The British HIV Association treatment guidelines for initial therapy for HIV-1 infection recommend the use of 2 nucleoside reverse-transcriptase inhibitors (NRTI) with a non-NRTI.1–3 Furthermore, the combination of tenofovir (TFV), emtricitabine (FTC), and efavirenz (EFV) has been shown to be superior in infected patients with high viral loads and is recommended as the preferred first-line regimen in the absence of primary baseline resistance.4,5
TFV, FTC, and EFV are today contained in a single tablet (Atripla) which is commonly prescribed to HIV-infected individuals as a single daily dose.
Interestingly, all 3 components of Atripla are characterized by sustained half-lives. In patients with virologic suppression, it has been shown that reducing dosing frequency to 5 days on therapy followed by 2 days of therapy resulted in similar virologic response rates through week 24, as remaining on standard daily dosing.1,6 This suggests that antiretrovirals with long half-lives, which lead to the presence of drug at a level high enough to exert antiviral activity, may allow for reduced dose frequency, increasing adherence and limiting toxicity and cost.
Furthermore, 2 of the components of Atripla, TFV and FTC, have been studied as HIV chemoprophylaxis, commonly referred to as pre-exposure prophylaxis (PrEP).7,8 The ideal PrEP dosing schedule of TFV/FTC is however still unclear. Importantly, PrEP agents should have limited toxicity and exhibit pharmacokinetic (PK) characteristics that support once daily or less frequent dosing.9,10
Tenofovir (administered as tenofovir disoproxil fumarate [TDF] and transformed to TFV during the absorption phase) and FTC are pro-drugs that require intracellular (IC) phosphorylation to their active anabolites. Whereas TFV is a monophosphate analogue and requires 2 phosphorylation steps to TFV-diphosphate (TFV-DP), FTC-triphosphate (FTC-TP) is formed by 3 endogenous enzymatic steps.5,11–13 TFV-DP and FTC-TP concentrations persist within cells and are characterized by prolonged half-lives (150 and 39 hours, respectively).5,13–15
Importantly, drug concentration values may not be sufficient to interpret ideal exposure to IC NRTI, as their antiviral activity is not only dependent on the levels of the DP/TP formed but also on the endogenous concentrations of the deoxyribonucleotide triphosphate (dNTPs) that the NRTI-TP competes with for incorporation into the proviral DNA.1,3
In this study, we have assessed the “pharmacokinetic forgiveness” of IC TFV-DP, FTC-TP, and plasma EFV over 228 hours (9.5 days) after drug intake cessation in HIV-negative healthy volunteers.
Male and nonpregnant nonlactating female participants were eligible for enrollment if they provided written informed consent and met the following criteria: age between 18 and 65 and body mass index 18–35 kg/m.2
Participants were excluded if they had any significant acute or chronic medical illness; abnormal physical examination, ECG, or clinical laboratory determinations; positive screen to HIV, hepatitis B, or C; current or recent (within 3 months) gastrointestinal disease; clinically relevant alcohol or drug use that the investigator felt would adversely affect compliance with trial procedures; exposure to any investigational drug or placebo within 3 months of first dose of study drug; use of any other drugs, including over-the-counter medications and herbal preparations, within 2 weeks before the first dose of study drug; and previous allergy to any of the constituents of the pharmaceuticals administered during the trial.
This was a 24-day, open-label, single-treatment arm PK study performed at the Pharmacokinetic Unit of the St. Stephen's Centre, Chelsea and Westminster Hospital, London. The study protocol was reviewed and approved by the Institute of Child Health/Great Ormond Street Hospital Research Ethics Committee, UK. All participants provided written informed consent, and the trial was conducted in accordance with the Declaration of Helsinki and applicable regulatory requirements (EudraCT 2009-018055-16).
At screening, participants had a clinical assessment and routine laboratory investigations performed. The safety and tolerability of study medications were evaluated throughout the study using the NIAID Division of AIDS table for grading the severity of adult and pediatric adverse events to characterize abnormal findings (published December 2004), vital signs, physical examinations, and clinical laboratory investigations.
After successful screening, participants were administered Atripla 1 tablet once daily in the evening for 14 days. Blood samples for PK assessment (plasma and IC) were taken before the final dose in the evening of day 14 (within 10 minutes before dose) and 2, 4, 8, and 12 hours postdose while the participants were admitted to the unit. After the 12-hour sample, participants were discharged from the unit, returning for samples over the ensuing 10 days at 24, 36, 48, 60, 84, 108, 132, 156, 180, 204, and 228 hours after the final dose.
