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Nuclear Receptor-Mediated Induction of CYP450 by Antiretrovirals: Functional Consequences of NR1I2 (PXR) Polymorphisms and Differential Prevalence in Whites and Sub-Saharan Africans

Svärd, Jenny MSc*; Spiers, J Paul BSc, PhD*; Mulcahy, Fiona MD, FRCPI; Hennessy, Martina PhD, FRCPI*

JAIDS Journal of Acquired Immune Deficiency Syndromes: December 15th, 2010 - Volume 55 - Issue 5 - p 536-549
doi: 10.1097/QAI.0b013e3181f52f0c
Basic and Translational Science
Free

Background: Antiretroviral therapy including HIV protease inhibitors and nonnucleoside reverse transcriptase inhibitors can both inhibit and induce expression of cytochrome P450s, potentially leading to drug interactions. However, information is lacking on the impact of genetic polymorphism on this interaction.

Methods: This study examines the prevalence of 33 polymorphisms in NR1I2 (pregnane X receptor [PXR]), CYP3A4, and CYP2B6 in 1013 white and sub-Saharan African patients with HIV; explores the inductive ability of 16 antiretrovirals on CYP3A4 and CYP2B6 promoter activity through nuclear receptors PXR and constitutive androstane receptor (CAR); and evaluates the influence of naturally occurring PXR genetic variants on antiretroviral activation.

Results: Seventeen polymorphisms were present at different frequencies between the two ethnicities. Darunavir, fosamprenavir, lopinavir, nelfinavir, tipranavir, efavirenz, and abacavir increased CYP3A4 and/or CYP2B6 promoter activity, some through constitutive androstane receptor but mainly through PXR. Addition of low-dose ritonavir enhanced levels of CYP promoter activity for several protease inhibitors. Some PXR variants displayed lower fosamprenavir- and lopinavir-induced CYP3A4 promoter activity than the PXR reference sequence, whereas efavirenz and nelfinavir induction was unchanged.

Conclusions: The presence of NR1I2 polymorphisms can alter the induction of CYP3A4 and CYP2B6 promoter activity, potentially adding to the unpredictable nature of antiretroviral drug interactions. These polymorphisms differ in prevalence between whites and sub-Saharan Africans.

From the *Department of Pharmacology & Therapeutics, Trinity College, Dublin, Ireland; and †Department of Genitourinary Medicine and Infectious Disease, St James's Hospital, Dublin, Ireland.

Received for publication March 5, 2010; accepted August 2, 2010.

The authors declare no conflict of interest.

Parts of the data in this publication have been presented at the following meetings: British Pharmacological Society Winter Meeting, Brighton, December 16-18, 2008-oral presentation: Nuclear receptor-mediated expression of CYP3A4 and CYP2B6 by antiretrovirals-implications for prediction of drug interaction potential (JS, PS, FM, MH); oral presentation: Genetic variability in PXR, MDR-1, CYP3A4, and CYP2B6 in HIV infected Caucasian and Sub-Saharan African patients-benefits from a cohort approach (JS, PS, FM, MH); HIV9, Glasgow, November 9-13, poster: Antivirals and nuclear receptor activation of CYP3A4 and 2B6 (JS, JPS, FM, MH); Infectious Diseases Society Ireland Annual Scientific Meeting, Dublin, June 11-13, 2008; oral presentation: Antivirals and nuclear receptor activation of CYP3A4 and 2B6 (JS, JPS, FM, MH).

Correspondence to: Martina Hennessy, PhD, FRCPI, Department of Pharmacology and Therapeutics, Trinity College Dublin, Trinity Centre for Health Sciences, St. James's Hospital, Dublin 8, Ireland (e-mail: mhenness@tcd.ie).

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INTRODUCTION

A number of pharmacologic factors influence drug metabolism, including genetic variability in metabolizing enzymes and their regulators as well as exposure to various xenobiotic compounds, which possess the capacity to modulate enzyme activity and/or expression. These factors become particularly important when complex drug regimens are used as is the case in HIV treatment. Knowledge of these risk factors for drug interactions is essential, especially in resource-poor settings where the infection is widespread but treatment options are limited.1

Nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) have in recent years emerged as coordinators of cholesterol,2 glucose and lipid homeostasis as well as inflammatory response.3 However, their roles as xenosensors and regulators of cytochrome P450 (CYP450) metabolizing enzymes remain of importance in pharmacokinetics, because several pharmaceuticals have been reported as activators of nuclear receptors (mainly PXR) with implications for drug interactions. Some HIV protease inhibitors (PIs) fall into this category. Ritonavir is a confirmed ligand of PXR4,5 and increased hepatic expression of PXR target genes of the CYP3A subclass has been demonstrated in amprenavir and nelfinavir-treated rats.6 Gupta et al7 used a reporter assay-based approach in an intestinal cell line for a number of single-concentration (10 μM) PIs, which all gave rise to significantly increased CYP3A4 promoter activation when cotransfected with PXR. Hariparsad et al8 demonstrated also that the nonnucleoside reverse transcriptase inhibitor (NNRTI) efavirenz can induce CYP3A4 promoter activity through PXR and indeed an increase in CYP3A4 activity is seen in patients on efavirenz treatment.9

Studies of naturally occurring polymorphic variants of the PXR encoding gene (NR1I2) have revealed not only changes in PXR expression and activity, but also effects on CYP3A4 expression and inducibility as demonstrated by Zhang et al,10 King et al,11 and Lamba et al.12 Hustert et al13 assessed the impact of six nonsynonymous coding polymorphisms and found significant changes in basal and/or induced transcriptional activity after treatment with rifampicin or corticosterone in four of them: G36R (106G>A), V140M (4374G>A), D163G (4444A>G) and A370T (8528A>G). Only one single nucleotide polymorphism (SNP) has been associated with alterations in antiretroviral (ARV) drug plasma concentrations; patients homozygous for -6994T (position 63396 relative to GenBank Accession AF364606 origin) had atazanavir trough levels below the minimum effective concentration.14 These NR1I2 polymorphisms, in combination with polymorphisms in target CYP450 genes, could have a high influence on interindividual variation in ARV drug metabolism; several SNPs in CYP3A4 have shown association with altered enzyme activity and/or expression levels. Furthermore, some researchers have demonstrated changes in efavirenz plasma levels among subjects with the -392A>G polymorphism,15,16 whereas other studies reported no effect on either efavirenz or nelfinavir.17,18 In the CYP2B6 gene a range of SNPs such as the well-studied 516G>T (Q172H)15,19,20 have been correlated to changes in plasma drug concentrations of efavirenz and/or nevirapine in patients. Fewer studies have focused on polymorphism in the gene encoding CAR, NR1I3, and only a small number of rare SNPs in this gene have been correlated to significant changes in nuclear receptor activity or expression.21

