Nevirapine (NVP) is a potent non-nucleoside reverse transcriptase inhibitor (NNRTI) and has been used widely for treating HIV-1-infected children as a component of highly active antiretroviral therapy (HAART) . It has been also used for the prevention of mother-to-child transmission of HIV-1 because of its rapid reduction in viral load and low cost [2,3].
NVP is mainly metabolized by hepatic CYP2B6 and CYP3A4 and is eliminated primarily in the urine in four different metabolites . Among them, 3-hydroxynevirapine and 2-hydroxynevirapine are the major metabolites, transformed by CYP2B6 and CYP3A4, respectively [4,5]. Several studies have shown the importance of the CYP2B6-G516T gene polymorphisms determining plasma efavirenz (EFV) concentrations in HIV-infected adults [6–12] and children . To our knowledge, two studies have examined the association between the CYP2B6-G516T gene polymorphisms and NVP pharmacokinetics in HIV-infected adults [8,14]; however, the numbers of participants were limited and the clinical responses to the different NVP concentrations were not evaluated.
Genetic variants in ATP-binding cassette, sub-family B, member 1 (ABCB1) (previously called multidrug resistance 1, MDR1) gene encoding for P-glycoprotein have been reported to be responsible for variability in nelfinavir (NFV) pharmacokinetics in children  and adults . Although NVP or EFV is not a substrate of P-glycoprotein , several studies have shown the clinical associations between ABCB1 genotypes and EFV pharmacokinetics , virologic outcome in HIV-infected adults receiving EFV containing HAART regimens , and the risk of NVP induced hepatotoxicity [18,19]. Furthermore, intracellular NVP concentrations inversely correlate with P-glycoprotein expression on peripheral blood mononuclear cells (PBMC) in patients receiving NVP . These data may suggest that NVP or its metabolites may be a substrate of P-glycoprotein. P-glycoprotein exists in different anatomical sites including the blood–brain barrier; however, currently no data are available regarding the association between ABCB1 genotype and NVP concentrations in cerebrospinal fluid (CSF).
The primary objective of this study was to evaluate the association between the CYP2B6 and ABCB1 gene polymorphisms and plasma concentrations of NVP and clinical responses to antiretroviral therapy in HIV-infected children who received NVP as a component of HAART. The secondary objective of this study was to evaluate the association between these gene polymorphisms and NVP levels in CSF in a subset of study patients.
Materials and methods
This was a retrospective study including a total of 126 participants who received NVP as a component of HAART regimens from the Pediatric AIDS Clinical Trial Group (PACTG) 366  (n = 52, 41%) and PACTG 377  (n = 74, 59%) cohorts. Detailed antiretroviral regimens and previous antiretroviral experience are summarized in Table 1. The participants were selected because they satisfied the following criteria: (1) received NVP as a component of HAART for > 24 weeks with reported excellent compliance to HAART; (2) virologic and immunologic data were available at baseline, weeks 12 and 24; and (3) pharmacokinetic data for NVP were available.
In addition, 14 pairs of CSF and plasma NVP levels were available from 11 patients with known or suspected HIV encephalopathy, presence of symptoms consistent with neurological decline attributable to HIV-related neurologic diseases from PACTG 366. The CSF and plasma NVP levels were obtained between 3 and 5 h after receiving a dose of NVP.
The baseline characteristics of the 126 patients and the subset of patients are summarized in Table 2. Written informed consent was obtained from the study participants when they enrolled in the studies.
