Journal of Antimicrobial Chemotherapy Advance Access published February 14, 2008
Journal of Antimicrobial Chemotherapydoi:10.1093/jac/dkn029
Impact of CYP2B6 983T>C polymorphism on non-nucleoside reverse
transcriptase inhibitor plasma concentrations in HIV-infected patients
Christoph Wyen1, Heidy Hendra1, Martin Vogel2, Christian Hoffmann3, Heribert Knechten4,
Norbert H. Brockmeyer5, Johannes R. Bogner6, Ju¨rgen Rockstroh2, Stefan Esser7, Hans Jaeger8,
Thomas Harrer9, Stefan Mauss10, Jan van Lunzen11, Nicole Skoetz12, Alexander Jetter13,
Christiane Groneuer1, Gerd Fa¨tkenheuer1, Saye H. Khoo14, Deirdre Egan14, David J. Back14 and
Andrew Owen14* on behalf of the German Competence Network for HIV/AIDS
1Department of Internal Medicine, University of Cologne, Ko¨ln, Germany; 2Department of Internal Medicine,
University of Bonn, Bonn, Germany; 3IPM Study Center, Hamburg/University of Schleswig Holstein, Kiel,
Germany; 4PZB, Aachen, Germany; 5Department of Dermatology, St Josef Hospital, Bochum, Germany;
6Department of Internal Medicine, University of Munich, Mu¨nchen, Germany; 7Department of Dermatology,
University of Essen, Essen, Germany; 8HIV Research and Clinical Care Centre Munich, Munich, Germany;
9Department for Internal Medicine 3, University Hospital Erlangen, University of Erlangen-Nuremberg,
Germany; 10Gemeinschaftspraxis Mauss und Kollegen, Du¨sseldorf, Germany; 11Infectious Diseases Unit,
University Medical Center Hamburg Eppendorf, Hamburg, Germany; 12ZKS, Ko¨ln, Germany; 13Division of
Clinical Pharmacology and Toxicology, Department of Internal Medicine, University Hospital Zu¨rich, Zu¨rich,
Switzerland; 14Department of Pharmacology and Therapeutics, University of Liverpool, 70 Pembroke Place,
Received 27 November 2007; returned 1 January 2008; revised 4 January 2008; accepted 8 January 2008
Objectives: The aim of this study was to investigate the frequency of CYP2B6 polymorphisms (accord-ing to ethnicity) and the influence of heterozygosity and homozygosity on plasma concentrations ofefavirenz and nevirapine.
Methods: Following written informed consent, 225 Caucasians and 146 Blacks were recruited from theGerman Competence Network for HIV/AIDS. Plasma concentrations of efavirenz and nevirapine wereassessed by HPLC, and genotyping for 516G>T, 983T>C and 1459T>C polymorphisms in CYP2B6 wasconducted by real-time PCR-based allelic discrimination.
Results: The minor allele frequency for 516G>T, 983T>C and 1459T>C was 0.29, 0 and 0.08 inCaucasians and 0.34, 0.07 and 0.02 in Blacks, respectively. Two Black patients with the 983C allelereceiving efavirenz were identified and both were withdrawn from therapy within 1 week of samplingdue to toxicity. In multivariate analyses, efavirenz and nevirapine plasma concentrations were signifi-cantly associated with 983T>C (P < 0.0001 and P 5 0.02, respectively), 516G>T (P < 0.0001 andP 5 0.002, respectively) and time of drug analysis post-dose (P < 0.0001 for both). Body mass indexwas independently related to efavirenz (P 5 0.04) but not nevirapine concentrations, and age wasrelated to nevirapine (P 5 0.05) but not efavirenz concentrations. Consistent with other studies,1459C>T was not associated with plasma concentrations of either drug (P > 0.05 for both drugs).
Conclusions: This is the first report that the 983T>C genotype ( part of the CYP2B6*18 haplotype)impacts on nevirapine plasma concentrations and the first study to assess the impact of 983C homo-zygosity on efavirenz concentrations. These data have implications for administration of non-nucleoside reverse transcriptase inhibitors to Black patients.
