Abstract: This study evaluated mid-dosing interval efavirenz plasma concentrations and the influence of CYP2B6 polymorphisms in relation to efficacy, tolerability, and adherence in 97 Rwandan HIV-infected children (3–16 years). Plasma drug concentrations and CYP2B6 polymorphisms were determined. Ten children were excluded for nonadherence. Large intersubject variability in efavirenz plasma concentrations was found. Of the 87 remaining, efavirenz concentrations were therapeutic, supratherapeutic, and subtherapeutic in 67%, 20%, and 14%, respectively. No associations were found between efavirenz concentrations and central nervous system disturbances or virologic failure. Minor allele frequencies were 0.32 (516G>T), 0.33 (785A>G), and 0.09 (983T>C). Polymorphisms in CYP2B6 were strongly associated with high efavirenz levels.
*Department of Pediatrics, Kigali University Teaching Hospital (KUTH), Kigali, Rwanda
†Academic Medical Centre, Kigali, Rwanda
‡Academic Medical Centre/Amsterdam Institute for Global Health and Development (AMC-AIGHD), Amsterdam, the Netherlands
§Department of Pharmacy and Nijmegen Institute for Infection, Inflammation and Immunity (N4i), Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands
‖Utrecht University Children's Hospital/University Medical Centre of Utrecht (UMCU), Utrecht, the Netherlands
¶Treatment and Research on HIV/AIDS Centre (TRAC-plus)/Outpatients Clinic, Kigali, Rwanda
#Royal Tropical Institute (KIT), Biomedical Research, Epidemiology Unit, Amsterdam, the Netherlands
**Department of Clinical Chemistry (AKC), Erasmus MC, Rotterdam, the Netherlands.
Correspondence to: Dr. Philippe R. Mutwa, MD, INTERACT, PO Box 2128, Kigali, Rwanda (e-mail: firstname.lastname@example.org).
Supported by the Netherlands–African Partnership for Capacity Development and Clinical Interventions Against Poverty-Related Diseases (NACCAP).
The authors have no conflicts of interest to disclose.
Part of the were presented at the 5th INTEREST Workshop; May 10–13, 2011; Dar es Salaam, Tanzania, and published as abstract for the same conference.
Received September 24, 2011
Accepted March 20, 2012
Current Rwandan national guidelines recommend 2 nucleoside reverse transcriptase inhibitors plus a nonnucleoside reverse transcriptase inhibitors for first-line antiretroviral therapy of HIV-infected children older than 3 years.1,2 Efavirenz is given as an alternative to nevirapine, when the child develops nevirapine-associated side effects or receives antituberculosis treatment.1,2 The drug is clinically and virologically effective, safe, and well tolerated in children.3,4 Its prolonged half-life enables once-daily dosing, which facilitates adherence to antiretroviral therapy.5
An efavirenz plasma concentration of 1.0–4.0 mg/L 8–20 hours after ingestion is recommended as appropriate for achieving viral suppression and for limiting side effects.6 Studies evaluating efavirenz pharmacokinetics in African children are sparse but indicated a high prevalence of virologic failure after exposure to efavirenz plasma concentrations <1.0 mg/L,7 and a disadvantage of efavirenz is the large inter- and intrapatient pharmacokinetic variability, both in adults and children.7–13 High variability in efavirenz plasma levels is associated with various factors, like ethnicity, body weight, dosing, nonadherence, and concomitant medication.14,15 Notably, because efavirenz is mainly metabolized by cytochrome P450 (CYP) 2B6, both elevated and decreased efavirenz plasma concentrations have been associated with CYP2B6-polymorphisms.12,16–19 Studies have shown a high prevalence of some genotypic variants in CYP2B6 enzyme in African adults that would lead to higher efavirenz plasma levels and potentially more toxicity.20,21
This study aimed to evaluate the mid-dosing interval efavirenz plasma concentrations and the influence of CYP2B6 polymorphisms in relation to treatment efficacy, tolerability, and adherence in Rwandan HIV-infected children.