Africans are underrepresented in clinical trials in general and likewise in genetic screenings of the previously mentioned genes, and most ARV dosage recommendations are based on results from studies with white subjects. The functional consequence of SNPs in nuclear receptors and their target genes in the context of activation potential by ARV drugs has not been investigated. It is not clear whether all PIs are inducers of PXR-mediated CYP3A4 expression, if this ability is shared by more NNRTIs other than efavirenz, by nucleoside reverse transcriptase inhibitors (NRTIs, generally not metabolized by CYP450 enzymes and hence not expected to influence their transcriptional regulation), or by newer classes such as entry inhibitors. Furthermore, it is not known if this effect is changed in any way in combination with low-dose ritonavir, used to “boost” the bioavailability of the partner PI. Additionally, many previous studies have used uniform concentrations that may or may not reflect clinical plasma concentrations. ARV induction of CYP2B6 (metabolizer of NNRTIs) has not been explored and neither has the importance of CAR as a CYP3A4/CYP2B6 induction pathway by ARVs.

The aims of this study were to 1) investigate the frequency distributions of a wide range of SNPs in the NR1I2 (PXR), CYP3A4, and CYP2B6 genes in white and sub-Saharan African patients with HIV as well as comparing sub-Saharan African SNP frequencies with published results from studies of black groups; 2) examine the ability of a wide range of ARVs across classes, both new and established and at concentrations derived from Cmax values from clinical studies, to induce promoter activity of CYP3A4 and CYP2B6 through nuclear receptors PXR or CAR pathways, to determine if the presence of low-dose ritonavir alters the response, and additionally validate these results in primary human hepatocytes by measuring mRNA and protein expression of CYP3A4 and CYP2B6 after exposure to ARVs; and 3) assess the impact of coding polymorphisms in NR1I2 (PXR) detected in the genotype screening on ARV induction of CYP3A4 and CYP2B6 promoter activity.

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MATERIALS AND METHODS

Study Population and Genotyping

One thousand thirteen subjects from the Dublin HIV Cohort (established in 2005 after obtaining ethical approval and encompassing HIV-positive patients from three major Dublin hospitals: St. James's Hospital, Mater Misericordiae University Hospital, and Beaumont Hospital) were included in the study (Table 1). The ethnicity distribution was approximately 65% white and 35% sub-Saharan Africans (self-reported). DNA was isolated from whole blood using QIAamp DNA Blood Midi Kit (QIAGEN, Hilden, Germany).

TABLE 1

TABLE 1

Thirty-two SNPs and one three-basepair insertion in NR1I2 (PXR), CYP3A4, and CYP2B6 were selected for screening based on previous association with altered expression levels or activity of the respective proteins, or potential to affect the same by virtue of its location in regulatory regions, transcription factor binding sites, or coding regions (Table 2). Genotyping was performed by KBioscience (Herts, UK) using patented KASPar technology (homogenous FRET-based system coupled with competitive allele-specific polymerase chain reaction; see www.kbioscience.co.uk/reagents/KASP.html). Polymerase chain reaction-restriction fragment length polymorphism was carried out in-house for one SNP per gene using previously described primers and enzymes (NR1I2 7635A>G,22CYP3A4 1221C>T,23CYP2B6 516G>T24) with 9% of the DNA samples as a quality control.

TABLE 2

TABLE 2

TABLE 2

TABLE 2

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Plasmids

The XREM-CYP3A4 luciferase construct was a gift from Professor Chris Liddle (University of Sydney, New South Wales, Australia) and the CYP2B6-PBREM/XREM luciferase construct was kindly donated by Professor Hongbing Wang (University of Maryland, College Park, MD). Dr. Steven Kliewer (University of Texas, Dallas, TX) provided the pSG5-hCAR and pSG5-hPXR plasmids, whereas Dr Oliver Burk (Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany) supplied the PXR polymorphism constructs in pcDNA3 (“PXRwt,” ie, the reference sequence, P27S, G36R, V140M, A370T). An internal standard, pRL-TK (expressing Renilla luciferase), was obtained from Promega (Madison, WI).

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Antiretroviral Drugs and Controls

Abacavir and fosamprenavir were gifts from GlaxoSmithKline (Middlesex, UK). Lopinavir, nelfinavir, nevirapine, and tenofovir were provided by Abbott (Abbott Park, IL), Pfizer (New York, NY), Boehringer Ingelheim (Ingelheim, Germany), and Gilead (Foster City, CA), respectively. Efavirenz was purchased from LGM Pharmaceuticals (Boca Raton, FL) and indinavir, ritonavir, and saquinavir from USP Reference Standards (Rockville, MD). The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health: atazanavir sulfate, maraviroc, lamivudine, tipranavir, and zidovudine. Darunavir was provided by Tibotec, Inc through the same program. Positive controls rifampicin and CITCO were purchased from SIGMA (St. Louis, MO).

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HepG2 Cell Culture and Transfections

HepG2 cells were cultured in Minimum Essential Medium Eagle supplemented with 10% fetal bovine serum, 2mM L-glutamine, and 100 U + 0.1 mg/mL penicillin-streptomycin (all from SIGMA). Cells were seeded into a 24-well format (4 × 105 cells per well) the day before transient transfection using Lipofectamine LTX (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions with the following DNA quantities (ng nuclear receptor/reporter construct/internal standard, optimized for maximal positive control induction): PXR/CYP3A4/pRL-TK 2/400/10 ng, PXR/CYP2B6/pRL-TK 10/400/25 ng, CAR/CYP3A4/pRL-TK 10/400/50 ng, or CAR/CYP2B6/pRL-TK 2/400/10 ng. The transfections were allowed to proceed for 8 to 9 hours in serum- and antibiotic-free medium.

