Cytochrome P450 2B6 516G→T is associated with plasma concentrations of nevirapine at both 200 mg twice daily and 400 mg once daily in an ethnically diverse population

Authors

Errata

This article is corrected by:

  1. Errata: ERRATUM Volume 10, Issue 10, 661, Article first published online: 2 September 2009

Dr Tabitha W. Mahungu, Department of HIV Medicine, Royal Free NHS Trust, Pond Street, London NW3 2QG, UK. Tel: +44 207 472 6232; fax: +44 207 472 6233; e-mail: wambura@gmail.com

Abstract

Objectives

The aim of the study was to characterize the impact of the cytochrome P450 2B6 (CYP2B6), CYP3A4, CYP3A5 and ATP-binding cassette sub-family B member 1 (ABCB1) polymorphisms on nevirapine plasma concentrations.

Methods

A total of 104 patients (82% male; 26% non-Caucasian) were genotyped for eight single nucleotide polymorphisms at four loci (CYP2B6, CYP3A4, CYP3A5 and MDR1). Nevirapine plasma concentrations were determined using high-performance liquid chromatography.

Results

Non-Caucasian ethnicity [5609 ng/mL (n=27) for non-Caucasians vs. 3771 ng/mL (n=77) for Caucasians; P<0.0001] and CYP2B6 516G→T [GG, 3574 ng/mL (n=50); GT, 4634 ng/mL (n=50); TT, 8170 ng/mL (n=4); Panalysis of variance (anova)=0.001] were significantly associated with a higher nevirapine trough concentration (Ctrough). The latter association was maintained with both 200 mg twice daily (bid) and 400 mg once daily (qd) dosing [GG, 3527 ng/mL (n=30); GT, 4525 ng/mL (n=32); TT, 7020 ng/mL (n=2); Panova=0.05 and GG, 3645 ng/mL (n=20); GT, 4861 ng/mL (n=17); TT, 9508 ng/mL (n=2); Panova=0.01, respectively]. In a multivariable analysis, CYP2B6 516G→T and non-Caucasian ethnicity remained significant predictors of nevirapine Ctrough but CYP2B6 516G→T homozygosity had the greatest effect (108% higher, 46% higher). No associations were found between nevirapine Ctrough and the remaining polymorphisms.

Conclusion

In this population, both non-Caucasian ethnicity and carriage of the variant allele of CYP2B6 516G→T were significant predictors of nevirapine Ctrough. The association between CYP2B6 516G→T and higher plasma nevirapine exposure was maintained at both bid and qd dosing.

Introduction

Nevirapine is a nonnucleoside reverse transcriptase inhibitor (NNRTI) that is an alternative component of first-line antiretroviral regimens in HIV-infected individuals [1]. It is commonly prescribed in resource-poor settings for the treatment of HIV-1-infected children and adults and in the prevention of mother-to-child transmission (PMTCT) [2]. The use of nevirapine is limited by a potentially fatal, immune-mediated hypersensitivity reaction which can manifest as hepatotoxicity, fever and/or rash [3, 4] and a fragile genetic barrier to the development of drug resistance [5]. Higher plasma levels of nevirapine have been reported to be associated with improved virological response and reduced selection of resistant mutations [6–8]. There are conflicting reports on the association between nevirapine plasma levels and the development of adverse events [9–11].

Previous studies have found ethnicity, gender, weight and underlying hepatic disease to be predictive of nevirapine plasma concentrations [12–14]. Nevirapine is primarily metabolized by the cytochrome P450 3A4 (CYP3A4) and 2B6 (CYP2B6) enzymes into its major metabolites 2-hydroxynevirapine and 3-hydroxynevirapine, respectively, with a minor contribution from CYP3A5 [15]. CYP3A4 is also involved in the biotransformation of nevirapine to 8-hydroxynevirapine and 12-hydroxynevirapine, both of which are minor metabolites [15]. There is increasing evidence that host genetic polymorphisms may in part explain the observed inter-individual variability of drug disposition and drug response [16]. The impact of the CYP2B6 516G→T single nucleotide polymorphism (SNP) on the pharmacokinetics and pharmacodynamics of efavirenz, another NNRTI, has been well described [17, 18]. A small number of studies have shown that the 516G→T SNP is also predictive of nevirapine plasma concentrations [19–22]. In these studies, the parameters under examination were nevirapine oral clearance [20], trough concentration (Ctrough) [20] and area under the curve (AUC) [19]. Recently, we reported that heterozygosity for the 983T→C SNP was associated with significantly higher nevirapine plasma levels in black patients [22].