Plasma Collection for Tenofovir, Emtricitabine, and Efavirenz Analysis
Blood samples for the determination of plasma PK measurements were collected into two 6 mL Vacutainer blood collection tubes (Lithium heparin) per time point.
Immediately after collection, the blood tube was inverted several times and then kept on ice or refrigerated until centrifugation. Within 30 minutes of blood collection, each blood sample was centrifuged for 10 minutes at 1200 g at 4°C. Plasma was then aliquoted equally into three 2.0 mL tubes (Sarstedt Germany) and stored at −20°C. Samples were shipped on dry ice to the Good Clinical Laboratory Practice-accredited (Good Clinical Laboratory Practice) Liverpool Biomedical Research Centre Bioanalytical Facility for analysis by liquid chromatography–tandem mass spectrometry (MS/MS).
Peripheral Blood Mononuclear Cell Isolation for TFV-DP and FTC-TP Analysis
Blood samples for the determination of IC PK measurements in PBMCs were collected in two 8-mL cell preparation tubes (CPT; Becton Dickinson Vacutainer, New Jersey) per time point. Tubes were gently mixed by inversion until centrifugation at room temperature (18–25°C) in a horizontal rotor (swing out bucket) centrifuge for 20 minutes at 1600 Relative Centrifugal Force. Centrifugation was performed within 60 minutes of venesection to maximize cell yield. After cell counting (Digital Bio Adam Microchip Automatic Cell Counter; NanoEnTek, Inc., Seoul, Korea) and after centrifugation for 15 minutes at 400 Relative Centrifugal Force to pellet the cells, the supernatant was removed and the cell pellet was then resuspended and lysed in 1.0 mL of ice-cold 70% methanol solution and agitated by vortex for 3–5 minutes before being stored at −80°C before liquid chromatography–MS/MSanalysis.
Quantification of Tenofovir and Emtricitabine from Plasma
Plasma samples (300 μL) were prepared in formic acid (300 μL; 0.02%) containing an internal standard, 2-chloroadenosine (Cl-A; 5 μg/mL; 20 μL), and loaded onto a 100-mg SPE BondElut C18 column (Agilent Technologies, UK Ltd., Berkshire, UK). Columns were conditioned with methanol (1 mL) and 0.02% formic acid (1 mL; pH 3). Samples (400 μL) were loaded onto the column and rinsed with 0.02% formic acid (200 μL). Analytes were eluted with methanol (100 μL), evaporated to dryness, reconstituted in 100 μL of mobile phase, and injected (10 μL) onto the high-performance liquid chromatography (HPLC) column.
Chromatography was performed on a Synergi polar C18 column (4 μm: 150 mm × 2.0 mm; Phenomenex, Cheshire, UK). Mobile phase A consisted of 0.1% formic acid in water and mobile phase B, 0.1% formic acid in acetonitrile (ACN). Initial conditions consisted of 99% mobile phase A, increasing in organic content to 40% B in 0.7 minutes maintained over 1.3 minutes and equilibrated back to the initial conditions over a total run time of 6 minutes. The flow rate was 400 μL/minute. The triple quadrupole mass spectrometer (TSQ Quantum Access; Thermo Electron Corporation, Hemel Hempstead, UK) was operated in positive ionization mode, and detection and quantification was performed using multiple reaction monitoring.
The assay was validated over a calibration range of 0.52–996 ng/mL (TFV) and 0.47–5368 ng/mL (FTC). Inter-day precision (coefficient of variation [CV%]) based on quality control (QC) samples was between 9%–12.2% (TFV) and 4.6%–6.9% (FTC), and accuracy (% bias) was 97.5%–100.8% (TFV) and 107%–114.2% (FTC). The percentage recovery for both analytes at the low medium and high levels were consistent, precise, and reproducible.
Quantification of Efavirenz from Plasma
Plasma EFV concentrations were quantified by a validated protein precipitation extraction method coupled with reverse phase HPLC–MS/MS, as previously described.4
The assay was validated over a calibration range of 8.58–10,203.9 ng/mL. Inter-day precision (CV%) based on quality control (QC) samples was between 2.9% and 5.3% and accuracy (% bias) was between 93.2% and 105.1%. The percentage recovery at the low medium and high levels were consistent, precise, and reproducible.
Quantification of IC Tenofovir-Diphosphate (TFV-DP) and Emtricitabine-Triphosphate (FTC-TP)
IC TFV-DP and FTC-TP concentrations were quantified by a validated protein precipitation method coupled with weak anion exchange HPLC–MS/MS.