Keywords: NNRTIs, pharmacogenetics, pharmacokinetics, metabolism, drug disposition
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
*Corresponding author. Tel: þ44-151-794-5919; Fax: þ44-151-794-5656; E-mail: aowen@liv.ac.uk
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
# The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org
Drug treatment in HIV disease is characterized by variable
Total genomic DNA was isolated using the QIAamp DNA minikit according to the manufacturer’s instructions. Following extrac-
responses, in terms of both efficacy and toxicity. Genetic and
tion, purity was assessed by comparing the A
environmental factors are important determinants of this varia-
DNA was quantified using PicoGreenw (PG) dsDNA Quantitation
bility, although the relative contributions are unclear and likely
Reagent (Molecular Probes, CA, USA) and normalized to 20 ng/mL.
to vary with different drugs (see Owen et al.1 for review).
Pre-amplification for exon 4, exon 9 and exons 7 and 8 (com-
Efavirenz and nevirapine are metabolized by CYP2B6
bined) was first conducted to discriminate from the CYP2B6 pseu-
(although CYP3A also contributes to nevirapine metabolism).
Single nucleotide polymorphisms (SNPs) and haplotype organiz-
methods.2 Genotyping for 516G.T, 983T.C and 1459C.T was
ation of CYP2B6 in Caucasians were originally described by
then performed on the resultant amplicons by real-time PCR
Lang et al.,2 who showed a reduced hepatic CYP2B6 protein
allelic discrimination using standard methodology (958C for
expression and activity in carriers of the 516G.T (rs3745274)
15 min followed by 40 cycles of 958C for 15 s and 608C for
and 1459T.C (rs3211371) polymorphisms. However, the
1 min) in a DNA Engine Opticonw 2 system (MJ Research Inc.,
association with protein expression and activity was not statisti-
USA). Full PCR conditions as well as primer and probe sequences
cally significant for 516G.T, although the authors acknowl-
edged the limitation of the sample size. With respect to HIV, therole of the 516G.T SNP in disposition and treatment responseto non-nucleoside reverse transcriptase inhibitors (NNRTIs) is
now well established.3 – 9 Moreover, a recent study indicated that
Plasma obtained from blood samples was heat-inactivated, and efa-
efavirenz dose reduction according to 516G.T genotype was a
virenz and nevirapine concentrations were determined (median time
post-dose was 10 h) using HPLC with UV-Detection using pre-
Recently, the 983T.C SNP (rs28399499) was described in
viously validated assays as described elsewhere.14,15 The Liverpool
Black populations. This polymorphism results in an amino acid
Laboratory participates in an external quality assurance scheme
change in the CYP2B6 protein (Ile328Thr), and heterozygosity
has been shown to impact upon efavirenz pharmacokinetics.11,12Furthermore, a laboratory-based assessment of this polymorph-
ism indicated that it may represent a null allele.11 Investigationof the 983T.C polymorphism in various populations has shown
All data are given as median (range), unless otherwise stated.
that the allele is absent in Caucasian populations, yet its fre-
Genotypes were tested for Hardy – Weinberg equilibrium by x2 test
of observed versus predicted (from allele frequency) genotype fre-
Ghanaians.13 However, to date, no homozygotes for this poly-
quencies. Normality of the data was assessed using a Shapiro – Wilk
test. Subsequently, univariate statistical analysis was conducted by
The aim of this study was to investigate the frequency of
simple linear regression (continuous data) or one-way analysis of
CYP2B6 polymorphisms (516G.T, 983T.C and 1459T.C)
variance (categorical data). Missing values were imputed byregression against efavirenz concentrations, and multivariate analysis
and the influence of heterozygosity and homozygosity for these
was conducted by multiple linear regressions using best subset
polymorphisms on plasma concentrations of efavirenz and nevir-
apine in a cohort of Caucasian and Black patients from theGerman Competence Network for HIV/AIDS. In addition, theassociation with gender, ethnicity, weight, height, alcohol con-sumption, smoking status, glutamic oxaloacetic transaminase
(GOT) and glutamic pyruvic transaminase (GPT) was also deter-
mined. Finally, a multivariate analysis was conducted using bestsubset analysis.