The role of p-glycoprotein [coded for by ATP-binding cassette sub-family B member 1 (ABCB1)] in the disposition of NNRTIs remains controversial [23, 24]. ABCB1 variants (3435C→T) have not been shown to be predictive of nevirapine pharmacokinetics but have been reported to be protective against hepatotoxicity in nevirapine hypersensitivity reactions [25, 26]. The impact of CYP3A5 and CYP3A4 variants on the pharmacokinetics of their substrates is also unclear [27]. There are no reported associations between these variants and nevirapine.

The primary aim of this study was to explore the impact of CYP2B6 (516G→T, 1459C→T and 983T→C), CYP3A4*1B (−392A→G), CYP3A5*3 (6986A→G) and ABCB1 (3435C→T, 2677G→T and 1236C→T) variants on the pharmacokinetics of nevirapine in an ethnically diverse population. Given the greater contribution of CYP3A4 to nevirapine biotransformation, we were particularly interested in exploring the impact of CYP2B6 polymorphisms on nevirapine exposure. The effects of age, weight, ethnicity and gender on nevirapine exposure were also investigated.

Patients and methods

Patients

Between April 2006 and April 2008, all patients on nevirapine-based regimens were approached and screened for this ethics-approved collaborative pharmacogenetic study between the Department of HIV Medicine, Royal Free NHS Trust, London and the Liverpool HIV Pharmacology Group, University of Liverpool, Liverpool. Patients were only recruited if they had achieved steady state on a nevirapine-based regimen and were known to be fully compliant. Patients on a concomitant ritonavir-boosted protease inhibitor were excluded. A total of 104 adult patients fulfilled the inclusion criteria. Whole blood was collected at Ctrough (immediately pre-dose) in ethylenediaminetetraacetic acid (EDTA) for DNA extraction and in lithium heparin for plasma drug concentration determination. Where this was not possible the projected Ctrough was calculated using population pharmacokinetic data. Participating patients recorded the time of the previous nevirapine dose and provided written informed consent.

Genotyping

Genomic DNA was extracted from whole blood using the QIAamp DNA blood mini-kit (Qiagen, Crawley, UK) according to the manufacturer's instructions. The extracted DNA was subsequently quantified using PicoGreen® dsDNA Quantitation Reagent (Molecular Probes, Sunnyvale, CA, USA) and normalized to a concentration of 20 ng/μL.

Eight SNPs at four loci [CYP2B6 (1459C→T, 516G→T and 983T→C), CYP3A4 (−392A→G), CYP3A5 (6986A→G) and ABCB1 (3435C→T, 2677G→T and 1236C→T)] were identified by real-time quantitative polymerase chain reaction (PCR)-based allelic discrimination. For CYP2B6 SNPs a pre-amplification was performed prior to real-time allelic discrimination in order to differentiate CYP2B6 from the pseudogene CYP2B7. For pre-amplification, 1 μL of DNA was added to a 10-μL reaction mix consisting of 1 μL of 10 × Buffer II (Applied Biosystems, Warrington, UK), 0.6 μL of MgCl2 (25 mM), 0.4 μL of forward primers (1 μM), 0.4 μL of reverse primers (1 μM), 0.2 μL of dNTPs (10 mM), 0.2 μL of AmpliTaq (Applied Biosystems) (5 u/ μL) and 6.2 μL of RNase-free water. The standard PCR reaction consisted of an initial 5-min denaturation step followed by 45 cycles of 95 °C for 30 s, 58 °C for 30 s and 72 °C for 45 s. This was followed by a 5-min step at 72 °C.