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Drug Exposure and Dual-Luciferase Reporter Assays

Transfected cells were washed with phosphate-buffered saline (SIGMA) and treatment initiated with drugs diluted in phenol red-free Minimum Essential Medium (Gibco, Carlsbad, CA) with 10% charcoal-stripped fetal bovine serum, 2 mM L-glutamine. and 100 U + 0.1 mg/mL penicillin-streptomycin (SIGMA); drug concentrations used correspond to reported mean (median for nelfinavir, ritonavir, and tipranavir) plasma Cmax values from clinical studies (PI concentrations were ritonavir “boosted” because this is how they are usually administered). PIs atazanavir 4 μM (3.211 μg/mL25), darunavir 10 μM (5.834 μg/mL26), fosamprenavir 13 μM (8.039 μg/mL27), indinavir 15 μM (10.65 μg/mL28), lopinavir 16 μM (9.69 μg/mL29), nelfinavir 6 μM (3.614 μg/mL30), ritonavir 1 μM (1.08 μg/mL31), saquinavir 4 μM (3.064 μg/mL),32 tipranavir 20 μM (22.5 μM33); NRTIs abacavir 5 μM (3.19 μg/mL34), lamivudine 7 μM (1.567 μg/mL35), tenofovir 1 μM (360 ng/mL36), and zidovudine 4 μM (1.067μg/mL37); NNRTIs efavirenz 10 μM (3.28 μg/mL38), and nevirapine 7.5 μM (1.93 μg/mL39); and entry inhibitor maraviroc 0.5 μM (144 ng/mL40). Additionally, the cells were exposed to a range of concentrations (0.1 μM, 1 μM, 5 μM, 10 μM, 20 μM) of selected CYP3A4 and/or CYP2B6 inducers (lopinavir, efavirenz, and abacavir) for construction of dose-response curves. Drugs were dissolved in ethanol, DMSO, or H2O. The following PIs were also tested in combination with low-dose (1 μM) ritonavir: atazanavir, darunavir, fosamprenavir, lopinavir, and saquinavir. Rifampicin (10 μM) and CITCO (100 nM) were included as positive controls for PXR and CAR, respectively, as well as vehicle controls representing the highest ethanol (0.17%) and DMSO (0.1%) final concentrations. After 48 hours, the cells were harvested and the Dual-Luciferase Reporter Assay System (Promega) used to measure transcription levels with the aid of a luminometer (ThermoScientific, Waltham, MA). Reporter construct responses (firefly) were normalized to internal standard (Renilla) and fold increases calculated relative to untreated controls.

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Assessing the Effect of Pregnane X Receptor Polymorphisms on Antiretroviral Induction of CYP3A4

Four PXR (NR1I2) polymorphisms, P27S (79C>T), G36R (106G>A), V140M (4374G>A), and A370T (8528A>G) were selected for in vitro assessment of their functional impact on ARV induction of CYP3A4 based on the following criteria: they were coding polymorphisms (resulting in an amino acid change) detected among the patients in the cohort and have been associated with altered activity. Hustert et al13 found increased corticosterone-induced PXR activity with the G36R variant and increased basal expression of CYP3A4 with V140M and A370T as well as a trend toward decreased rifampicin-activated expression with the latter two PXR variants and increased corticosterone activation with P27S. This SNP has also been reported in a patient with reduced nifedipine clearance.10

The P27S, G36R, V140M, and A370T constructs along with a PXR reference sequence plasmid were subsequently used for cotransfections with XREM-CYP3A4. Cells were exposed to rifampicin, lopinavir, fosamprenavir, nelfinavir, efavirenz, and tenofovir (to represent PIs, NNRTIs, and NRTIs) at the same concentrations as in previous experiments and Dual-Luciferase Reporter assays as described previously.

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Primary Human Hepatocytes: mRNA and Protein Expression of CYP3A4 and CYP2B6

Fresh primary human hepatocytes (from two white male donors supplied by Biopredic International, Rennes, France) in 24-well plates (350,000 cells/well) were exposed in duplicate to 0.1% DMSO, 10 μM rifampicin, 100 nM CITCO, or varying concentrations of lopinavir, efavirenz, or abacavir (0.1 μM, 1 μM, and 10 μM) for 48 hours in phenol red-free William's E incubation medium (Biopredic). Total RNA and protein was isolated using TRIsure (Bioline, London, UK). A total of 1 μg DNaseI-treated (SIGMA) RNA was reverse-transcribed to cDNA using random hexamers (Bioline) and M-MLV RT (SIGMA), and real-time polymerase chain reaction subsequently performed with QuantiTect SYBR Green PCR Kit and Primer Assays (QIAGEN) for human CYP3A4, CYP2B6, and β-actin (housekeeping gene) with Applied Biosystems 7900HT.

Isolated protein was denatured by boiling in sample buffer (6% sodium dodecylsulfate, 100 mM Tris-HCl pH 6.8, 20% glycerol, 0.4% bromophenol blue) and loaded onto 10% sodium dodecylsulfate-polyacrylamide gels. Western blots were carried out using Amersham Hybond-P membranes (GE Healthcare, Buckinghamshire, UK). Primary rabbit antihuman CYP3A4 (CR3340, 1:1000 dilution) and CYP2B6 (CR3290, 1:500 dilution) were from Biomol/Enzo Life Sciences, Exeter, UK, whereas horseradish peroxidase-conjugated swine antirabbit secondary antibody was purchased from Dako Denmark A/S (Glostrup, Denmark) and mouse antihuman β-actin antibody (internal standard) from Santa Cruz, Heidelberg, Germany. Blots were visualized by enhanced chemiluminescence detection as described by Haan and Berrman.41

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Statistics

Differences in allele frequencies between whites and sub-Saharan Africans were compared by the χ2 test (SPSS Version 15.0; SPSS Inc, Chicago, IL) and P values were calculated by Fisher exact test (two-sided). Allele frequencies of sub-Saharan Africans were also compared with available frequency data from African Americans using the same method. Haploview Version 4.2 (www.broadinstitute.org/mpg/haploview)42 was used for construction of linkage disequilibrium (LD) plots and Hardy-Weinberg exact tests with Bonferroni correction performed within both ethnic groups. Haplotype analysis was performed using HAP (http://research.calit2.net/hap/).43

Data from reporter assays, real-time polymerase chain reaction and Western blots were normalized to internal standards and analyzed by one-way analysis of variance with Dunnett post hoc analysis, whereas results from reporter assays with ritonavir combinations were compared with single PIs by Mann-Whitney t tests (GraphPad Prism Version 5). EC50 values from dose-response experiments were calculated using the same software (nonlinear fit, sigmoidal dose-response curves). All reporter assay experiments were performed in duplicate at least three independent times and presented as means ± standard deviation. P ≤ 0.05 was regarded as indication of a significant difference for all experiments.

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RESULTS

Patient Demographics

The demographics of the whites and sub-Saharan Africans subgroups in the cohort differ in terms of gender and probable route of transmission (Table 1). Three fourths of the white population are male, and the risk groups “men who have sex with men” and “intravenous drug users” are well represented. Among the sub-Saharan Africans, consisting mainly of first-generation immigrants, the gender distribution is the opposite, and the route of transmission is primarily through heterosexual contact. The high proportion of women in this group reflects detection of HIV infection through the national antenatal screening program.