When x2 test of observed versus predicted genotype frequencieswas conducted, all polymorphisms were found to be in Hardy –Weinberg equilibrium. The frequencies of 516G.T, 983T.Cand 1459C.T are given in Figure 1.
In the entire cohort, efavirenz plasma concentrations were 2077
Whole blood and plasma were provided by the German Competence
(487 – 21 486) ng/mL. In the univariate analysis, 516G.T [1779
Network for HIV/AIDS. Three hundred and seventy-one patients
(530 – 26 018), 2299 (487 – 11 198) and 6248 (1345 – 23 592) ng/
(225 Caucasians and 146 Blacks), with age range from 21 to 82(median ¼ 43) years, who were on stable efavirenz- (n ¼ 186) or
mL for GG, GT and TT, respectively; P , 0.0001], 983T.C
nevirapine- (n ¼ 185) containing HAART for at least 3 months,
[2068 (530 – 11 198), 2076 (1269 – 6835) and 23 418 (20 818 –
were included in the study. Median (range) CD4 counts were 487
26 018) ng/mL for TT, TC and CC, respectively; P , 0.0001],
(24 – 1690) cells/mm3, and 88% had undetectable plasma viral RNA.
body mass index (BMI) (R2 ¼ 0.02; P ¼ 0.05), alcohol con-
Ethics approval was granted by the Ethics Committee of the
sumption (R2 ¼ 0.03; P ¼ 0.04) and time post-dose of drug
Ruhr-Universita¨t Bochum, Germany, and local Ethics Committee
analysis (R2 ¼ 0.22; P , 0.0001) were all significantly associ-
approval was obtained at each site. Written informed consent was
ated with efavirenz plasma concentrations (Table 1). Following
multivariate analysis, only 516G.T (P , 0.0001), 983T.C
Figure 1. The impact of 516G.T (a and b), 1459C.T (c and d), and 983T.C (e and f ) polymorphisms in CYP2B6 on efavirenz (a, c and e) and nevirapine(b, d and f ) plasma concentrations according to ethnicity (see text for experimental procedures). The genotype frequencies within each group are also given.
(487 – 26 018) and 1974 (688 – 23 592) ng/mL for male and
(P , 0.0001) remained statistically significant (Table 1).
female, respectively], ethnicity [2143 (669 – 11 198) and 1963
(487 – 26 018) ng/mL for Caucasian and Black, respectively],
(792 – 4357) ng/mL for CC and CT, respectively], gender [2168
The CYP2B6 gene is highly polymorphic with numerous SNPs
and associated haplotypes. The association of the 516G.T SNPwith efavirenz and nevirapine pharmacokinetics is well estab-
lished, but a recent study indicated that the 516TT genotype was
not associated with the time to failure of efavirenz-containing
regimens or increases in the CD4 cell count.16 This polymorph-
ism did predict CNS adverse effects at week 1 of therapy, but
exposure.16 Nonetheless, a recent manuscript described the use
of 516G.T (along with 499C.G and 785A.G) in successful
dose reduction of efavirenz in a Japanese cohort.10 The cost-
effectiveness of this approach now requires further investigation.
Genetic variability has been assessed in different ethnicities
and a number of novel functional variants discovered,11,17
among these, the 983T.C polymorphism is a suspected null
allele. With the increasing use of NNRTIs in developing
countries, it is imperative that the functional significance of
these alleles on NNRTI therapy in different sub-populations be
assessed. The 983T.C polymorphism has only previously been
identified in Hispanic and African populations,11,12 with allele
frequencies of 1.1% in Hispanic Americans and up to 7.5% in
some African countries.13 We observed this allele with a fre-
quency of 2.6% in Blacks residing in Germany.