For the real-time assay, 2 μL of DNA was added to a 23-μL reaction mix consisting of 12.5 μL of 2 × Abgene Absolute qPCR mix (Abgene, Epsom, UK), 1.25 μL of 20 × Primer mix (Abgene), 1.25 μL of 20 × Probe mix (Abgene) and 8 μL of RNase-free water. The real-time PCR reaction consisted of an initial 15-min denaturation step at 95 °C followed by x cycles of 95 °C for 15 s and 60 °C for 1 min [x=35 for CYP2B6 (1459C→T and 516G→T); x=40 for CYP3A4 (−392A→G) and CYP3A5 (6986A→G); x=45 for ABCB1 (3435C→T, 2677G→T and 1236C→T); x=50 for CYP2B6 (983T→C)]. All primer and probe sequences are available from the corresponding author on request.

Quantification of drug levels

Plasma obtained from blood samples was heat-inactivated and nevirapine concentrations were determined using high-performance liquid chromatography (HPLC) with UV detection using previously validated assays as described elsewhere [24]. The Liverpool laboratory participates in an external quality assurance scheme (KKGT, the Hague, the Netherlands).

Statistical analysis

Statistical calculations were performed with Statistical Product and Service Solutions for Windows (spss), version 16.0 (SPSS Inc., Chicago, IL, USA). Nevirapine plasma concentrations were log-transformed for analysis as they were not normally distributed, as assessed by the Shapiro–Wilk test; therefore, all averages are presented as the geometric mean. A one-way analysis of variance (anova) was used to determine whether there were significant differences in nevirapine Ctrough among the three genotypes. In this analysis Panova refers to the statistical significance of the differences among all three groups after simultaneous comparison. An independent t-test was used when two genotypes or two variables were compared. The correlation of nevirapine Ctrough (log-transformed) with age and weight was evaluated with Pearson's test for correlation. All variables with P<0.10 in univariable analysis were included in a multivariable linear regression analysis where plasma concentrations were fitted on a log scale. Estimates of effect size and 95% confidence intervals from the model were then back-transformed from their natural log values.

Results

Nevirapine Ctrough and baseline factors

Patients' baseline characteristics are summarized in Table 1. There was considerable inter-individual variability in nevirapine plasma concentrations at Ctrough, ranging from a minimum value of 499 ng/mL to a maximum value of 12 843 ng/mL. In univariate analysis, non-Caucasian ethnicity (5609 vs. 3771 ng/mL in Caucasians; P<0.0001) was significantly associated with a higher nevirapine Ctrough and a trend towards higher exposures was observed in women (5080 vs. 4002 ng/mL in men; P=0.06). There were no significant differences in nevirapine Ctrough between the different dosing regimens [200 mg twice daily (bid): 4082 ng/mL; 400 mg once daily (qd): 4340 ng/mL; 600 mg qd: 4416 ng/mL; P=0.84]. These findings are summarized in Figure 1. There was no correlation between nevirapine Ctrough and age (P=0.85) or weight (P=0.27).

Table 1.   Baseline characteristics at the time of pharmacokinetic sampling
  1. 3TC, lamivudine; ABC, abacavir; bid, twice daily; HAART, highly active antiretroviral therapy; MSM, men who have sex with men; NRTI, nucleoside reverse transcriptase inhibitor; PI, protease inhibitor; qd, once daily; TDF, tenofovir; ZDV, zidovudine.