Amplification and detection of CYP2B6 and other gene polymorphisms using real-time polymerase chain reaction
Genomic DNA was extracted from PBMC using the QIAamp DNA Blood Mini Kits (Qiagen, Valencia, California, USA). Novel assays using specific primers and fluorescently labeled probes were designed to distinguish CYP2B6 (NG_000008) and CYP2B7 (pseudogene). For each analysis, 2 μl of genomic DNA (10-20 ng/μl), 2 μl of Hot Start Reaction Mix (Roche Diagnostics, Indianapolis, Indiana, USA), and 3 mmol/l MgCl2 in a 20 μl reaction volume were used for real-time PCR in LightCycler with the LightCycler FastStart DNA Master Hybridization Probe kit (Roche Diagnostics). For the CYP2B6-G516T genotype (rs3745274), an asymmetric PCR was performed using 0.5 μmol/l of forward primer, 0.1 μmol/l of reverse primer, and 0.2 μmol/l of SimpleProbe (IT Biochem, Salt Lake City, Utah, USA). For the CYP2B6-C1459T genotype (rs3211371), an amplification reaction was performed using 0.5 μmol/l of each primer and 0.2 μmol/l of each fluorescent probe (IT Biochem). The following primers and probes were used; CYP2B6-G516T (312 bp): FP 5′-GCGTGACGTGCTGGTACATA-3′, RP 5′-ACCTGGCCGAATACAGA-3′, SimpleProbe 5′-Fluorescein-SPC-TAATGGACTGGAAGAGGAAGGTG-3′, CYP2B6-C1459T (181 bp): FP 5′-CCAGCCCCGTGGCCCCA-3′, RP 5′-TTGCGGGGAGTCAGAGCCATTG-3′, AP 5′-CAGGAAGCGGATCTGGTATG-Fluorecein-3′, SP 5′-LC Red640- GGGGTATTTTGCCCACACCACA-Phosphate. For the PCR reaction, we used an initial denaturation step of 96°C for 10 min, followed by 45 cycles of 96°C for 30 s, 60°C (for the CYP2B6-G516T) or 62°C (for the CYP2B6-C1459T) for 10 s, and 72°C for 10 s, and a melting curve cycle at 96°C for 30 s, 40°C for 2 min, followed by 1°C/s for melting analysis to 80°C. The peaks in the melting curves for wild type and mutant were 62°C and 53°C in the CYP2B6-G516T assay and 60°C and 54°C in the CYP2B6-C1459T assay. These assays were validated using DNAs previously genotyped and provided by Dr Richard Kim at Vanderbilt University. For the ABCB1-C3435T (rs1045642) and CYP3A4-A392G (rs2740574) genotyping, previously developed real-time PCR assays were used .
Among the 126 patients, 40 patients (32%) had intensive pharmacokinetics collected over 8 h at week 4 of treatment and the remaining 86 patients (68%) had sparse pharmacokinetic data available (3.7 samples/patient) during HAART. The intensive pharmacokinetic analysis has been previously presented [22,23]. In the patients with intensive pharmacokinetic evaluations, oral clearance (CL/F) was determined as dose/area under the curve (AUC) and was adjusted to body surface area (CL/F per m2). The AUC was determined at steady-state using the linear trapezoidal method from the predose concentration (C0) to the last measured concentration (Clast); AUC0-last. The remaining AUC, AUClast-12, was determined by the trapezoidal method from the predicted C12. The C12 was determined from the log-linear regression the terminal slope (ke) and the Clast, C12 = Clast*e(−ke*(12−Tlast)). In one patient, a terminal slope could not be determined and C0 was used as an estimate from C12. In patients with sparse data, pharmacokinetic analysis was performed using the program NONMEM Ver.1 (Globomax, Maryland, USA) . The mean ratio of CL/F by methods (noncompartmental/population post-hoc) was 1.002 (95% confidence interval, 0.976–1.030) and the mean differences by these methods were 5.9%, indicating no bias between the two methods. The NVP levels in CSF were also evaluated in 14 samples from the subset of 11 patients in the PACTG 366 cohort at week 12 (n = 11) and week 48 (n = 3). Paired single samples in plasma and CSF were collected between 3 and 5 h after a dose of NVP.
Measurement of plasma HIV-1 RNA and CD4+ T cells
Plasma HIV-1 RNA (HIV-1 RNA) was quantified using the Roche Amplicor HIV-1 Monitor assay (Roche Molecular Systems, Alameda, California, USA) with a detection limit of 400 copies/ml. The absolute numbers and percentages of CD4+ T cells were determined in PACTG certified laboratories by flow cytometry.
