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HIV-infected children are treated with tenofovir in combination with other, potentially interacting, antiretroviral agents. We report the pharmacokinetic parameters of tenofovir in combination with efavirenz, darunavir-ritonavir, or atazanavir-ritonavir in HIV-infected children. HIV-infected patients 8 to 18 years of age receiving a tenofovir (300 mg)-based regimen containing efavirenz (300 or 600 mg) once daily (group 1), darunavir (300 or 600 mg)-ritonavir (100 mg) twice daily (group 2), or atazanavir (150 to 400 mg)-ritonavir (100 mg) once daily (group 3) were enrolled. Plasma samples were collected over a 24-h time interval. The 90% confidence intervals (90% CI) of the geometric means for the area under the plasma concentration-time curve (AUC) and the minimum concentration of drug in serum (Cmin) of each antiretroviral were computed and checked for overlap with intervals bracketing published values obtained in adult or pediatric studies demonstrating safety and/or efficacy. Group 1 efavirenz plasma concentrations were observed to be higher in patients receiving fixed-dose combination tablets compared with subjects receiving the individual formulation. In group 2, tenofovir and darunavir exposure was observed to be lower than expected. In group 3, tenofovir and atazanavir administered concomitantly produced exposures similar to those published for adult patients. The 90% CI of AUC and Cmin for tenofovir overlapped the target range for all combinations. Informal comparisons of treatment groups did not indicate any advantage to any combination with respect to tenofovir exposure. Further study of exposures achieved with antiretrovirals administered in combination is warranted.
Tenofovir disoproxil fumarate (TDF) is a nucleotide reverse transcriptase inhibitor commonly prescribed for the treatment of HIV infection due to its convenience of once-daily dosing, availability in fixed-dosage combinations (FDCs), potency, and safety. Tenofovir disoproxil fumarate is available as a single 300-mg tablet, branded as Viread (6). It is also available as an FDC tablet with emtricitabine, branded as Truvada, and as an FDC tablet with emtricitabine and efavirenz, branded as Atripla (4, 5). Tenofovir has demonstrated potent antiviral efficacy with a low risk of the development of resistance when used in combination therapy (3, 13). In clinical trials, it was generally well tolerated with a low risk of lipoatrophy and lipid abnormalities compared with other nucleoside analogues. Although the risk of adverse renal effects is low, concerns of nephrotoxicity exist based upon preclinical data, case reports, and observational studies (15). Tenofovir is currently recommended as first-line therapy in combination with a nucleoside reverse transcriptase inhibitor and a non-nucleoside reverse transcriptase inhibitor or protease inhibitors in HIV-1-infected adults and adolescents (11). Recent approval in patients 12 to 18 years of age was based on a pharmacokinetic (PK) study in only 8 HIV-infected patients; thus, a critical need for tenofovir pharmacokinetic data in the pediatric and adolescent population still exists. Furthermore, HIV-infected children and adolescents are often treated with tenofovir in combination with other, potentially interacting, antiretroviral agents. Tenofovir exposure can be increased in the presence of protease inhibitors but is not affected by the non-nucleoside reverse transcriptase inhibitor efavirenz (2, 8, 16). However, availability of the FDC tablet, Atripla, may result in high plasma concentrations of tenofovir and/or efavirenz compared with concentrations seen in adults due to children receiving a larger weight-based dose. The primary objective of this study was to evaluate the pharmacokinetic parameters of tenofovir in combination with efavirenz, darunavir-ritonavir, or atazanavir-ritonavir in HIV-infected children and adolescents, with specific concern for identifying large, unexpected, and potentially clinically deleterious interactions.