Age (years) [median (range)]41 (28, 68)
Weight (kg) [median (range)]72 (49, 132)
Ethnicity [n (%)]
Caucasian77 (74)
Non-Caucasian
 Black African18 (17)
 Other9 (9)
Transmission risk [n (%)]
 Blood2 (2)
 Heterosexual25 (24)
 MSM75 (72)
 Other2 (2)
Gender [n (%)]
 Female19 (18)
 Male85 (82)
Dose [n (%)]
 200 mg bid64 (62)
 400 mg qd39 (37)
 600 mg qd1 (1)
HIV viral load <50 copies/mL at time of sampling [n (%)]104 (100)
Concomitant PI [n (%)]
No104 (100)
Naïve to HAART on commencing
nevirapine [n (%)]25 (24)
Duration on nevirapine (weeks)
[median (range)]155 (5, 578)
NRTI backbone [n (%)]
 ABC/3TC20 (19)
 ZDV/3TC30 (29)
 TDF/3TC19 (18)
 TDF/FTC16 (15)
Other19 (18)
Figure 1.

 The impact of ethnicity, gender and dosing regimen on nevirapine plasma concentrations. bid, twice daily; qd, once daily.

Genotype frequencies

When a χ2 test of observed vs. predicted genotype frequencies was conducted, all polymorphisms were found to be in Hardy–Weinberg equilibrium. The frequencies of these SNPs and their impact on nevirapine exposure in this population are shown in Table 2.

Table 2.   Nevirapine plasma concentration by genotype
SNPNevirapine plasma
concentration
[ng/mL; geometric
mean (SD)]
n (%)
ABCB1
  1. ABCB1, ATP-binding cassette sub-family B member 1; CYP, cytochrome P450; SD, standard deviation; SNP, single nucleotide polymorphism.

3435C→TCC4748 (2152)23 (22)
CT4122 (2366)57 (55)
TT3825 (1711)24 (23)
ABCB1GG4189 (2392)36 (35)
2677G→TGT3287 (1402)28 (27)
TT4938 (2246)39 (38)
ABCB1CC4872 (2038)32 (31)
1236C→TCT3454 (1685)44 (42)
TT4736 (2687)28 (27)
CYP3A4AA4072 (2200)31 (31)
−392A→GAG5061 (2620)18 (18)
GG4009 (2023)52 (51)
CYP3A5AA5180 (2755)11 (11)
6968A→GAG4689 (2325)19 (18)
GG3920 (2047)73 (71)
CYP2B6GG3574 (1418)50 (48)
516G→TGT4634 (2348)50 (48)
TT8170 (2762)4 (4)
CYP2B6CC4339 (2347)82 (80)
1459C→TCT3624 (1129)21 (20)
TT
CYP2B6TT4167 (2198)103 (99)
983T→CTC58061 (1)
CC

Nevirapine Ctrough and CYP3A4, CYP3A5 and ABCB1 genotype

Neither CYP3A4*1B −392A→G [AA, 4072 ng/mL (n=31); AG, 5061 ng/mL (n=18); GG, 4009 ng/mL (n=52); Panova=0.23] nor CYP3A5*3 6968A→G [AA, 5180 ng/mL (n=11); AG, 4689 ng/mL (n=19); GG, 3920 ng/mL (n=73); Panova=0.13] was predictive of nevirapine plasma concentrations. There was also no gene–dose association between ABCB1 variants and nevirapine plasma concentrations: 3435C→T [CC, 4748 ng/mL (n=23); CT, 4122 ng/mL (n=57); TT, 3825 ng/mL (n=24); Panova=0.33], 2677G→T [GG, 4189 ng/mL (n=36); GT, 3287 ng/mL (n=28); TT, 4938 ng/mL (n=39); Panova=0.005] and 1236C→T [CC, 4872 ng/mL (n=32); CT, 3454 ng/mL (n=44); TT, 4736 ng/mL (n=28); Panova=0.004].