Measurement of liver enzymes
Alanine transferase (ALT) and asparatate transferase (AST) were quantified as a part of the study protocols. Abnormal liver enzymes (≥ Grade 2) were defined as AST or ALT: ≥ 5 times higher than upper normal limits.
The Kruskal–Wallis test was used to determine whether the following parameters differed among the genotypes: (1) CL/F for NVP; (2i) clinical parameters including age, baseline HIV-1 RNA, and CD4+ T-cell count and percentage. The Wilcoxon sum rank test was used in the above comparisons when two of the three genotypes were compared. The Spearman correlation test was used to evaluate the correlation between NVP CL/F and age. Multivariate analyses for NVP CL/F and change in CD4+ T-cell percentage were performed to evaluate the contribution of covariates including CYP2B6-G516T genotype (T/T genotype vs. others), concomitant protease inhibitor [NFV + ritonavir (RTV) vs. NFV or RTV], ABCB1-C3435T genotype (T/T genotype vs. others), race/ethnicity (African American vs. others), and gender (male vs. female). Fisher's exact test was used to make pairwise comparisons between genotypes and binominal values. The Hardy–Weinberg test was performed for the whole study population and race/ethnicity, and the observed and expected allele and genotype numbers were compared using the chi-squared test for fit of data to the Hardy-Weinberg equilibrium. All P-values calculated were two-sided and a P-value of < 0.05 was considered to be statistically significant.
Patients and frequency of CYP2B6-G516T genotype
Thirty-nine percent (49 of 126) of the patients had the CYP2B6-516-G/G genotype (wild-type), 50% (63 of 126) had the G/T genotype (heterozygous mutant) and 11% (14 of 126) had the T/T genotype (homozygous mutant). No significant differences were found among these three groups with respect to baseline characteristics (P = 0.16–0.86) (Table 2). The CYP2B6-516-T allele frequency was 0.37 in the whole cohort. The allelic frequency did not differ significantly by race/ethnicity (P > 0.37); the allelic frequency in White was 0.26 (n = 19), 0.38 in African American (n = 78) and 0.37 in Hispanic (n = 27). The overall frequency of the CYP2B6-G516T genotypes and the frequency of the CYP2B6-G516T genotype in each race/ethnicity were in Hardy–Weinberg equilibrium (P = 0.41 and P = 0.16–0.80, respectively). The frequency of all genetic polymorphisms evaluated in the study by race/ethnicity is listed in Table 3.
Children with the CYP2B6-516-T/T genotype demonstrate lower NVP CL/F and higher AUC compared to those with the -G/G and -G/T genotypes. A comparative analysis of NVP pharmacokinetic parameters was performed among the patients with the CYP2B6-G516T genotypes. NVP CL/F in children with the CYP2B6-516-T/T genotype (1.6 l/h per m2) was significantly decreased compared to those with the -G/G (2.3 l/h per m2; P = 0.001) and -G/T genotype (2.1 l/h per m2; P = 0.003) (Fig. 1). Furthermore, children with the CYP2B6-516-T/T genotype had a higher median AUC for NVP (95.0 μg*h/ml) compared with those with the -G/G (57.8 μg*h/ml; P = 0.004) and -G/T genotype (58.3 μg*h/ml; P = 0.003). No significant difference was observed when CL/F rates or AUC for NVP were compared between children with the -G/G (P = 0.53) and -G/T genotype (P = 0.49).
Seven patients (5.5%) had the CYP2B6-1459-C/T genotype (heterozygous mutant) and the rest of patients had the CYP2B6-1459-C/C genotype (wild type). The mean NVP CL/F did not differ between the C/T genotype and the C/C genotype (2.8 vs. 2.5 l/h per m2, respectively; P = 0.95). Similarly, NVP CL/F was not different among children with the CYP3A4-A392G genotypes (P = 0.51).
Other factors including age (r = 0.06; P = 0.49) and race/ethnicity (P = 0.75) were not associated with NVP CL/F; however, NVP CL/F in males (2.7 l/h per m2; n = 64) was higher in those in females (2.4 l/h per m2; n = 62) (P = 0.02).