International Maternal Pediatric and Adolescent AIDS Clinical Trials Group (IMPAACT) protocol 1058 evaluated the pharmacokinetics of tenofovir (300 mg)-based regimens containing efavirenz (300 or 600 mg) once a day (QD) (group 1), darunavir (300 or 600 mg)-ritonavir (100 mg) twice daily (BID) (group 2), or atazanavir (150 to 400 mg)-ritonavir (100 mg) QD (group 3). The study was designed as an observational pharmacokinetic assessment of antiretroviral combinations used clinically in the pediatric and adolescent population. The protocol did not prescribe therapy, did not provide medications, and did not dictate subject management. Eligible subjects included stable HIV-infected children ≥8 to 18 years of age with a body surface area (BSA) of ≥0.85 m2 receiving at least 2 weeks of the appropriate antiretroviral therapy before the pharmacokinetic evaluation was performed. Subjects in all groups were allowed to receive either Viread or Truvada, and subjects in group 1 were also allowed to receive Atripla as part of the tenofovir-containing regimen. No protease inhibitors were allowed in group 1. Subjects were excluded if they had any clinical or laboratory toxicity that was grade 2 or higher at screening (except for patients in arm 3 receiving atazanavir-ritonavir, who were excluded if total bilirubin was grade 3 or higher), had a hemoglobin level of <8.5 gm/dl, or were receiving a drug that might interact with the drugs of interest. A negative pregnancy test was required at the time of enrollment for females of childbearing capacity. The study was approved by the institutional review board at each site. Informed consent was obtained from each subject's parent or legal guardian, and assent was signed when appropriate. HIV-1 RNA level, CD4, and Tanner stage data were collected in order to characterize the population under study. Serum creatinine clearance was calculated using the Schwartz equation. Any adverse events occurring from study enrollment until completion of the pharmacokinetic analysis were reported on an expedited basis according to the Division of AIDS (DAIDS) table for grading the severity of adult and pediatric adverse events (http://rcc.tech-res-intl.com/). Sample sizes were selected to have power to identify situations in which tenofovir in combination with other antiretrovirals led to pharmacokinetic parameter values that were outside the interval (T/1.25, 1.25 · T), where T is the published target PK parameter value for tenofovir received alone. In the original protocol, eight strata of eight patients each were defined by details of dosage administered and patient BSA and were to have been recruited within the IMPAACT network. The protocol specified formal details for early termination in the case of underenrollment. Simulation was used to establish the adequacy of a stratum size of 8 to have good power (>95%) to identify cases where the 90% confidence intervals (90% CI) for AUC lay completely outside the interval (T/1.25, T · 1.25), with false-positive probability bounded at 7%.
Pharmacokinetic evaluations were performed in a general clinical research center or clinic setting. Antiretroviral drugs were administered in an open-label fashion, with or without food, at time zero. Blood samples were collected before dosing and 1, 2, 4, 6, 8, 12, and 24 h after dosing. Patients who took efavirenz before bedtime had to change to the morning for at least 1 week prior to the pharmacokinetic study visit. Plasma was transferred to a cryovial tube and stored frozen at or below −20°C until analysis. Plasma samples were analyzed for antiretroviral concentrations within 6 weeks from time of delivery to the laboratory. Prior to dosing on the day of the pharmacokinetic analysis, each subject was asked to empty his or her bladder. The total urine output was collected over the intervals of 0 to 4, 4 to 12, and 12 to 24 h after dosing. An aliquot of 5 ml of urine at each time interval was retained and stored at −20 or −70°C until shipment to the lab for analysis of tenofovir.
Efavirenz, darunavir, and atazanavir plasma concentrations were determined by an internally validated and externally cross-validated high-performance liquid chromatography (LC) with UV detection method. Following liquid-liquid extraction in 2 ml of tert-butyl methyl ether (TBME) at basic pH, samples were separated via reversed-phase liquid chromatography on a YMC C8 (4.6 by 100 mm, 3 μm) analytical column under isocratic conditions (55% 20 mM Na acetate, pH 4.88; 45% acetonitrile), with a total run time of 25 min. UV detection at 212 nm provided adequate sensitivity with minimal interference from endogenous matrix components. The assay is linear over a concentration range of 25 to 20,000 ng/ml. For efavirenz, the interassay precisions for low- and high-quality controls were 4.9 and 5.0%, while the accuracies were 2.7 and 7.9%, respectively. For atazanavir, the interassay precisions for low- and high-quality controls were 7.0 and 4.4%, while the accuracies were 11.6 and 3.4%, respectively. For darunavir, the interassay precisions for low- and high-quality controls were 4.9 and 4.7%, while the accuracies were 5.5 and 2.9%, respectively.
Plasma concentrations of tenofovir were determined by a validated LC-tandem mass spectrometry assay. Following a solid-phase extraction (SPE) procedure with Waters Oasis MCX 30-mg (1-cc) cartridges, analytes and their stable isotope internal standards were separated via reversed-phase liquid chromatography on a Waters Atlantis dC18 (2.1 by 100 mm, 3 μm) analytical column under isocratic conditions (90% 0.1% trifluoroacetic acid [TFA]; 10% acetonitrile), with a total run time of 4 min. Detection and quantitation of tenofovir were achieved by multireaction monitoring (MRM) using the following transitions for protonated daughters: [M+H]+ m/z 288.2 > 176.1. The assay is linear in the range of 10 to 5,000 ng/ml using a 200-μl aliquot of human plasma. The precisions for the standard curves (percent coefficient of variation [%CV]) ranged from 1.2 to 3.6%. Interassay precisions for low- and high-quality controls were 6.5 and 2.3% respectively, while the accuracies were −1.6 and −1.1% respectively.