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NR1I2 (PXR), CYP3A4, and CYP2B6 Polymorphism Frequencies

Of 33 polymorphisms examined, 17 were found at significantly different (P < 0.05) allelic frequencies in whites compared with sub-Saharan Africans: in NR1I2: -25564G>A, -25385C>T, -24756G>A, and -24381A>C in the promoter region; 79C>T and 106G>A in exons; and -6994C>T, 7635A>G, and 8055C>T in introns as well as position 11156A>C in the 3′ untranslated region (Table 2). P < 0.05 was also reached with CYP3A4 polymorphisms -11128insTGT (three basepair insertion), -392A>G, and 683C>T and similarly with CYP2B6 SNPs 516G>T, 785A>G, 983T>C, and 1459C>T. For all these SNPs, homozygotes of the minor alleles were present (albeit often in small numbers) with the exception of NR1I2 106G>A and CYP3A4 683C>T. Remaining SNPs were either absent of the populations or found at a very low prevalence and any difference between whites and sub-Saharan Africans could not be determined. Genotype distributions were in Hardy-Weinberg equilibrium (HWE) with the exception of 7635A>G (NR1I2, PXR) among the sub-Saharan Africans. In CYP2B6, positions 516 and 785 were in LD among both ethnicities (r2 = 0.82 and 0.91 for whites and sub-Saharan Africans, respectively), which is in accordance with other studies44,45 and shown in Figures 1C and 1D, as were NR1I2 SNP pairs -25385/-24381 (r2 = 0.96) and 8055/11156 (r2 = 0.96) in the white population, consistent with HapMap data as well as Dring et al.22 Sub-Saharan Africans exhibited different LD patterns for the same gene; 52/8528 showed strong LD (r2 = 0.70); however, because HapMap data are not available for position 8528 (rs59152710), this could not be confirmed (see Fig. 1A-B). These ethnicity-specific patterns are reflected in their contrasting block structures of the haplotype reconstruction, shown in Table 4. None of the CYP3A4 polymorphisms detected displayed LD.

TABLE 4

TABLE 4

FIGURE 1

FIGURE 1

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Comparison of Sub-Saharan African Allelic Frequencies With African Americans

Three allelic frequencies of sub-Saharan Africans observed in this study were found to be significantly different to those of black subjects in other studies: -24756G>A and 8055C>T in NR1I2 (PXR) compared with a study by Zhang et al10 as well as -392A>G in CYP3A4 compared with data from a publication by Haas et al15 (Table 2). All three minor allele frequencies were higher among sub-Saharan Africans than African Americans. Of the remaining SNPs with available data on black populations, four SNPs were not significantly different between the two groups, whereas five SNPs failed to reach statistical significance as a result of low numbers of the minor allele.

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Antiretroviral-Induced Pregnane X Receptor-Mediated CYP3A4 and CYP2B6 Promoter Activity

Fosamprenavir, lopinavir, nelfinavir, and tipranavir showed the ability to induce PXR-mediated CYP3A4 promoter activity significantly in reporter assays, producing fold increases of the following magnitudes compared with untreated: 13.5 ± 3.9, 7.5 ± 2.7, 5.6 ± 2.3, and 9.9 ± 3.4 (Fig. 2A). CYP2B6 promoter activity was also increased by lopinavir (11.4 ± 10.0) as well as by darunavir (6.1 ± 0.4; Fig. 2B). Efavirenz increased both CYP3A4 and CYP2B6 by 5.7 ± 3.3 and 4.7 ± 2.3-fold, respectively, whereas abacavir increased only CYP2B6 by 2.3 ± 0.6-fold (Fig. 2C-D). EC50 values of lopinavir and efavirenz for induction of CYP3A4 promoter activity were calculated to 1.3μM (95% confidence interval, 124 nM to 13.4 μM) and 23.3 μM (95% confidence interval, 592 nM to 917 μM), respectively. However, the same drugs did not reach maximal response for CYP2B6 promoter activity; hence, EC50 values were estimated at 72 mM in both cases (Figs. 4A-D). Higher drug concentrations than 20 μM were not tested as a result of cytotoxicity (as established by cytotoxicity assays, data not shown).

FIGURE 2

FIGURE 2

FIGURE 3

FIGURE 3

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Antiretroviral-Induced Constitutive Androstane Receptor-Mediated CYP3A4 and CYP2B6 Promoter Activity

In reporter assays using CAR-transfected HepG2, CYP2B6 promoter activity was increased by fosamprenavir, lopinavir, and tipranavir with fold increases of 3.4 ± 3.2, 3.0 ± 1.3, and 4.8 ± 2.4 compared with untreated controls (Fig. 3B) but unchanged by non-PI ARVs (Fig. 3D). None of the PIs tested had an effect on CAR-mediated CYP3A4 transcriptional activity (Fig. 3A). However, it was induced after treatment with abacavir (2.5 ± 1.0, Fig. 2C). EC50 of abacavir was 1.7 μM (95% confidence interval, 200 nM to 13.9 μM) for CYP3A4 induction and estimated at 48 mM for lopinavir-induced CYP2B6 promoter activity, because maximal induction was not reached with this protease inhibitor (Fig. 4E-F). No change in promoter activity was seen with vehicle controls (data not shown).

FIGURE 4

FIGURE 4

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Effect of Coadministration With Ritonavir

Promoter activity of CYP3A4 by lopinavir and saquinavir was increased (P < 0.05) when these PIs were combined with 1 μM ritonavir (fold increases compared with single drugs with PXR were 1.7 ± 0.5 and 2.7 ± 1.0, respectively, and with CAR 2.5 ± 0.9 and 2.4 ± 1.1, respectively) as well as fosamprenavir/ritonavir in the CAR-mediated assay (2.4 ± 0.5). Saquinavir was the only drug tested that increased CYP2B6 promoter activity when low-dose ritonavir was added through PXR fold increase 5.7 ± 1.4 (Table 3).

TABLE 3

TABLE 3

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Impact of NR1I2 (pregnane X receptor) Polymorphisms on CYP3A4 Induction

One hundred thirty-five patients within the cohort were found to have SNPs in the NR1I2 gene leading to PXR amino acid substitutions P27S, G36R, V140M, and A370T. Interestingly, none of these patients carried more than one. The effect of these exonic polymorphisms on ARV induction of CYP3A4 was assessed. Transfection of HepG2 cells with V140M and A370T mutation constructs both resulted in lower CYP3A4 promoter activity after rifampicin stimulation in comparison with the reference PXR sequence. A comparable effect is seen with the same PXR variants in the presence of fosamprenavir and lopinavir, in which induction is significantly (P < 0.05) lower. Variants P27S and G36R showed trends toward reduced rifampicin and fosamprenavir induction but did not reach statistical significance. Nelfinavir and efavirenz-induced CYP3A4 promoter activity was not altered by PXR variants P27S, G36R, V140M, or A370T. Tenofovir, included as a negative control because it did not increase CYP3A4 promoter activity using the PXR reference sequence construct, similarly had no effect in experiments with PXR variants (Fig. 5).