Children with the CYP2B6-516-T/T genotype have greater CD4+ T-cell percentage increases
Next, changes in CD4+ T-cell percentage were compared for children with the CYP2B6-G516T genotypes from baseline to weeks 12 and 24. The baseline CD4+ T-cell percentages were not different among the CYP2B6-G516T genotypes (P = 0.44, Table 2). Children with the CYP2B6-516-T/T genotype had the greatest increase in CD4+ T-cell percentages (+9.0%) compared to those with the -G/G (+3.2%, P = 0.008) and -G/T genotype (+5.0%, P = 0.04) at week 12. This trend continued when increase in CD4+ T-cell percentages in children with the CYP2B6-516-T/T genotype (+10.5%) was compared with those in children with the -G/G (+4.7%, P = 0.01) and -G/T genotype (+8.2%, P = 0.06) at week 24 (Fig. 2).
Virologic response in children with the CYP2B6-G516T genotype
The percentage of patients who reached HIV-1 RNA < 400 copies/ml were compared for children among the CYP2B6-G516T genotypes. The baseline HIV-1 RNA was not different among the three groups (P = 0.86, Table 2). At week 12, 63% (31 of 49), 54% (34 of 63) and 79% (11 of 14) of children with CYP2B6-516-G/G, -G/T and -T/T genotypes, respectively, achieved HIV-1 RNA levels < 400 copies/mL (P = 0.20). A similar lack of significant difference was observed among the groups at weeks 24 (P = 0.24).
Concomitant protease inhibitors and nevirapine oral clearance
All participants received a concomitant protease inhibitor including NFV (n = 67), RTV (n = 36) and NFV + RTV (n = 22). NVP CL/F was significantly lower in children who received NFV + RTV (1.9 l/h per m2) in comparison with those who received NFV only (2.7 l/h per m2, P = 0.005), but not those who received RTV (2.5 l/h per m2, P = 0.10). No significant differences in NVP CL/F were observed between children in PACTG 366 and those in PACTG 377 (P = 0.11).
ABCB1 genotypes and nevirapine pharmacokinetics
We further evaluated whether the ABCB1-C3435T genotype alters NVP pharmacokinetics. Although there was a trend that children with the ABCB1-3435-T/T genotype (homozygous mutant, n = 15) had higher mean NVP CL/F (3.3 l/h per m2) in comparison with those with the -C/C genotype (2.4 l/h per m2; n = 53) and the -C/T genotype (2.4 l/h per m2, n = 58), no statistical significance was observed (P = 0.39). Of note, no association was observed between the ABCB1-C3435T genotype and the CYP2B6-G516T genotype in this study population (P = 0.98).
Multivariate analyses to predict nevirapine oral clearance and change in CD4+ T-cell percentage
A multivariate analysis for NVP CL/F showed that the CYP2B6-G516T genotype (T/T genotype, P = 0.02) and concomitant PIs (NFV + RTV, P = 0.05) and were independently associated with NVP CL/F (Table 4). Similarly, a multivariate analysis for change in CD4+ T-cell percentage showed that the CYP2B6-G516T genotype (T/T genotype) was associated with a change in CD4+ T-cell percentage from baseline to week 12 (P = 0.03, Table 5) with a similar trend at week 24 (P = 0.08). Thus, the CYP2B6-G516T genotype remains an important determinant of NVP CL/F and early immunologic recovery even after controlling for other factors.
Incidence of adverse effects in children with the CYP2B6 and ABCB1 polymorphisms
Abnormal liver enzymes due to NVP use was observed in eight patients (6.3%) during the 24 weeks of HAART. Four patients (50%) had the CYP2B6-516-G/G genotype, two patients (25%) had the CYP2B6-516-G/T genotype and two patients (25%) had the CYP2B6-516-T/T genotype. Additionally, four patients (50%) had the ABCB1-3435-C/C genotype and the rest of four patients (50%) had the ABCB1-3435-C/T genotype.