MATERIALS AND METHODS
Ninety-seven children (3–16 years) on efavirenz-based regimens from the Department of Paediatrics, Kigali University Teaching Hospital and the Treatment and Research AIDS Centre; Kigali, Rwanda, participated in the study. Efavirenz was prescribed once daily as a (halved) 600-mg tablet and/or capsules containing 200 and/or 50 mg in weight band doses according to Rwandan 2009 National Guidelines and World Health Organization's 2006 Pediatric Recommendations.1,22 No children were on antituberculosis treatment or other comedication interfering with the efavirenz-based regimen. All legal guardians gave informed written consent. Children 12 years and older provided additional consent. The study was approved by the Rwandan National Ethical Committee.
Demographic data were collected using patient's medical record and a case report form with standardized questionnaires. All caregivers and children if 12 years and older were interviewed, and all children underwent physical examination.
Adherence was assessed using questionnaires, self-reports, and monthly pharmacy refill forms. For children with undetectable efavirenz plasma concentrations, adherence was also assessed by determination of lamivudine plasma levels. For evaluation of safety and acceptability, a complaint checklist was developed indicating whether the child had experienced adverse events (AEs) in the past month. AEs were stated as unlikely, possibly, probably, or definitely related to the drugs.
Blood Sampling and Laboratory Investigations
The study visit was planned 8–20 hours after the last efavirenz intake to collect 1 blood sample for determination of mid-dosing interval efavirenz plasma concentrations, CYP2B6 genotype testing, hematology, biochemistry, CD4 count, and HIV-1 RNA concentration (viral load, VL).
The bioanalyses of efavirenz and (if applicable) lamivudine were done at the Department of Pharmacy, Radboud University Nijmegan, Medical Centre, Nijmegen, the Netherlands using 2 validated high-performance liquid chromatography assays with UV detection.23,24
Genotype testing for CYP2B6 was performed at the Pharmacogenetics Core Laboratory, Department of Clinical Chemistry, Erasmus Medical Centre, Rotterdam, the Netherlands, using validated polymerase chain reaction–restriction fragment length polymorphism assays with primers 5'-CTGTTGCAGTGGACATTTG-3' and 5'-ATCTCACTCCTGCACTCAC-3' for 1459C>T, with digestion of the 460 base pair product with BglII (New England Biolabs), yielding 460 base pair for wild type and 258 and 202 base pair for variant alleles. For 785A>G primers, 5'-GACAGAAGGATGAGGGAGGAA-3' and 5'-CTCCCTCTGTCTTTCATTCTGT-3' were used: the 640 base pair product was digested with BstN1 (New England Biolabs) for 2 hours at 60°C. The fragments for wild-type alleles were 312, 136, 109, and 83 base pair and for variant alleles 295, 136, 109, 83, and 17 base pair. For 516G>T, primers 5'-GGTCTGCCCATCTATAAAC-3' and 5'-CTGATTCTTCACATGTCTGCG-3' were used: the product of 526 base pair was digested with Bsr1 (New England Biolabs), yielding fragments for wild-type of 268, 241, and 17 base pair and for variant alleles 509 and 17 base pair. All 3 polymerase chain reaction–restriction fragment length polymorphism assays were validated by direct sequencing.
Assays for hematology, biochemistry, CD4 counts, and VL (lower limit of detection 40 copies/mL) were done at the National Reference Laboratory at Kigali, Rwanda. Virologic failure was defined as VL ≥400 copies per milliliter.
Logistic regression models were used for associations between efavirenz plasma concentrations and VL suppression (<40 copies/mL) and CD4 count (≥350 cells/mm3). Separate univariate logistic regression analyses were done to determine correlations between efavirenz plasma concentrations and reported side effects. Analyses were conducted with STATA version 10.
The median [interquartile range (IQR)] age of the 97 children (55% female) was 12.0 (10.0–13.7) years. Efavirenz was combined with lamivudine and zidovudine (70%), stavudine (28%), or tenofovir (1%). The average efavirenz dose was 11.4 (10.0–12.9) mg/kg, and median efavirenz duration was 3.7 (1.5–4.9) years. Five of 97 children (5%) were on efavirenz for ≥2 but <6 months. Median (IQR) CD4 cell count was 643 (444–925) cells per cubic millimeter.
All children were in good clinical condition although the majority was moderately wasted and stunted with median (IQR) weight-for-age z-score −1.3 (−2.4 to −0.3) and height-for-age z-score −1.2 (−2.2 to −0.6). One (1%), 6 (6%), 17 (18%), 17 (18%), 36 (37%), and 20 (21%) children were in weight bands 10–14.9, 15–19.9, 20–24.9, 25–29.9, 30–39.9, and ≥40 kg, receiving 200-, 250-, 300-, 350-, 400-, and 600-mg efavirenz, respectively.