For urine analysis of tenofovir, the SPE procedure was slightly different from the validated plasma SPE procedure due to the pH difference between plasma (Biological Specialties Corp., Colmar, PA) and urine and the extreme concentrations which accompanied the urine samples. A majority of samples required a 10:1 dilution of plasma to urine samples, essentially making it a spiked plasma solution. If the dilution did not allow the measured concentrations to fall within the predetermined curve (10 to 5,000 ng/ml) of the assay, samples were reanalyzed using a 20:1 dilution of plasma to urine samples. Otherwise, the assay was followed as instructed by the validated tenofovir and emtricitabine extraction for plasma with LC-tandem mass spectrometry.
Pharmacokinetic parameters of tenofovir, efavirenz, darunavir, and atazanavir were determined using noncompartmental methods (WinNonlin version 5.2; Pharsight Corp., Mountain View, CA). The tenofovir dose of 136 mg in a 300-mg dose of tenofovir disoproxil fumarate was used in all calculations of tenofovir parameters. The area under the plasma concentration-time curve (AUC24 for tenofovir, efavirenz, and atazanavir; AUC12 for darunavir) was calculated using the linear-up/log-down trapezoidal rule. Maximum plasma concentration (Cmax) and time to maximum concentration (Tmax) were taken directly from the observed concentration-time data. Oral clearance (CL/F) was calculated as dose/AUCτ. Terminal apparent distribution volume (Vz/F) was calculated as dose divided by the product of the elimination rate constant (λz) and the area under the concentration-time curve for the dosing interval (AUCτ). The elimination rate constant was determined by linear regression of the terminal elimination phase concentration-time points; elimination half-life (t1/2) was calculated as ln (2)/λz. For the calculations of urinary pharmacokinetics, the amount and percentage of a TDF dose recovered were calculated by summation of drug excretion (concentration times urine volume) and comparison to the administered dose. Renal clearance (CLrenal) was calculated by the matched Xu/AUC ratio, where Xu is the amount of drug excreted in the urine.
Statistical comparisons examined whether the 90% confidence intervals of the geometric mean (GM) AUC and Cmin for each antiretroviral were within 25% of those parameters observed in previous studies demonstrating safety and/or efficacy (7, 10, 12, 14, 16). The target ranges for tenofovir AUC and Cmin were 2.3 to 3.6 mg · h/liter and 0.05 to 0.08 mg/liter, respectively. The target ranges for efavirenz AUC and Cmin were 32 to 124 mg · h/liter and 0.9 to 3.6 mg/liter, respectively. The target ranges for darunavir AUC and Cmin were 51 to 80 mg · h/liter and 3.1 to 4.9 mg/liter, respectively. The target ranges for atazanavir AUC and Cmin were 15 to 75 mg · h/liter and 0.3 to 1.0 mg/liter, respectively. If the estimated 90% CI for AUC or Cmin fell entirely outside the target range, it was considered evidence that dosing in the particular combination should be reevaluated.
Fifty HIV-infected patients were enrolled into the study and intensive pharmacokinetic data were available from 47 patients: 17 in group 1, 13 in group 2, and 17 in group 3. Three patients did not participate in the pharmacokinetic analysis. Demographic characteristics of the study population are shown in Table 1. Median (range) serum creatinine and creatinine clearance were 0.6 (0.3 to 1.1) mg/dl and 154 (74.8 to 267.6) ml/min/1.73 m2, respectively, suggesting that no patients were experiencing renal dysfunction.