Cerebrospinal fluid nevirapine concentrations and ABCB1 genotypes
NVP CSF: plasma ratios were analyzed with the ABCB1 and CYP2B6 genotypes from 14 pairs of CSF and plasma NVP levels. NVP CSF: plasma ratios were higher in patients with the ABCB1-3435-C/T (0.62, n = 8) or -T/T (0.62, n = 1) compared with those with the ABCB1-3435-C/C genotype (0.43, n = 5) (P = 0.01, Fig. 3). No significant difference was observed when the ratios were compared with the CYP2B6-G516T genotype (P = 1.00).
To our knowledge, these are the first data demonstrating the association between the CYP2B6-G516T gene polymorphisms, and NVP pharmacokinetics and clinical outcomes in children. Although several reports have shown the effect of the CYP2B6-G516T genotype on EFV pharmacokinetics in HIV-1 infected adults [6–12] and children , the actual influence of the CYP2B6-G516T genotypes on clinical responses has been evaluated in only a few studies [10,13]. In contrast to our findings, these reports found no association between the CYP genotypes and clinical outcomes. The explanations for the differences between the current study for NVP and our earlier study of EFV in children  are unclear. It has been shown that the intracellular accumulation rate for NVP in human lymphocytes (a ratio of intracellular and extracellular concentrations of NVP: 0.005) is significantly lower than that of EFV (1.3) [20,25]. Although the toxicity of higher levels of intracellular NVP have not been evaluated, Pilon et al. demonstrated that high levels of concentrations of EFV in lymphocytes induce dose-dependent, caspase- and mitochondria-dependent apoptosis . Additionally, the minimum intracellular concentrations of NVP and EFV to achieve sufficient viral suppression and immune recovery may be different. It is possible that the NVP dose of 120 mg/m2 per dose every 12 h provides intracellular levels of drug that approximate a critical threshold for optimal response. The current recommended maintenance dose for NVP in the pediatric population is 120–200 mg/m2 per dose every 12 h in the United States Guidelines  and 160–200 mg/m2 per dose every 12 h by World Health Organization . Therefore, the dose of NVP used in this study (120 mg/m2 per dose every 12 h) might have provided for a better clinical response in children with the CYP2B6-516-T/T genotype compared to those with the CYP2B6-516-G/G or G/T genotype who potentially received sub-maximal NVP dosing.
Our data demonstrated that the NVP CSF: plasma ratios were associated with the ABCB1-C3435T genotypes, but not variants of CYP2B6-G516T, which were strongly associated with plasma NVP levels in the current study. P-glycoprotein is known to be expressed on the blood–brain barrier  and plays an important role in protecting the brain by limiting the uptake of P-glycoprotein substrates at the apical surface of brain epithelium. The explanation of our finding is that those with the ABCB1-3435-C/T or -T/T genotype express less P-glycoprotein on the surface of the brain epithelium, resulting in transporting less substrate from epithelium to blood, which leads to higher concentrations of NVP in epithelium and CSF compared with the plasma NVP concentrations if NVP is a substrate of P-glycoprotein. Although our number of CSF samples was limited, our findings provide additional support for NVP or NVP metabolites being substrates of P-glycoprotein .
In our previous study of EFV, we found for the CYP2B6-516-G/G genotype that children aged < 5 years exhibited a greater difference in EFV CL/F than children ≥ 5 years . This difference was only significant for the CYP2B6-516-G/G genotype that is associated with the greatest expression of hepatic CYP2B6 when compared with children with the -G/T or -T/T genotypes. In the current study, however, no significant difference was observed when we divided the patients by age. Although no association was found between the CYP3A4-A392G genotype and NVP CL/F in children in the current study, other variants in CYP genes including CYP3A4 or CYP2D6 may alter NVP metabolism . Additionally, the developmental maturation of each hepatic isoenzyme is unknown and could result in different NVP pharmacokinetics as children age from infancy through to adolescence.