FIGURE 5

FIGURE 5

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Primary Human Hepatocytes: mRNA and Protein Expression of CYP3A4 and CYP2B6

The effect of lopinavir, efavirenz, and abacavir on mRNA and protein expression in primary human hepatocytes was determined. CYP3A4 mRNA levels were increased significantly only by 10 μM efavirenz (fold increase 2.8 ± 0.7), although a trend toward increased expression is seen with increasing concentrations of abacavir; however, the lowest concentration 0.1 μM gives rise to significantly lower CYP3A4 mRNA expression compared with vehicle control (Fig. 6A). CYP2B6 mRNA was increased by 10 μM efavirenz (30.1 ± 12.8) and 10 μM abacavir (3.2 ± 0.4) (Fig. 6B). Determination of protein content by Western blot showed increased CYP3A4 by 10 μM efavirenz (7.6 ± 4.7) and CYP2B6 by 10 μM lopinavir (1.8 ± 0.4) (Fig. 6C-D). Representative Western blots are shown in Figures 6E and F.

FIGURE 6

FIGURE 6

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DISCUSSION

In this study, the frequency distribution of SNPs in genes selected for their established or likely impact on ARV drug metabolism was examined in a cohort of more than 1000 white and sub-Saharan African patients with HIV. The ability of 16 different ARVs at clinically relevant concentrations to induce promoter activity of CYP3A4 or CYP2B6 through nuclear receptors PXR and CAR was evaluated as well as the effect of low-dose ritonavir in combination with a subset of the PIs. The impact on ARV-induced promoter activity of CYP3A4 by four exonic, nonsynonymous NR1I2 (PXR) polymorphisms detected among the patients was subsequently assessed. Real-time polymerase chain reaction and Western blot analysis were also conducted with primary human hepatocytes to detect changes in CYP3A4 and CYP2B6 mRNA and protein expression after drug exposure.

The genotyping results of this study strengthen allele frequency data from smaller studies (ranging between 48 and 511 patients) of comparable groups (NR1I2,10,13,33,46CYP3A4,15,17,18,47-51 and CYP2B615,51,52). However, few studies have included a large number of subjects from sub-Saharan Africa. Our study, containing 357 sub-Saharan Africans revealed a higher prevalence in this subgroup of the CYP3A4 promoter polymorphism -392A>G as well as CYP2B6 SNPs 516G>T, 983T>C, and 785A>G compared with the white patients. Also, more than half of the NR1I2 (PXR) SNPs screened for the “minor alleles” linked to altered expression or activity were more common among sub-Saharan Africans. Although no ancestry informative markers53 were included in the screening, allele frequencies similar to data from previous studies (where available) are reassuring that the differences between ethnicities are authentic. The one deviation from HWE (NR1I2 7635A>G) among sub-Saharan Africans can be explained by a degree of genetic diversity within this subcontinent: when the patients were divided into northeast, northwest, and southern regions, this SNP was in HWE in all three groups (however, 8055C>T deviated from HWE in the southern group). Increased plasma concentrations of NNRTIs54 and predisposition to toxicity (typically cardiovascular and renal events55) seen among patients of African origin is mainly attributed to genetic variation in CYP2B6, although multiple polymorphisms in the PXR gene may also be a contributing factor. The only SNP in NR1I2 (PXR) clinically associated with alterations in ARV drug levels to date, -6994C>T, which reduced atazanavir concentrations among homozygotes,14 was nearly twice as common in the white population. Furthermore, some significant differences in allele frequency were noted between our sub-Saharan African patients and mixed black groups (NR1I2/PXR: Zhang et al,10CYP3A4: Haas et al15; see Table 2). This indicates that predictions of drug efficacy and toxicity in African patients with HIV based on data from black study populations should perhaps be interpreted with caution.

Results from Dual-Luciferase Reporter assays indicate that PXR has a more pronounced role than CAR in mediating ARV-induced promoter activity of CYP3A4 and CYP2B6 in HepG2 cells. Its dominance over CAR as an induction pathway may be explained by a higher degree of ligand promiscuity resulting from a larger and more flexible ligand binding pocket,56 but as suggested by others,57 it is also possible that the constitutive activation of CAR in immortalized cell lines58 renders the process of identifying activators of this nuclear receptor more difficult. Nevertheless, this study found fosamprenavir, lopinavir, tipranavir, and abacavir to have CAR-activating abilities.

The majority of the drugs found to have inductive abilities were from the PI subclass; however, it does not appear to be a general characteristic because some PIs did not give rise to any significant increase of CYP3A4/CYP2B6 promoter activity at the concentrations tested. This is consistent with earlier results from Dussault et al,4 who were also unable to detect PXR activation by indinavir and saquinavir at 10 μM. However, this publication also presented negative results for nelfinavir, which in our study increased PXR-mediated CYP3A4 promoter activity significantly at 6 μM. Conversely, Gupta et al7 reported PXR activation by ritonavir, saquinavir, indinavir, and atazanavir whose effects did not differ significantly from untreated control subjects in our study. These discrepancies may be the result of the use of lower concentrations (with the exception of indinavir, which was used at 15 μM) and a different cell line, HepG2 (human hepatocarcinoma, widely used for reporter assays) versus CV-1 (African green monkey kidney cells) and LS180 (human colorectal adenocarcinoma) in the other studies. The degree of efavirenz induction of CYP3A4 through the PXR pathway is comparable to luciferase reporter assays performed by Hariparsad et al8 in HepG2, in which a three- to fourfold increase was reached. A somewhat surprising result was the finding that abacavir increased both PXR-mediated CYP2B6 promoter activity as well as CAR-mediated CYP3A4 promoter activity, considering that as an NRTI, it is subjected to very limited CYP450 metabolism and therefore an unlikely candidate for involvement in drug interactions through this pathway. Nevertheless, the inductive abilities of efavirenz and abacavir testify that these characteristics are not exclusive to PIs. The finding that low-dose ritonavir enhanced the response to most PIs, despite demonstrating no ability to activate PXR alone at the same concentration, is interesting. This dualistic effect of enzyme inhibition and promoter activation is likely to contribute to the complexity of ritonavir-associated drug interactions.