In agreement with previous studies, we observed a significant
gene dose effect between 516G.T and efavirenz and nevirapine
plasma concentrations. In addition, 983T.C genotype was also
associated with plasma concentrations of both drugs. Importantly,
homozygote patients in the efavirenz arm discontinued therapy 1
week after blood was collected for this study (but before druganalysis) due to CNS toxicity. Previous studies have illustrated that
Associations with a P value less than 0.15 in the univariate analysis and
heterozygosity of this polymorphism ( present in the CYP2B6*18
therefore included in the multivariate analysis are shown in bold, as are
allele) is associated with efavirenz concentrations.12 However, no
those associations with a P value less than 0.05 in the multivariate analysis.
homozygotes for this polymorphism have been previouslyreported. Therefore, this is the first study to illustrate the phenoty-
(R2 ¼ 0.02), nor age (R2 ¼ 0.001) was significantly associated
pic consequence of homozygosity for efavirenz and to show an
with efavirenz plasma concentrations (Table 1).
association with nevirapine plasma concentrations. Further studiesare now required to determine the impact of 983T.C on long-term efficacy and toxicity of this important class of drug.
In the entire cohort, nevirapine plasma concentrations were 5589(149– 21 026) ng/mL. In the univariate analysis, 516G.T [5184
(1894 – 11 158), 6132 (149– 11 689) and 6699 (4164 – 21 026) ng/
We would like to thank Doris Reichelt, Mu¨nster, Hartmut
mL for GG, GT and TT, respectively; P ¼ 0.007], 983T.C
Klinker, Wu¨rzburg, Matthias Stoll and Reinhold Schmidt,
[5483 (864– 21 026) and 8685 (4890 – 10 294) for TT and TC,
Hannover Practice Adam/Schewe/Weitner, Hamburg, Practice
respectively; P ¼ 0.02], age (R2 ¼ 0.03; P ¼ 0.03), and time
Go¨lz/Moll, Berlin, Hans-Ju¨rgen Stellbrink, Hamburg, Practice
post-dose of drug analysis (R2 ¼ 0.09; P ¼ 0.0001) were all sig-
Dupke/Carganico/Baumgarten, Berlin, Ansgar Rieke, Koblenz
nificantly associated with nevirapine plasma concentrations
and Mark Oette, Du¨sseldorf for enrolling patients into the study.
(Table 1). Following multivariate analysis, 516G.T (P ¼ 0.002),
Furthermore, we would like to thank Gislea Kremer and Ellen
983T.C (P ¼ 0.02), age (P ¼ 0.05), and time post-dose
Rund, Ko¨ln for administrative assistance and Winfried Siffert,
(P ¼ 0.0004) all remained statistically significant (Table 1).
Essen for collecting and providing the blood samples of the
Neither 1459C.T [5670 (864 – 21 026) and 5243 (2230 –
German Competence Network for HIV/AIDS. We are extremely
7436) ng/mL for CC and CT, respectively], gender [5893 (149 –
grateful to all patients who contributed to the study.
11 689) and 5483 (864 – 21 026) ng/mL for male and female,respectively], ethnicity [5893 (1894 – 11 689) and 5138 (149 –21 026) ng/mL for Caucasian and Black, respectively], BMI
(R2 ¼ 0.01), GOT (R2 ¼ 0.004), GPT (R2 ¼ 0.006), alcoholconsumption (R2 ¼ 0.002), nor smoking status (R2 ¼ 0.005)
This work was funded by the UK Department of Health
was significantly associated with nevirapine plasma concen-
Biomedical Research Centre for Microbial Diseases, the German
(Bundesministerium fu¨r Bildung und Forschung; grant 01 KI
isoenzyme on efavirenz plasma concentrations in HIV-infected sub-
0771). H. H. was supported by a scholarship of the Else Kroener
jects. Clin Infect Dis 2005; 40: 1358– 61.
8. Rotger M, Colombo S, Furrer H et al. Influence of CYP2B6 poly-
morphism on plasma and intracellular concentrations and toxicity ofefavirenz and nevirapine in HIV-infected patients. PharmacogenetGenomics 2005; 15: 1 – 5.
9. Tsuchiya K, Gatanaga H, Tachikawa N et al. Homozygous
A. O., S. H. K. and D. J. B. have received research funding from
CYP2B6 *6 (Q172H and K262R) correlates with high plasma efavirenz
Boehringer Ingelheim, GlaxoSmithKline, Abbott Laboratories,
concentrations in HIV-1 patients treated with standard efavirenz-containing regimens. Biochem Biophys Res Commun 2004; 319:
Pharmaceuticals. G. F. has received honoraria for lectures and
10. Gatanaga H, Hayashida T, Tsuchiya K et al. Successful efavir-
advisory boards from Abbott, Boehringer Ingelheim, Bristol
enz dose reduction in HIV type 1-infected individuals with cytochrome
Myers Squibb, Gilead, GlaxoSmithKline, Novartis, Pfizer,
P450 2B6 *6 and *26. Clin Infect Dis 2007; 45: 1230 – 7.