ACN (1 mL) was added to each of the samples of IC lysate (1 mL in 70% methanol). The samples were vortexed and centrifuged (13,000 g, 6 minutes). The sample supernatant was transferred into glass tubes and internal standard, 2-chloroadenosine 5′-triphosphate (Cl-ATP; 4 μg/mL; 20 μL), was added. Samples were evaporated to dryness and reconstituted in 150 μL of ammonium formate (5 mM), vortexed, and injected (20 μL) onto the HPLC column.
Chromatography was performed on a BioBasic AX column (5 μm: 50 mm × 2.1 mm; Thermo Scientific, Hertfordshire, UK) with a pH gradient. Mobile phase A consisted of 10 mM ammonium acetate in ACN/water (30:70, vol/vol), pH 6, and mobile phase B consisted of 1 mM ammonium acetate in ANC/water (30:70, vol/vol), pH 10.5. Initial conditions consisted of 90% mobile phase A, increasing in organic content to 50% B in 0.51 minutes maintained over 1.25 minutes, then increased again to 100% B, and maintained for 4.75 minutes then equilibrated back to the initial conditions over a total run time of 12 minutes. The flow rate was 250 μL/min. The triple quadrupole mass spectrometer (TSQ Quantum Access MAX; Thermo Electron Corporation) was operated in positive ionization mode, and detection and quantification was performed using selective reaction monitoring.
The assay was validated over a calibration range of 0.059–10.91 ng/mL (TFV-DP) and 0.38–103.47 ng/mL (FTC-TP). Inter-day precision (CV%) based on quality control (QC) samples was between 6.3%–11% (TFV-DP) and 6%–18.6% (FTC-TP) and accuracy (% bias) was 97.5%–100.8% (TFV-DP) and 98%–100.3% (FTC-TP). The percentage recovery for both analytes at the low medium and high levels were consistent, precise, and reproducible.
PK and Statistical Analysis
The calculated PK parameters for plasma TFV, FTC, EFV, and IC TFV-DP and FTC-TP were the concentration measured 24 hours after the observed dose (C24h), the maximum observed concentration (Cmax), and the area under the concentration–time curve (AUC) from 0 to t (in hours).
The half-life was determined from the elimination phase within the normal dosing interval of 0–24 hours and as a terminal elimination half-life to the last measurable concentration within. All PK parameters were calculated using actual blood sampling times and noncompartmental modeling techniques (WinNonlin Phoenix, version 6.1; Pharsight Corp, Mountain View, CA). Descriptive statistics, including geometric mean (GM) and 90% confidence intervals (CI), were calculated for plasma TFV, FTC, EFV, and IC TFV-DP and FTC-TP PK parameters. Interindividual variability in drug PK parameters was expressed as a coefficient of variation [CV (SD/mean) × 100].
Demographic and Clinical Characteristics
Sixteen participants completed the study. Median (range) age, weight, and body mass index were 33 (22–58) years, 80 (54–109) kg, and 25 (20–34) kg/m2, respectively. Six participants were women, 12 were white, 2 were black, 1 of Asian origin, and 1 defined himself as other. The study drugs were well tolerated and no grade 3 or 4 adverse events were reported.
Tenofovir and Emtricitabine Plasma PK
Plasma TFV and FTC PK parameters are illustrated in Table 1 and concentration–time curves in Figure 1A, B), respectively. GM and 90% CI plasma terminal elimination half-life to 228 hours of TFV was 31 hours (24–45 hours) and longer than the 0–24 hours half-life (14 hours). GM and 90% CI plasma terminal elimination half-life to 228 hours of FTC was 37 hours (33–43 hours) and longer than the 0–24 hours half-life (6 hours). There was a marked interindividual variability in both plasma TFV and FTC PK parameters (range, 25%–56%).
Efavirenz Plasma PK
Plasma EFV PK parameters are illustrated in Table 1 and concentration–time curves in Figure 1C. GM and 90% CI plasma terminal elimination half-lives to 24 hours was 28 hours (21–47 hours), to 48 hours was 51 hours (43–74 hours), and to 228 hours was 92 hours (84–108 hours).
Five of 16 participants had concentrations below the suggested MEC of 1000 ng/mL 48 hours postdose and 50% maintained concentrations greater than the suggested MEC 84 hours postdose. There was high interindividual variability in EFV PK parameters (range, 40%–77%).
Tenofovir-DP and Emtricitabine-TP IC PK
IC TFV-DP and FTC-TP PK parameters are illustrated in Table 1 and concentration–time curves in Figure 2A, B, respectively.
TFV-DP GM and 90% CI IC terminal half-life to 228 or the last measured concentration was 164 hours (152–190 hours).