Nevirapine Ctrough and CYP2B6 genotype

516G→T was significantly associated with higher nevirapine Ctrough in all patients: GG, 3574 ng/mL (n=50); GT, 4634 ng/mL (n=50); TT, 8170 ng/mL (n=4); Panova=0.001. This trend was maintained when the sample was stratified by dosing interval: GG, 3527 ng/mL (n=30); GT, 4525 ng/mL (n=32); TT, 7020 ng/mL (n=2); Panova=0.05 in patients on 200 mg bid and GG, 3645 ng/mL (n=20); GT, 4861 ng/mL (n=17); TT, 9508 ng/mL (n=2); Panova=0.01 in patients on 400 mg qd. This is summarized in Figure 2. There was no association between 1459C→T [CC, 4339 ng/mL (n=82); CT, 3624 ng/mL (n=21); P=0.15] or 983T→C [TT, 4167 ng/mL (n=103); TC, 5806 ng/mL (n=1); P=0.52) and nevirapine plasma concentrations.

Figure 2.

 The impact of 516G→T on nevirapine plasma concentrations in (a) all patients, (b) patients on 200 mg twice daily (bid) and (c) patients on 400 mg once daily (qd).

In a multivariate analysis with ethnicity, gender and CYP2B6 G516T genotype as covariates, the effect of non-Caucasian ethnicity (46% higher) and heterozygous (22% higher) and homozygous (108% higher) carriage of the mutant allele of CYP2B6 516G→T remained significant predictors of nevirapine Ctrough, with homozygosity for the mutant allele of CYP2B6 associated with the greatest effect (Table 3).

Table 3.   Factors influencing nevirapine plasma concentrations
All patientsNevirapine plasma
concentration [ng/mL;
geometric mean (SD)]
Effect
(% change)
(95% CI)
Univariate PEffect
(% change)
(95% CI)
Multivariate P
  1. Numbers in bold represent significant effects.

  2. bid, twice daily; CI, confidence interval; CYP, cytochrome P450; qd, once daily; SD, standard deviation.

Age (per 10 years)−1 (−12, +11)0.85
Weight (per 10 kg)−4 (−11, +4)0.27
Dose200 mg bid4082 (1879)    
400 mg qd4340 (2653)+6 (−14, +31)0.56
600 mg qd4416+8 (−61, +202)0.88  
GenderMale4002 (1925)    
Female5080 (2976)+27 (+2, +64)0.060.50
CaucasianYes3771 (1684)    
No5609 (2757)+49 (+20, +84)<0.0001+46 (+11, +91)0.006
CYP2B6 516G→T (all patients)GG3574 (1418)    
GT4634 (2348)+30 (+7, +57)0.008+22 (+1, +47)0.04
TT8170 (2762)+128 (+40, +275)0.001+108 (+28, +239)0.003

Discussion

In this analysis of an ethnically diverse cohort stable on nevirapine-based regimens, we found a significant gene – dose effect between CYP2B6 516G→T and nevirapine Ctrough. In a multivariate analysis, non-Caucasian ethnicity remained a significant predictor of nevirapine Ctrough but homozygosity for CYP2B6 516G→T had the greatest effect. Previous studies have shown that nevirapine plasma concentration at the end of the dosing interval (Cmin) is significantly lower in patients on 400 mg qd compared with those on 200 mg bid [28]. In this cohort, no significant association was found between nevirapine Ctrough and dosing regimen. Furthermore, the observed association between CYP2B6 516G→T and higher nevirapine Ctrough was maintained when the sample population was stratified by dosing interval. In the present study, only one patient had the CYP2B6 983T→C mutation (a heterozygote); therefore, the low frequency of this SNP coupled with it being present only in black populations compromised the robustness of the analysis. Despite the significant contribution of CYP3A4 to the metabolism of nevirapine, no association was found between CYP3A4 −392A→G and nevirapine plasma concentrations.

A recent analysis of the impact of CYP2B6 and ABCB1 variants on the time to virological failure or central nervous system (CNS) event in individuals on efavirenz-based regimens failed to find consistent associations between different ethnic groups [29]. This same group has previously reported an association between CYP2B6 516G→T and the development of CNS events at week 1 [17] and an association between ABCB1 3435C→T and the decreased likelihood of virological failure in patients on efavirenz-based regimens [30]. A recent retrospective study on a paediatric cohort on nevirapine-based regimens reported a significant association between CYP2B6 516G→T genotype and immunological responses at weeks 12 and 24 [20]. This finding is yet to be validated in adult cohorts.