Roche, Schering Plough and Tibotec. The other authors have
11. Klein K, Lang T, Saussele T et al. Genetic variability of CYP2B6
in populations of African and Asian origin: allele frequencies, novelfunctional variants, and possible implications for anti-HIV therapy withefavirenz. Pharmacogenet Genomics 2005; 15: 861 – 73.
12. Rotger M, Tegude H, Colombo S et al. Predictive value of
known and novel alleles of CYP2B6 for efavirenz plasma concen-
1. Owen A, Pirmohamed M, Khoo SH et al. Pharmacogenetics of
trations in HIV-infected individuals. Clin Pharmacol Ther 2007; 81:
HIV therapy. Pharmacogenet Genomics 2006; 16: 693 – 703.
2. Lang T, Klein K, Fischer J et al. Extensive genetic polymorphism
13. Mehlotra RK, Bockarie MJ, Zimmerman PA. CYP2B6 983T.C
in the human CYP2B6 gene with impact on expression and function in
polymorphism is prevalent in West Africa but absent in Papua New
human liver. Pharmacogenetics 2001; 11: 399 –415.
Guinea: implications for HIV/AIDS treatment. Br J Clin Pharmacol
3. Haas DW, Bartlett JA, Andersen JW et al. Pharmacogenetics of
nevirapine-associated hepatotoxicity: an Adult AIDS Clinical Trials
14. Almond LM, Hoggard PG, Edirisinghe D et al. Intracellular
Group collaboration. Clin Infect Dis 2006; 43: 783 – 6.
plasma pharmacokinetics of efavirenz in HIV-infected individuals.
4. Haas DW, Ribaudo HJ, Kim RB et al. Pharmacogenetics of efa-
J Antimicrob Chemother 2005; 56: 738 – 44.
virenz and central nervous system side effects: an Adult AIDS Clinical
15. Clarke SM, Mulcahy FM, Tjia J et al. Pharmacokinetic inter-
Trials Group study. AIDS 2004; 18: 2391– 400.
actions of nevirapine and methadone and guidelines for use of nevira-
5. Ribaudo HJ, Haas DW, Tierney C et al. Pharmacogenetics
pine to treat injection drug users. Clin Infect Dis 2001; 33: 1595– 7.
of plasma efavirenz exposure after treatment discontinuation: an
16. Haas DW, Smeaton LM, Shafer RW et al. Pharmacogenetics of
Adult AIDS Clinical Trials Group Study. Clin Infect Dis 2006; 42:
long-term responses to antiretroviral regimens containing efavirenz
and/or nelfinavir: an Adult AIDS Clinical Trials Group study. J Infect Dis
6. Ritchie MD, Haas DW, Motsinger AA et al. Drug transporter and
metabolizing enzyme gene variants and nonnucleoside reverse-
17. Lang T, Klein K, Richter T et al. Multiple novel nonsynonymous
transcriptase inhibitor hepatotoxicity. Clin Infect Dis 2006; 43: 779 – 82.
CYP2B6 gene polymorphisms in Caucasians: demonstration of pheno-
7. Rodriguez-Novoa S, Barreiro P, Rendon A et al. Influence
typic null alleles. J Pharmacol Exp Ther 2004; 311: 34 –43.
of 516G.T polymorphisms at the gene encoding the CYP450-2B6
B u n d e s s t r a f g e r i c h t T r i b u n a l p é n a l f é d é r a l T r i b u n a l e p e n a l e f e d e r a l e T r i b u n a l p e n a l f e d e r a l G esc h äf ts num m er: BG .2 0 0 5. 1 5 Entscheid vom 16. Juni 2005 Beschwerdekammer Bundesstrafrichter Emanuel Hochstrasser, Vorsitz, KANTON AARGAU, KANTON BERN, Bestimmung des Gerichtsstandes i.S. A