FTC-TP GM and 90% CI IC terminal half-life to 228 hours of FTC-TP was 39 hours (36–45 hours). There was marked interindividual variability in TFV-DP and FTC-TP PK parameters (range, 64%–79%).
We report here the PK of plasma TFV, FTC, and IC TFV-DP and FTC-DP, after the administration of TDF/FTC/EFV (300/200/600 mg once daily, respectively) over 228 hours, after drug intake cessation in 16 healthy volunteers, both men and women, as we aimed to investigate the decay of individual antiretroviral concentrations when administered in combination.
Our data fully characterize the PK forgiveness of TFV/FTC/EFV, as no other previous study measured the decay of the combination of drug concentrations for up to 10 days after drug intake cessation.
As the clinical pharmacology of TFV and FTC depends on their IC concentrations, our study showed that they are both characterized by prolonged terminal half-lives (164 hours/6.8 days and 39 hours/1.6 days, respectively). Therefore, when combined with EFV, which was shown to have a terminal half-life of 92 hours/3.8 days, they indeed provide a PK forgiving regimen. However, information on optimal target concentrations of phosphorylated active NRTI anabolites for HIV treatment and prevention is lacking, and studies investigating the pharmacology of these agents in depth are warranted.
Importantly, EFV concentrations showed a wide interindividual variability throughout the whole decay phase. This is likely to be because of the CYP2B6 polymorphism, and slow metabolizers are likely to show longer EFV half-lives.6
Furthermore, the GM EFV terminal half-life reported here is slightly longer than previously shown during drug development7,8 and in accordance with what was measured in HIV-infected individuals stopping the drug.9,10
In the context of treatment, this could be beneficial in the case of suboptimal adherence, especially in complex clinical situations such as antiretroviral treatment initiation at high viral loads. It may also explain the superiority of the studied combination that has emerged from prospective randomized clinical trials.5
In patients with chronic diseases, poor adherence to medications has been shown to be common.2,16 When adherence to antiretroviral treatment is reduced, drug concentrations are not sufficient to suppress HIV replication and to control plasma viral load. Therefore, poor adherence to antiretrovirals accelerates the development of drug-resistant HIV.5,17 Although it is clear that omitting numerous drug doses leads to these consequences, it is unclear whether delayed dosing or omitting a limited number of doses can affect drug efficacy for long term. Therefore, identifying antiretroviral agent persistence is important to increase knowledge and ensure prolonged viral load suppression. PK forgiveness is also very important from the perspective of HIV prevention, as ideal PrEP agents should be characterized by PK attributes that allow for once daily or less frequent dosing.1,7,9
Importantly, the interindividual variability of IC TFV-DP and FTC-TP concentrations was wide, increasing the challenge of establishing and maintaining optimal drug exposure. Reasons for this high variability could be because of differences in the activity of key drug transporters (ie, polymorphisms of the drug transporter genes) responsible for TFV and FTC IC intake or to differences in the activity of the IC kinases responsible for drug phosphorylation.7,18 To understand optimal dosing schedules of TDF/FTC as PrEP, however, further data are required to elucidate their ability to accumulate and persist in tissues (ie, vaginal or rectal compartments).
One of the limitations of this study is the lack of knowledge of target triphosphate/diphosphate concentrations. Although decay in concentrations of EFV can be related to the suggested minimum effective concentrations (1000 ng/mL) used to interpret therapeutic drug monitoring (TDM) results in clinical, practice, where this test is available,9,19 concentration cut-off values of active IC phosphate anabolites have not been established.
A further limitation, when looking at the studied triple combination in the context of treatment, is that HIV-negative volunteers rather than HIV-infected patients were enrolled to avoid withholding antiretroviral drug required for a clinical indication. Finally, a third limitation is the time of drug intake and the achievement of steady state: the ideal time to reach steady state might be longer than 14 days; however, data are available showing that TFV-DP and FTC-TP reach an IC accumulation plateau after few doses.11–13,20 Whether the presence of HIV infection has an impact on antiretroviral drug exposure is debatable;5,13–15,21 however, for some antiretrovirals, the latter seem to differ between individuals with and without the infection.
Nonetheless, in both areas of long life treatment and HIV infection prevention, it is reassuring to have data describing maintenance of “high” drug concentrations that allow for missed doses, even if further information is required to understand how many doses can be missed to avoid therapeutic failure or to ensure protection from HIV. In conclusion, our study has explored the PK forgiveness of the combination TDF/FTC/EFV and measured their terminal half-lives, which showed to be prolonged.
The authors thank the St Stephen's AIDS Trust Research Team for their hard work and the volunteers who took part in the study.
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