin problems such as rash (adjusted β 1847, 95% CI 137.9 to 3556.1; p =0.035) and the presence of body pain in the past 30 days (adjusted β 475, 95% CI, 117.2 to 832.8; p = 0.009). The SNPs CYP2B6 516G>T (adjusted β 2414, 95% CI 1876.6 to 2951.4; p <0.001), CYP2B6 835 G >C (adjusted β 2877, 95% CI 1498 to 4256; p <0.001) and number of SNPs per patient (adjusted β 570, 95% CI 362 to 778; p <0.001). Some of the important limitations worth mentioning in this study included. First, the use of NVPbased ART regimens in Kenya and other countries, especially developed countries, has been considerably reduced in the recent past, meaning that this study could be relevant to a restricted number of patients. Second, standardized tools for measuring pharmacoecological variables are not available, limiting the generalizability of this study outcomes. Third, this was a cross-sectional study, which only permitted the description of the relationship between pharmacogenetic and pharmacoecological factors and NVP/EFV plasma concentrations and not a causal conclusion. Such outcomes can be confirmed in a longitudinal study. These limitations notwithstanding, the

following conclusions can be drawn. These data establish prolonged immunologic/virologic response to ART among patients continuing on therapy. The prevalence of anemia, thrombocytopenia and leucopenia was low with minimal renal and hepatotoxicity impairment observed. Wide interindividual variability were observed in NVP and EFV plasma concentrations with a large proportion of patients falling outside of therapeutic window, proposing potentially an increased risk of treatment failure or toxicity. This study demonstrates the importance of therapeutic drug monitoring in determining the ARV treatment outcome. Six SNPs of the CYP2B6 gene (CYP2B6 329G>T, CYP2B6 637T>C, CYP2B6 785A>G, CYP2B6 18492C>T, CYP2B6 21563C>T and 15582C>T) and one CAR (540T>C) could potentially act as additional independent predictors of NVP and EFV plasma concentrations beyond that provided by the CYP2B6 c.516G>T and CYP2B6 983T>C polymorphism. Host pharmacoecological factors influence ART drug adherence impacting on the NVP and EFV plasma concentrations.