All patients received tenofovir 300 mg once daily. Figure 1 illustrates the mean and standard error of the mean tenofovir concentration-time curves for all groups. In group 1, one patient received efavirenz (200 mg), four patients received efavirenz (400 mg), and 12 patients received efavirenz (600 mg) once daily. In group 2, two patients received darunavir (300 mg) and 11 patients received darunavir (600 mg) in combination with ritonavir (100 mg) twice daily. In group 3, three patients received atazanavir (150 mg) and 14 patients received atazanavir (300 mg) in combination with ritonavir (100 mg) once daily. Table 2 describes the median (range) tenofovir, efavirenz, darunavir, and atazanavir pharmacokinetics. In group 1, GMs (90% CI) for tenofovir AUC and Cmin were 2.9 (2.5 to 3.4) mg · h/liter and 0.07 (0.05 to 0.09) mg/liter, with the 90% CI Cmin slightly higher than the upper target range of 0.08 mg/liter. GMs (90% CI) for efavirenz AUC and Cmin were 88.4 (65 to 120) mg · h/liter and 2.7 (1.8 to 4.0) mg/liter, respectively, with the 90% CI Cmin slightly higher than the upper target range of 3.6 mg/liter. In group 2, GMs (90% CI) for tenofovir AUC and Cmin were 3.0 (2.5 to 3.6) mg · h/liter and 0.06 (0.05 to 0.08) mg/liter, respectively. GMs (90% CI) for darunavir AUC and Cmin were 60.3 (48.7 to 74.7) mg · h/liter and 2.7 (2.0 to 3.6) mg/liter, respectively. Darunavir 90% CI for AUC and Cmin were lower than the target range of 51 mg · h/liter for AUC and 3.1 mg/liter for Cmin. In group 3, GMs (90% CI) for tenofovir AUC and Cmin were 3.6 (3.1 to 4.2) mg · h/liter and 0.07 (0.06 to 0.09) mg/liter. Tenofovir 90% CI for AUC and Cmin were slightly higher than the target range of 3.6 mg · h/liter for AUC and 0.08 mg/liter for Cmin. GMs (90% CI) for atazanavir AUC and Cmin were 36.9 (33 to 42) mg · h/liter and 0.5 (0.4 to 0.7) mg/liter, respectively.
Urine collected over the 24-hour dosing period, to determine the renal clearance of tenofovir, was available from only 18 of the 47 patients: 7 patients in group 1, 5 patients in group 2, and 6 patients in group 3. The mean ± standard deviation (SD) amount and percentage of tenofovir recovered in the urine from 18 patients were 29.98 ± 15.0 mg and 10% ± 5%, respectively. A trend toward a low tenofovir renal clearance was seen for patients in group 3 compared with groups 1 and 2. However, the sample size of patients in which tenofovir renal clearance could be determined in each group was too small for statistical analyses. No adverse events or toxicities related to study medications were reported during the study.
IMPAACT P1058 was designed to evaluate possible interactions between tenofovir and other antiretrovirals including efavirenz, atazanavir, and darunavir in HIV-infected children. In the presence of efavirenz, only the GM (90% CI) for tenofovir Cmin was slightly above the target upper limit of 0.08 mg/liter. The Cmin was higher than the upper limit for only 3 of 17 subjects, 2 of whom weighed approximately 33 kg and were the smallest subjects enrolled. The third subject had extremely high concentrations of tenofovir and efavirenz, suggesting that the dose may have been doubled before the pharmacokinetic visit. In contrast, the GM (90% CI) for efavirenz Cmin was above the target upper limit of 3.6 mg/liter. Although the 90% CI for efavirenz AUC was within the target range, efavirenz exposure was high in our patient population. Eighty-eight percent of patients (n = 15) received efavirenz according to weight-based dosing approved by the Food and Drug Administration. However, over half of these patients had an elevated efavirenz Cmin and AUC. Six patients with high efavirenz exposure were receiving the FDC Atripla tablet, which may have altered drug absorption in this patient population. A controlled two-way crossover study comparing the pharmacokinetics of efavirenz, tenofovir, and emtricitabine from the Atripla formulation versus the individual formulations in the pediatric and adolescent population should be performed to answer this question.
The GMs (90% CI) for tenofovir AUC and Cmin in the presence of darunavir-ritonavir were within the target range. The GMs (90% CI) for darunavir AUC and Cmin were below the target range. In healthy adults, tenofovir AUC and Cmin increase 22% and 37%, respectively, in the presence of darunavir-ritonavir whereas darunavir AUC and Cmin increase 21% and 24% respectively (12). According to these data, we expected tenofovir and darunavir Cmin and AUC to be higher than the upper limit of the target range. However, tenofovir exposure was adequate in our patient population and darunavir exposure was low. Current dosing recommendations for darunavir-ritonavir in children 6 to <18 years of age is darunavir (375 mg)-ritonavir (50 mg) BID for patients weighing ≥20 to <30 kg, darunavir (450 mg)-ritonavir (60 mg) BID for patients weighing ≥30 to <40 kg, and darunavir (600 mg)-ritonavir (100 mg) BID in patients weighing ≥40 kg. Patients in our study received darunavir (300 or 600 mg) and ritonavir (100 mg) twice daily since the 75-mg darunavir tablet was not commercially available during the course of the study. Darunavir Cmin was low for 54% of patients (n = 7), five of which were dosed according to guidelines. Two patients with low darunavir exposure were dosed lower than the weight-based recommendation. The lack of increase in tenofovir exposure seen in our patients may have resulted from low darunavir exposure. Our data suggest that higher doses of darunavir may be necessary in pediatric patients receiving tenofovir; however, the small sample size of this group warrants a larger study to confirm our findings.