Median (IQR) time of plasma sampling was 15.9 (14.8–16.8) hours after the last efavirenz intake. Twenty-two children (22.7%) had subtherapeutic efavirenz plasma concentrations (<1.0 mg/L). Ten children with undetectable plasma concentrations of both efavirenz and lamivudine were considered nonadherent and excluded from further analysis. Eighty percent of them had reported to be adherent by self-report. From the remaining 87 children, the median (IQR) efavirenz plasma concentrations were 2.05 (1.41–3.17) mg/L. Twelve children (13.8%) had subtherapeutic efavirenz plasma concentrations (< 1.0 mg/L), whereas 58 (66.7%) had therapeutic (1.0–4.0 mg/L) and 17 (19.5%) supratherapeutic efavirenz plasma levels (>4.0 mg/L). Four children had a concentration of >12.0 mg/L. A large intersubject variability in efavirenz mid-dosing interval plasma concentrations was found (coefficient of variation of 107%).
Efficacy, Adherence, and Safety
Sixty-one of 87 (70%) remaining children were fully virologically suppressed (<40 copies/mL), and 26 (30%) were not. Seventeen (20%) had a VL ≥400 copies per milliliter. No association was found between virologic response and efavirenz plasma concentration groups [odds ratio (OR): 1.7; 95% confidence interval (CI): 0.7 to 4.3; Table 1]. However, the proportion of children with virologic failure was higher in the subtherapeutic and therapeutic groups (>20%) compared to the group with supratherapeutic levels (6%). The majority of children (87%) self-reported to be well adherent (intake: >95% of prescribed doses in the previous month) including children with treatment failure. Adherence was not found to be associated with mid-dosing interval efavirenz plasma concentrations (OR: 1.1; 95% CI: 0.3 to 4.1).
Of 41 AEs reported, all were grade 1 or 2. Fourteen percent reported ≥1 central nervous system (CNS) side effect, including insomnia, nightmares, concentration disturbances, and dizziness (all grade 1). We did not find a significant relationship between efavirenz plasma concentrations and CNS side effects (OR: 0.8; 95% CI: 0.3 to 4.1). Skin problems reported by another 14% were atypical and not compatible with allergic reactions to antiretroviral drugs. Seventeen children (20%) had elevated aspartate aminotransferase and alanine aminotransferase concentrations. No serious AEs were reported.
Efavirenz Plasma Concentrations and Pharmacogenetics
Genotyping results are shown in Table 2. The CYP2B6 516GT genotype was present in 46% (37 of 80), 516GG in 45% (36 of 80), and 516TT genotype in 9% (7 of 80) children. CYP2B6 785AA, 785AG, and 785GG genotypes were present in 45% (37 of 83), 46% (38 of 83), and 9% (8 of 83) children, respectively. The CYP2B6 785A>G and the CYP2B6 516G>T gene polymorphisms were mostly found together in our population [CYP2B6*6, *19, *20, or *26 (http://www.cypalleles.ki.se)]. The minor allele frequency for 516G>T, 785A>G, and 983T>C was 0.32, 0.33, and 0.09, respectively. All polymorphisms were in Hardy–Weinberg equilibrium (P > 0.05).
Median efavirenz concentrations were strongly associated with CYP2B6 516G>T, 785A>G, and 983T>C polymorphisms (Table 2). Six of 7 children (86%) with the CYP2B6 516TT homozygous genotype had supratherapeutic (>4.0 mg/L) mid-dosing interval efavirenz plasma concentrations, whereas no individual in this group had an efavirenz plasma concentration <1.0 mg/L. Four of 36 516GG children (11%) had subtherapeutic efavirenz plasma concentrations, and none of the children had a plasma level >4.0 mg/L. Similar data were observed for the CYP2B6 785A>G and 983T>C polymorphisms.
In this study, a high level of intersubject variability in mid-dosing interval efavirenz plasma concentrations was observed. Only two-thirds of the children had efavirenz concentrations within the therapeutic range. High variability (ie, 107%) resulted in 14% children with subtherapeutic levels and 20% with potentially toxic plasma levels. Virologic failure was found in nearly a fifth of children with detectable efavirenz levels. Although the difference was not significant in this small study, a higher proportion of virologic failure was observed in children with (sub)therapeutic efavirenz levels.