NVP use is associated with some important adverse effects including hepatotoxicity. Although NVP is not a substrate of P-glycoprotein , the ABCB1-C3435T genotype has been reported to be associated with the incidence of hepatotoxicity in HIV-1 infected adults receiving NVP-containing HAART regimens [18,19]. Another study has shown that NVP intracellular concentrations correlate inversely with P-glycoprotein expression in PBMC , suggesting that NVP or its metabolites may be a substrate of P-glycoprotein. Other factors associated with hepatotoxicity include female gender, higher baseline CD4+ T-cell counts  and HLA-DRB1*0101 . In our current study, the incidence of increased liver enzymes (≥ grade 2) was low (6%) and the degree of abnormality was relatively mild making such analyses impossible. Among the eight patients who experienced increased liver enzymes, two (25%) patients were female and the mean CD4+ T-cell percentage at baseline of 23% did not differ from the resuming study population (25%; P = 0.51). Of note, none of the patients who experienced increased liver enzymes had the ABCB1-3435-T/T or CYP2B6-1459-C/T genotype, which is consistent with recent observation in adults .
Although the development of NVP-resistant virus in women and infected infants after receiving a single dose of NVP is a serious concern , NVP is being used worldwide not only as an agent for mother-to-child transmission of HIV infection in developing countries , but also as first line antiretroviral therapy in resource-limited settings . EFV is another NNRTI that is widely used in developed countries; however, it does not have a pediatric formulation and no data are available on the appropriate dosage for children < 3 years old. To give an appropriate therapeutic dosage of NVP, especially in infants and younger children, regardless of the settings, the CYP2B6-G516T variant is an important genotype to be evaluated to predict the NVP pharmacokinetics.
There are several limitations of the current study. First, this was a retrospective study and the number of patients was relatively small; therefore, our findings will need to be validated in other larger cohorts. Second, although the allelic frequency of the CYP2B6-G516T polymorphism has been reported to be higher in African Americans in comparison with Whites, or Asians [6,9,33], no association between race/ethnicity and the frequency of the CYP2B6-G516T allele was observed in the current study, most likely due to the limited sample size, especially the small numbers of Whites. Lastly, potentially important genotypes that could alter NVP pharmacokinetics including the CYP2B6-A785G in exon 5 and CYP2B6-T983T in exon 7 combining with the CYP2B6-G516T [9,11,34] were not investigated in this study.
In conclusion, CYP2B6-G516T gene polymorphisms affect NVP CL/F and clinical outcomes in HIV-1-infected children receiving NVP-containing HAART regimens. Children with the CYP2B6-516-T/T genotype may experience particular benefit from NVP-containing HAART regimens because of the favorable pharmacokinetic profile in this population. Combined with our previous EFV data, these findings support the importance of CYP2B6-G516T genotypes on NNRTI clearance and suggest that CYP2B6 gene polymorphisms may be useful in implementing strategies designed to provide optimal treatment outcomes for children receiving NNRTI-containing HAART regimens.
The authors acknowledge the PACTG sites and study participants for their time and dedication to these studies, Dr Kumud Singh and Neurita Salva at the University of California, San Diego for the help and assistance performing real-time PCR, Dr. Happy Araneta at the University of California, San Diego for help in performing statistical analyses, and Dr Richard Kim for providing control samples for the CYP2B6 genetic variants.
Informed consent was obtained from study participants. This study followed the human experimentation guidelines of the US Department of Health and Human Services and the University of California, San Diego (UCSD) review board.
Sponsorship: This study was supported by the Pediatric AIDS Clinical Trials Group/International Maternal, Perinatal, Adolescent AIDS Clinical Trials Group, by Grants from the National Institute of Allergy and Infectious Diseases [(U01A141089 and 5K23AI-56931 to A.S., AI- 41089, AI- 39004, AI- 27563, AI- 33835, AI- 41110); AI- 36214 (Virology Core UCSD Center for AIDS Research), AI- 32921] and by Bristol-Myers Squibb.
Presented in part: Fourteenth Conference on Retroviruses and Opportunistic Infections, Los Angeles, California, February 2007 (Poster: #735).
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Keywords:© 2007 Lippincott Williams & Wilkins, Inc.
ATP-binding cassette, sub-family B, member 1 (ABCB1); cerebrospinal fluid; children; CYP2B6; immunologic response; nevirapine; pharmacogenomics