In healthy adults, tenofovir AUC and Cmin increase approximately 37% and 29%, respectively, when tenofovir is given with atazanavir and ritonavir (1). In contrast, we found that the GMs (90% CI) for tenofovir AUC and Cmin in the presence of atazanavir-ritonavir were only slightly above the target upper limit and the median tenofovir AUC and Cmax were similar to what is described for patients not receiving concurrent atazanavir and ritonavir (6). However, tenofovir AUC and Cmin were higher than those reported in 22 HIV-infected young adults ≥18 to <25 years of age who received concomitant atazanavir and ritonavir (9). The authors suggested that the lower tenofovir exposure seen in their adolescent population may be due to faster tenofovir clearance (49.2 liters/h). The lower clearance (40.7 liters/h) observed in our patient population may explain the slightly higher tenofovir AUC in our patients. The atazanavir exposure in that study was similar to the results found in our study, suggesting that the atazanavir dosage used in the current study is appropriate.
Tenofovir pharmacokinetics did not differ in group 1 compared with group 2 or group 3 as illustrated in Fig. 1. This finding was unexpected, as many protease inhibitors modestly alter tenofovir plasma concentrations by −15% to +37% in adults (17). In group 2, it is likely that low darunavir concentrations did not increase the tenofovir concentrations significantly to detect an interaction. In group 3, the 90% CI for tenofovir AUC and Cmin were slightly above the target range, but this difference was not significant when the parameters were compared using a nonparametric statistical analysis. It is possible that a more noticeable difference may be found with a larger sample size. It has been debated whether the increase in tenofovir plasma level in the presence of protease inhibitors is due to a decrease in tenofovir renal clearance or an increase in tenofovir oral absorption; therefore, we attempted to collect urine over the dosing interval to determine tenofovir renal clearance. However, this collection was not possible in every patient due to difficulties of collecting urine overnight outside the clinic setting or due to patients' voiding without notifying clinic personnel. Based upon our limited data, the median tenofovir renal clearance varied with the different concomitant antiretrovirals. While patients receiving efavirenz and darunavir-ritonavir had median tenofovir renal clearance (117 ml/min/m2 and 120 ml/min/m2, respectively) that was comparable to that reported in 18 HIV-infected children receiving a median tenofovir dose of 208 mg/m2 (132 ml/min/m2) (7), tenofovir renal clearance was decreased (75.3 ml/min/m2) in our patients receiving atazanavir-ritonavir. These results should be confirmed in a larger study.
In conclusion, none of the 90% CI AUC and Cmin values for the drugs assayed were entirely outside the target range, meaning no drugs met the protocol-defined criteria for dosing reevaluation. Efavirenz exposure was high, although the majority of patients received the FDA-approved dose. Tenofovir and darunavir plasma concentrations were lower than expected when used in combination, suggesting that in this patient population, tenofovir exposure should be evaluated in the presence of higher doses of darunavir-ritonavir. Tenofovir and atazanavir administered concomitantly produced atazanavir exposure similar to that seen in adults without the expected increase in tenofovir exposure. However, there was a trend toward a decrease in tenofovir renal clearance in those patients receiving atazanavir-ritonavir compared with those patients receiving efavirenz or darunavir-ritonavir. Taken together, these data suggest that administering tenofovir (300 mg) once daily to HIV-infected patients 8 to 18 years of age in combination with efavirenz, darunavir-ritonavir, or atazanavir-ritonavir should provide exposure levels similar to those seen in HIV-infected adults. However, if individual patients experience adverse side effects or reduced clinical outcomes while taking these agents in combination, clinicians may consider monitoring systemic exposure of the antiretrovirals.
The project described was supported by grant number U01AI068632 from the National Institute of Allergy and Infectious Diseases and contract number N01-DK-9-001/HHSN267200800001C from the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD).
Statistical support was provided by the Statistical and Data Analysis Center at Harvard School of Public Health, under National Institute of Allergy and Infectious Diseases cooperative agreement #1 U01 AI068616 with the IMPAACT Group. Support for P.J.-P. was provided with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract no. HHSN272200800014C.
The content of this report is solely our responsibility and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health.
Published ahead of print on 13 June 2011.