When investigating the effect of PXR polymorphism variants on rifampicin-activated CYP3A4 expression in HepG2 cells, we found a lower induction with V140M and A370T. Similar results were presented by Hustert et al13 using LS174T cells, in which the difference between the PXR reference sequence and variants did not reach statistical significance. A lowered response was also seen in our experiments with the same PXR variants using fosamprenavir and lopinavir, but not with efavirenz. This could suggest an ARV drug class-specific effect. However, the same is not found with nelfinavir and efavirenz failing to show significant changes with variant constructs, which is perhaps more likely to be the result of a lower inductive power at this concentration. It is plausible that any CYP-inductive effect imposed by lopinavir treatment could be diminished among the patients in the cohort carrying one of these SNPs, potentially changing their drug metabolism in comparison with other patients.

Validation of the results in primary human hepatocytes exposed to lopinavir, efavirenz, and abacavir confirmed significant increases in both mRNA and protein expression for CYP3A4 as well as CYP2B6 mRNA by efavirenz. CYP2B6 was also increased at an mRNA level by abacavir and at a protein level by lopinavir. Paradoxically, abacavir and lopinavir also decrease CYP3A4 and CYP2B6 mRNA expression, respectively.

A limitation to this study arises from the difficulty in determining accurate correlations between in vivo and in vitro drug concentrations; confounding factors include plasma protein binding. Our reporter assay experiments were performed in 10% serum and the concentrations used were plasma Cmax values from clinical studies. It is possible that these estimates exceed the actual concentrations, although they are in keeping with the concentration range frequently used in in vitro reporter assays, which is a well-evaluated tool for predicting in vivo CYP3A4 induction.59 Great variability has also been reported in ARV drug concentrations in patients.60 Nevertheless, it would be of great value to validate this study clinically to establish the relationship between genotype and phenotype. In this large cohort, however, in which the patients are on complex drug regimens, it would be difficult to differentiate an effect of individual drugs.

In conclusion, we have shown that there are distinctive differences in prevalence of polymorphisms in genes of relevance for ARV drug interactions between white and sub-Saharan African populations. This could affect the extent of PXR- and CAR-mediated CYP3A4/CYP2B6 induction by ARVs, potentially influencing the bioavailability and/or toxicity of the inducing drug as well as coadministered drugs metabolized by these enzymes. Some SNPs in coding regions of the NR1I2 (PXR) gene examined in this study are indeed functionally relevant and may have a considerable impact on ARV pharmacokinetics among carriers.

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ACKNOWLEDGMENTS

We thank the Dublin HIV Cohort Steering Committee (Prof Bill Powderly, Prof Fiona Mulcahy, Prof Bill Hall, Dr. Mary Codd, Prof Colm Bergin, Dr. Susan Clarke, Dr. Grainne Courtney, Dr. Fiona Lyons, Prof Sam McConkey, Dr. Jack Lambert, Dr. Gerard Sheehan, Dr. Patrick Mallon, Dr. Suzie Coughlan, Dr. Jeff Connell, Dr. Martina Hennessy, and data coordinator Alan Macken) for access to cohort patient blood samples. We are also grateful to Dr. Eoin Cotter and staff at the Catherine McAuley Centre (Mater Misericordiae University Hospital, Dublin) for help with DNA extractions, to Drs. Ross McManus and Anthony Ryan (Trinity College, Dublin) for guidance with genotype analysis and to Dr. Kathleen Bennett (Trinity College, Dublin) for advice regarding statistical analysis.

