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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: [email protected] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
# 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: [email protected] 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.
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of 516G.T polymorphisms at the gene encoding the CYP450-2B6

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