In pediatric studies from South Africa and Burkina Faso, a large intersubject variability was also found (137% and 143%) and two-thirds of the children with persistent subtherapeutic efavirenz levels experienced virologic failure.7,10,15 In these studies, results were not corrected for adherence. In 2 other pediatric studies, subtherapeutic efavirenz exposure was also linked to suboptimal antiviral efficacy.7,25
Variability in efavirenz plasma concentrations can occur due to different factors. Low concentrations of antiretrovirals can result from poor adherence and subsequently lead to virologic failure.26,27 The majority of children reported to be fully adherent by self-report. The fact that nearly half of the children with subtherapeutic efavirenz levels also had undetectable lamivudine concentrations confirms that self- or proxy-reporting of adherence is unreliable and therapeutic drug monitoring provides more accurate information on adherence.
We also explored the influence of CYP2B6 polymorphisms on efavirenz plasma concentrations. The 516G>T polymorphism frequency of 0.32 was comparable to other reports,17 and a significant correlation between these polymorphisms and mid-dosing interval efavirenz plasma concentrations was found. This is consistent with other studies, like the ACTG-trial (one-third African patients), a Zimbabwean study in adults, and with the study by ter Heine et al12,21,28 in a Dutch pediatric population including 89% black children, which showed that clearance was 30% lower for 516GT compared with 516GG patients. The Zimbabwean study recommended a dose reduction of 35% for 516TT patients.
We found that CYP2B6 785AA patients had significantly lower efavirenz levels compared with 785GG or 785AG patients. The 785A>G allele frequency was 0.33 in our study population, which was not earlier explored in pediatric populations but are comparable to findings by Mukonzo et al in Ugandan adults.20 The increase in CYP2B6 activity by the additional 785A>G polymorphism appears not to overcome the decrease in CYP2B6 expression or activity produced by the 516G>T variant.29,30 Therefore, these findings indicate that the 516G>T effect is more relevant. In addition, the 983T>C polymorphism was also associated with efavirenz plasma concentrations. The prevalence is comparable to African adult populations.16,21,31
CNS side effects were not significantly associated with mid-dosing interval efavirenz plasma concentrations comparing groups with efavirenz concentrations of <1.0, 1.0–4.0, >4.0 mg/L, which is in agreement with pediatric studies from different continents.15,17,25 Reported skin problems were mild and not typical for efavirenz-associated drug reactions, which usually occur in the first week after initiation.2
One of the limitations of this study was the small number of children. It was also not possible to observe the ingestion of efavirenz the evening before study visit to reduce the number of nonadherent participants, nor were we able to collect full pharmacokinetic curves for every individual as this is a more reliable estimation of pharmacokinetic parameters. However, many pediatric studies have this shortcoming. Sparse blood sampling in children is recommended, and hence mid-dosing concentration levels are frequently used.7,10,15 Currently, only one pharmacokinetic study of efavirenz in African children is published with full pharmacokinetic curves.9
This study presented a large variability of efavirenz mid-dosing interval concentrations in Rwandan HIV-1–infected children, when they were dosed according to the national guidelines. The World Health Organization recently moved to higher pediatric weight band–based doses, these recommendations should continue to be reviewed for risks of high efavirenz concentrations and potential toxicity problems. Both adherence and CYP2B6 genetic polymorphisms are major factors influencing variability in mid-dosing interval plasma concentrations. Significantly higher efavirenz levels are correlated to CYP2B6 516TT, 785GG, and 983CC individuals. Therefore, children with these CYP2B6 variants may have an increased chance of toxicity.
The authors would like to thank all the patients and families from all the centers (Kigali Teaching Hospital and Treatment and Research for AIDS Center) participating in this study and the team of the INTERACT Project, the Rwandan Ministry of Health, Utrecht University Children's Hospital/University Medical Centre, the Netherlands. Laboratory technicians from the Department of Pharmacy, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands are kindly acknowledged for the analysis of the plasma efavirenz and lamivudine concentrations; and technicians from the Department of Clinical Chemistry, Erasmus Medical Centre, Rotterdam, the Netherlands are thanked for the CYP2B6 polymorphism analyses.
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