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REFERENCES

1. Dooley KE, Flexner C, Andrade AS. Drug interactions involving combination antiretroviral therapy and other anti-infective agents: repercussions for resource-limited countries. J Infect Dis. 2008;7:948-961.
2. Li T, Chen W, Chiang JY. PXR induces CYP27A1 and regulates cholesterol metabolism in the intestine. J Lipid Res. 2007;2:373-384.
3. Moreau A, Vilarem MJ, Maurel P, et al. Xenoreceptors CAR and PXR activation and consequences on lipid metabolism, glucose homeostasis, and inflammatory response. Mol Pharm. 2008;1:35-41.
4. Dussault I, Lin M, Hollister K, et al. Peptide mimetic HIV protease inhibitors are ligands for the orphan receptor SXR. J Biol Chem. 2001;36:33309-33312.
5. Luo G, Cunningham M, Kim S, et al. CYP3A4 induction by drugs: correlation between a pregnane X receptor reporter gene assay and CYP3A4 expression in human hepatocytes. Drug Metab Dispos. 2002;7:795-804.
6. Huang L, Wring SA, Woolley JL, et al. Induction of P-glycoprotein and cytochrome P450 3A by HIV protease inhibitors. Drug Metab Dispos. 2001;5:754-760.
7. Gupta A, Mugundu GM, Desai PB, et al. Intestinal human colon adenocarcinoma cell line LS180 is an excellent model to study pregnane X receptor, but not constitutive androstane receptor, mediated CYP3A4 and multidrug resistance transporter 1 induction: studies with anti-human immunodeficiency virus protease inhibitors. Drug Metab Dispos. 2008;6:1172-1180.
8. Hariparsad N, Nallani SC, Sane RS, et al. Induction of CYP3A4 by efavirenz in primary human hepatocytes: comparison with rifampin and phenobarbital. J Clin Pharmacol. 2004;11:1273-1281.
9. Fellay J, Marzolini C, Decosterd L, et al. Variations of CYP3A activity induced by antiretroviral treatment in HIV-1 infected patients. Eur J Clin Pharmacol. 2005;12:865-873.
10. Zhang J, Kuehl P, Green ED, et al. The human pregnane X receptor: genomic structure and identification and functional characterization of natural allelic variants. Pharmacogenetics. 2001;7:555-572.
11. King CR, Xiao M, Yu J, et al. Identification of NR1I2 genetic variation using resequencing. Eur J Clin Pharmacol. 2007;6:547-554.
12. Lamba J, Lamba V, Strom S, et al. Novel single nucleotide polymorphisms in the promoter and intron 1 of human pregnane X receptor/NR1I2 and their association with CYP3A4 expression. Drug Metab Dispos. 2008;1:169-181.
13. Hustert E, Zibat A, Presecan-Siedel E, et al. Natural protein variants of pregnane X receptor with altered transactivation activity toward CYP3A4. Drug Metab Dispos. 2001;11:1454-1459.
14. Siccardi M, D'Avolio A, Baietto L, et al. Association of a single-nucleotide polymorphism in the pregnane X receptor (PXR 63396C->T) with reduced concentrations of unboosted atazanavir. Clin Infect Dis. 2008;9:1222-1225.
15. Haas DW, Ribaudo HJ, Kim RB, et al. Pharmacogenetics of efavirenz and central nervous system side effects: an Adult AIDS Clinical Trials Group study. AIDS. 2004;18:2391-2400.
16. Saitoh A, Singh KK, Powell CA, et al. An MDR1-3435 variant is associated with higher plasma nelfinavir levels and more rapid virologic response in HIV-1 infected children. AIDS. 2005;4:371-380.
17. Fellay J, Marzolini C, Meaden ER, et al. Response to antiretroviral treatment in HIV-1-infected individuals with allelic variants of the multidrug resistance transporter 1: a pharmacogenetics study. Lancet. 2002;9300:30-36.
18. Haas DW, Smeaton LM, Shafer RW, et al. Pharmacogenetics of long-term responses to antiretroviral regimens containing eavirenz and/or nelfinavir: an Adult Aids Clinical Trials Group Study. J Infect Dis. 2005;11:1931-1942.
19. Rodriguez-Novoa S, Barreiro P, Rendon A, et al. Influence of 516G>T polymorphisms at the gene encoding the CYP450-2B6 isoenzyme on efavirenz plasma concentrations in HIV-infected subjects. Clin Infect Dis. 2005;9:1358-1361.
20. Rotger M, Colombo S, Furrer H, et al. Influence of CYP2B6 polymorphism on plasma and intracellular concentrations and toxicity of efavirenz and nevirapine in HIV-infected patients. Pharmacogenet Genomics. 2005;1:1-5.
21. Ikeda S, Kurose K, Jinno H, et al. Functional analysis of four naturally occurring variants of human constitutive androstane receptor. Mol Genet Metab. 2005;1-2:314-319.
22. Dring MM, Goulding CA, Trimble VI, et al. The pregnane X receptor locus is associated with susceptibility to inflammatory bowel disease. Gastroenterology. 2006;2:341-348.
23. Roy JN, Barama A, Poirier C, et al. Cyp3A4, Cyp3A5, and MDR-1 genetic influences on tacrolimus pharmacokinetics in renal transplant recipients. Pharmacogenet Genomics. 2006;9:659-665.
24. Lang T, Klein K, Fischer J, et al. Extensive genetic polymorphism in the human CYP2B6 gene with impact on expression and function in human liver. Pharmacogenetics. 2001;5:399-415.
25. von HN, Babacan E, Lennemann T, et al. The steady-state pharmacokinetics of atazanavir/ritonavir in HIV-1-infected adult outpatients is not affected by gender-related co-factors. J Antimicrob Chemother. 2008;3:579-582.
26. Sekar VJ, Lefebvre E, De PE, et al. Pharmacokinetic interaction between darunavir boosted with ritonavir and omeprazole or ranitidine in human immunodeficiency virus-negative healthy volunteers. Antimicrob Agents Chemother. 2007;3:958-961.
27. Luber AD, Brower R, Kim D, et al. Steady-state pharmacokinetics of once-daily fosamprenavir/ritonavir and atazanavir/ritonavir alone and in combination with 20 mg omeprazole in healthy volunteers. HIV Med. 2007;7:457-464.
28. Saah AJ, Winchell GA, Nessly ML, et al. Pharmacokinetic profile and tolerability of indinavir-ritonavir combinations in healthy volunteers. Antimicrob Agents Chemother. 2001;10:2710-2715.
29. Klein CE, Chiu YL, Cai Y, et al. Effects of acid-reducing agents on the pharmacokinetics of lopinavir/ritonavir and ritonavir-boosted atazanavir. J Clin Pharmacol. 2008;5:553-562.
30. Justesen US, Hansen IM, Andersen AB, et al. The long-term pharmacokinetics and safety of adding low-dose ritonavir to a nelfinavir 1,250 mg twice-daily regimen in HIV-infected patients. HIV Med. 2005;5:334-340.
31. Kearney BP, Mathias A, Mittan A, et al. Pharmacokinetics and safety of tenofovir disoproxil fumarate on coadministration with lopinavir/ritonavir. J Acquir Immune Defic Syndr. 2006;3:278-283.
32. Bittner B, Riek M, Holmes B, et al. Saquinavir 500 mg film-coated tablets demonstrate bioequivalence to saquinavir 200 mg hard capsules when boosted with twice-daily ritonavir in healthy volunteers. Antivir Ther. 2005;7:803-810.
33. McCallister S, Valdez H, Curry K, et al. A 14-day dose-response study of the efficacy, safety, and pharmacokinetics of the nonpeptidic protease inhibitor tipranavir in treatment-naive HIV-1-infected patients. J Acquir Immune Defic Syndr. 2004;4:376-382.
34. Yuen GJ, Weller S, Pakes GE. A review of the pharmacokinetics of abacavir. Clin Pharmacokinet. 2008;6:351-371.
35. Narang VS, Lulla A, Malhotra G, et al. Pharmacokinetic profiling and bioequivalence evaluation of 2 lamivudine tablet formulations after single oral administration in healthy human Indian volunteers. J Acquir Immune Defic Syndr. 2005;5:566-569.