There is growing interest in the use of the CYP2B6 516G→T genotype in the individualization of efavirenz doses in an attempt to reduce CNS side effects [31, 32]. The effect of genotype-based dosing of nevirapine is yet to be determined. In resource-limited countries, first-line regimens normally consist of two first-generation nucleoside reverse transcriptase inhibitors (NRTIs) co-administered with nevirapine. In these settings, treatment interruption is not uncommon. Published reports from Uganda cite discontinuation rates of up to 13% [33]. Reasons for unplanned treatment discontinuation include drug toxicity and unreliable and unaffordable drug supplies. Clinical scenarios requiring planned treatment discontinuation include short courses of antiretroviral therapy in PMTCT and the need to treat opportunistic infections with antimicrobial agents that have unfavourable drug interactions with nevirapine. As there are limited second-line regimens in these settings, there is a push to preserve first-line options for as long as possible. Abrupt discontinuation of nevirapine-based regimens can be associated with the development of NNRTI resistance as the long half life of nevirapine can lead to functional monotherapy. The British HIV Association (BHIVA) guidelines suggest continuing dual NRTI therapy for 7 days or substituting the NNRTI with a protease inhibitor. Given the variability in nevirapine exposure demonstrated here, it is clear that different populations require different treatment discontinuation strategies. As this study has shown, both CYP2B6 516G→T and non-Caucasian ethnicity are independent predictors of nevirapine exposure. There might therefore be a role for CYP2B6 G516T genotyping in non-Caucasian populations to allow the identification of individuals likely to require a longer course of NRTIs to avoid nevirapine monotherapy should they discontinue their regimens electively, especially when alternative regimens are limited.

The need for a better understanding of the mechanisms underpinning nevirapine hypersensitivity remains. As this study was performed in a cohort without a history of serious adverse events requiring treatment discontinuation, we cannot draw any conclusions about toxicity. Studies on the female Brown Norway rat have hypothesized that 12-hydroxynevirapine, a minor metabolite of nevirapine, and 4-carboxynevirapine, its successive metabolite, are involved in nevirapine hypersensitivity [34–36]. However, a recent case–control study in patients with nevirapine hypersensitivity failed to show an association between the occurrence of an adverse event and plasma concentrations of nevirapine or its five metabolites [37]. Further exploration of the pathways involved in the formation of these and successive metabolites may hold the key to understanding nevirapine hypersensitivity.

To conclude, in this study we have quantified the effects of ethnicity and the CYP2B6 516G→T polymorphism on nevirapine exposure in an ethnically diverse cohort. This adds to the current evidence base implicating CYP2B6 polymorphisms in the variability of nevirapine exposure. Only a well-powered prospective trial will allow us to address the clinical utility of prospective genotyping by assessing the impact of these variants on clinical outcomes.

Acknowledgements

We thank the HIV-infected patients who volunteered to participate in this study. This work was funded by the UK NIHR Biomedical Research Centre for Microbial Diseases in Liverpool and Boehringer Ingelheim.

Transparency declarations

AO, SK and DJB have received research funding from Boehringer Ingelheim, GlaxoSmithKline, Abbott Laboratories, Pfizer, AstraZeneca, Tibotec, Merck and Roche Pharmaceuticals. MAJ and MY have received research funding from Bristol Myers Squibb, Boehringer Ingelheim, GlaxoSmithKline, Abbott Laboratories, Pfizer, Tibotec, Merck, Gilead Sciences and Roche Pharmaceuticals. CJS has received speaker and consultancy honoraria from Bristol Myers Squibb, Abbott Laboratories, Gilead Sciences, Roche, Tibotec and GlaxoSmithKline. The other authors have nothing to declare. The University of Liverpool has spun out therapeutic drug monitoring of HIV drugs to Delphic Diagnostics Ltd; both DJB and SK serve as nonexecutive directors of Delphic.

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