36. Droste JA, Verweij-van Wissen CP, Kearney BP, et al. Pharmacokinetic study of tenofovir disoproxil fumarate combined with rifampin in healthy volunteers. Antimicrob Agents Chemother. 2005;2:680-684.
37. Marier JF, Manthos H, Kebir S, et al. Comparative bioavailability study of zidovudine administered as two different tablet formulations in healthy adult subjects. Int J Clin Pharmacol Ther. 2006;5:240-246.
38. Liu P, Foster G, LaBadie RR, et al. Pharmacokinetic interaction between voriconazole and efavirenz at steady state in healthy male subjects. J Clin Pharmacol. 2008;1:73-84.
39. Tarinas A, Tapanes RD, Gonzalez D, et al. Bioequivalence study of two nevirapine tablet formulations in human-immunodeficiency-virus-infected patients. Farm Hosp. 2007;3:165-168.
40. MacArthur RD, Novak RM. Reviews of anti-infective agents: maraviroc: the first of a new class of antiretroviral agents. Clin Infect Dis. 2008;2:236-241.
41. Haan C, Behrmann I. A cost effective non-commercial ECL-solution for Western blot detections yielding strong signals and low background. J Immunol Methods. 2007;1-2:11-19.
42. Barrett JC, Fry B, Maller J, et al. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics. 2005;2:263-265.
43. Halperin E, Eskin E. Haplotype reconstruction from genotype data using Imperfect Phylogeny. Bioinformatics. 2004;12:1842-1849.
44. Leger P, Dillingham R, Beauharnais CA, et al. CYP2B6 variants and plasma efavirenz concentrations during antiretroviral therapy in Port-au-Prince, Haiti. J Infect Dis. 2009;6:955-964.
45. Haas DW, Gebretsadik T, Mayo G, et al. Associations between CYP2B6 polymorphisms and pharmacokinetics after a single dose of nevirapine or efavirenz in African americans. J Infect Dis. 2009;6:872-880.
46. Bosch TM, Deenen M, Pruntel R, et al. Screening for polymorphisms in the PXR gene in a Dutch population. Eur J Clin Pharmacol. 2006;5:395-399.
47. Lamba JK, Lin YS, Thummel K, et al. Common allelic variants of cytochrome P4503A4 and their prevalence in different populations. Pharmacogenetics. 2002;2:121-132.
48. Dai D, Tang J, Rose R, et al. Identification of variants of CYP3A4 and characterization of their abilities to metabolize testosterone and chlorpyrifos. J Pharmacol Exp Ther. 2001;3:825-831.
49. Matsumura K, Saito T, Takahashi Y, et al. Identification of a novel polymorphic enhancer of the human CYP3A4 gene. Mol Pharmacol. 2004;2:326-334.
50. Garsa AA, McLeod HL, Marsh S. CYP3A4 and CYP3A5 genotyping by pyrosequencing. BMC Med Genet. 2005;19.
51. Rodriguez-Antona C, Sayi JG, Gustafsson LL, et al. Phenotype-genotype variability in the human CYP3A locus as assessed by the probe drug quinine and analyses of variant CYP3A4 alleles. Biochem Biophys Res Commun. 2005;1:299-305.
52. Wyen C, Hendra H, Vogel M, et al. Impact of CYP2B6 983T>C polymorphism on non-nucleoside reverse transcriptase inhibitor plasma concentrations in HIV-infected patients. J Antimicrob Chemother. 2008;4:914-918.
53. Kosoy R, Nassir R, Tian C, et al. Ancestry informative marker sets for determining continental origin and admixture proportions in common populations in America. Hum Mutat. 2009;1:69-78.
54. Stohr W, Back D, Dunn D, et al. Factors influencing efavirenz and nevirapine plasma concentration: effect of ethnicity, weight and co-medication. Antivir Ther. 2008;5:675-685.
55. Tedaldi EM, Absalon J, Thomas AJ, et al. Ethnicity, race, and gender. Differences in serious adverse events among participants in an antiretroviral initiation trial: results of CPCRA 058 (FIRST Study). J Acquir Immune Defic Syndr. 2008;4:441-448.
56. Watkins RE, Wisely GB, Moore LB, et al. The human nuclear xenobiotic receptor PXR: structural determinants of directed promiscuity. Science. 2001;5525:2329-2333.
57. Wang J, Sonnerborg A, Rane A, et al. Identification of a novel specific CYP2B6 allele in Africans causing impaired metabolism of the HIV drug efavirenz. Pharmacogenet Genomics. 2006;3:191-198.
58. Kawamoto T, Sueyoshi T, Zelko I, et al. Phenobarbital-responsive nuclear translocation of the receptor CAR in induction of the CYP2B gene. Mol Cell Biol. 1999;9:6318-6322.
59. Kozawa M, Honma M, Suzuki H. Quantitative prediction of in vivo profiles of CYP3A4 induction in humans from in vitro results with a reporter gene assay. Drug Metab Dispos. 2009;6:1234-1241.
60. Molto J, Blanco A, Miranda C, et al. Variability in non-nucleoside reverse transcriptase and protease inhibitors concentrations among HIV-infected adults in routine clinical practice. Br J Clin Pharmacol. 2007;6:715-721.
61. Murayama N, Nakamura T, Saeki M, et al. CYP3A4 gene polymorphisms influence testosterone 6beta-hydroxylation. Drug Metab Pharmacokinet. 2002;2:150-156.
    62. Kang YS, Park SY, Yim CH, et al. The CYP3A4*18 genotype in the cytochrome P450 3A4 gene, a rapid metabolizer of sex steroids, is associated with low bone mineral density. Clin Pharmacol Ther. 2009;3:312-318.
      63. Eiselt R, Domanski TL, Zibat A, et al. Identification and functional characterization of eight CYP3A4 protein variants. Pharmacogenetics. 2001;5:447-458.
        64. NCBI/The HapMap Project: Samples from Yoruba in Ibadan, Nigeria. Available at: http://www.ncbi.nlm.nih.gov/SNP/snp_viewTable.cgi?pop=1412. Accessed August 15, 2008.
          65. GeneCards Human Gene Database. Available at: http://www.genecards.org/cgi-bin/carddisp.pl?gene=CYP3A4&search=rs4987159&snp=322&snp_sort_mode=140#snp. Accessed November 17, 2009.
            66. Rotger M, Tegude H, Colombo S, et al. Predictive value of known and novel alleles of CYP2B6 for efavirenz plasma concentrations in HIV-infected individuals. Clin Pharmacol Ther. 2007;4:557-566.
              67. Rotger M, Colombo S, Furrer H, et al. Does tenofovir influence efavirenz pharmacokinetics? Antivir Ther. 2007;1:115-118.
                68. Gatanaga H, Hayashida T, Tsuchiya K, et al. Successful efavirenz dose reduction in HIV type 1-infected individuals with cytochrome P450 2B6 *6 and *26. Clin Infect Dis. 2007;9:1230-1237.
                  69. Jinno H, Tanaka-Kagawa T, Ohno A, et al. Functional characterization of cytochrome P450 2B6 allelic variants. Drug Metab Dispos. 2003;4:398-403.
                    70. Kirchheiner J, Klein C, Meineke I, et al. Bupropion and 4-OH-bupropion pharmacokinetics in relation to genetic polymorphisms in CYP2B6. Pharmacogenetics. 2003;10:619-626.
                      71. Motsinger AA, Ritchie MD, Shafer RW, et al. Multilocus genetic interactions and response to efavirenz-containing regimens: an adult AIDS clinical trials group study. Pharmacogenet Genomics. 2006;11:837-845.
                        72. Bertrand J, Treluyer JM, Panhard X, et al. Influence of pharmacogenetics on indinavir disposition and short-term response in HIV patients initiating HAART. Eur J Clin Pharmacol. 2009;7:667-678.
                          Keywords:

                          antiretroviral therapy; HIV protease inhibitor; drug interactions; polymorphism; PXR; CYP450

                          © 2010 Lippincott Williams & Wilkins, Inc.