Adams, Jessica L. PharmD, BCPS*; Sykes, Craig MSc*; Menezes, Prema PhD†; Prince, Heather M.A. PA-C†; Patterson, Kristine B. MD†; Fransen, Katrien MSc‡; Crucitti, Tania PharmD, Clin Biol, PhD‡; De Baetselier, Irith MSc‡; Van Damme, Lut MD, PhD§; Kashuba, Angela D.M. BScPhm, PharmD, DABCP*,†
Recent studies of the fixed dose combination of oral tenofovir disoproxyl fumarate (TDF) and emtricitabine (FTC), marketed as Truvada, for the prevention of HIV infection have had discrepant results. The iPrEX study of daily TDF/FTC in men who have sex with men demonstrated 44% protective efficacy overall and 73% protective efficacy in subjects reporting >90% adherence.1 The Partners PrEP study arm of daily TDF/FTC in the uninfected partners of serodiscordant heterosexual couples demonstrated 75% protective efficacy with 81% adherence as measured by detectable plasma drug concentrations.2,3 Additionally, the TDF2 study of TDF/FTC in heterosexual men and women, demonstrated 62% efficacy with 80% adherence as measured by plasma drug concentrations.4 In contrast, the FEM-PrEP study, which evaluated the efficacy of daily TDF/FTC in preventing HIV infection in high-risk heterosexual women, was stopped early because of futility.5 Although study subjects reported >90% adherence to study drug therapy, tenofovir (TFV) and FTC were detected in the plasma of only 21% of infected women at the visit in which she first had evidence of infection and only 26% at the last visit with no evidence of infection.5 Only 37% and 35% of uninfected controls had detectable drug concentrations at these same visits.
These studies demonstrate that consistent adherence is critical for efficacy and may particularly be for women. However, it is difficult to objectively measure adherence in real-time during clinical trials, and self-reported adherence often overestimates true adherence.6,7 Concentrations of TFV and FTC in plasma, do not accumulate significantly over time (plasma half-lives are 17 and 10 hours, respectively) and thus cannot be used to differentiate between single, intermittent, and consistent dosing8 but can be used as measures of short-term adherence.
Conversely, TFV-diphosphate (TFV-DP) and FTC-triphosphate (FTC-TP) in peripheral blood mononuclear cells (PBMCs) have half-lives of 6.25 and 1.6 days, respectively.9,10 Therefore, their accumulation has been considered a better measure of adherence, although no algorithms yet exist for interpretation of the results, and the isolation of PBMCs is both expensive and difficult. However, recently Rower et al11 demonstrated that the concentration of TFV-DP in the red blood cells (RBCs) of 5 subjects correlated with TFV-DP concentration in PBMCs. Therefore, RBCs may be an alternate vehicle for measuring these compounds with long half-lives.
During the FEM-PrEP study, investigators collected monthly samples of “upper layer packed cells” (ULPCs) for future virology investigations. The layer of blood cells that remained after centrifugation and plasma removal from a 10-mL EDTA tube is estimated to contain approximately 106–7 PBMCs and 109–10 RBCs.12 To determine whether these samples could be used to measure TFV-DP and FTC-TP as a surrogate for longer-term adherence, we conducted an investigation to determine the intra- and interindividual variability of TFV-DP and FTC-TP in ULPC samples, to evaluate the correlation between TFV-DP and FTC-TP concentrations in ULPC and PBMC samples, and to determine the stability of the ULPC samples as collected and processed at the FEM-PrEP study sites.
Study Design and Population
Ten HIV-infected adults taking 200 mg of FTC and 300 mg of TDF daily as a part of their antiretroviral (ARV) regimen (either as Truvada or in Atripla) were recruited from the UNC Healthcare Infectious Diseases Clinic in Chapel Hill, NC. The study was conducted under a general blood draw protocol, approved by the UNC Biomedical Institutional Review Board, and all study activities were carried out in accordance with the ethical standards of the Helsinki Declaration of 1975, as revised in 2000. Subjects with an undetectable viral load for 6 months and provider-endorsed adherence were specifically chosen for recruitment.
Sample Collection and Processing
Subjects were admitted to the North Carolina Translational and Clinical Sciences (NC TraCS) Institute Clinical and Translational Research Center (CTRC). Blood was collected in 10-mL K2EDTA tubes (BD Diagnostics, Franklin Lakes, NJ) and 8-mL CPT tubes (BD Diagnostics, Franklin Lakes, NJ) at 0, 1, 2, 4, and 6 hours after a witnessed dose or 12, 13, 14, 16, and 18 hours after a subject verified dose taken the night before admission. Subjects took their doses of TDF/FTC within an hour of the time that they usually take their home dose.
Within 4 hours after collection, K2EDTA tubes stored at room temperature were processed by centrifugation at 800g at 21°C for 10 minutes, as was done at the urban FEM-PrEP study sites. The resultant plasma was centrifuged again and then aliquoted into labeled cryovials, and stored at −80°C. The total blood cells remaining were counted by trypan blue exclusion on a Countess Cell Counter (Invitrogen, Grand Island, NY) and then frozen at −80°C. To simulate conditions at a rural FEM-PrEP study site, at one time point per subject, the ULPC samples were split between 2 cryovials: one was immediately processed and cells stored at −80°C and the other refrigerated for 14 hours before processing and storage at −80°C.
Within 2 hours after collection, CPT tubes were centrifuged at 1300g for 30 minutes at room temperature with the brake off. The resulting PBMC-containing upper layer was removed, combined with 2 mL of cold phosphate buffered saline used to wash cells off the gel layer of the CPT tube, and centrifuged at 350g for 10 minutes at 4°C. After discarding the supernatant, the cell pellet was resuspended in RBC lysis buffer and allowed to sit at room temperature for 2 minutes and then 10 mL of cold phosphate buffered saline was added and the cells were again centrifuged at 300g for 5 minutes at 4°C. Cells were counted using Trypan blue exclusion and a Countess Cell Counter. After counting, cells were lysed with 300 μL of 70:30 of methanol:water solution and placed on ice for 15 minutes before storage at −80°C until analysis.
Because the majority of the PBMCs would be expected to reside within the top portion of the ULPC, further analysis was performed to determine if TFV-DP and FTC-TP concentrations would differ depending on where the sample was obtained. Therefore, aliquots of 0.5 mL were taken from the top and bottom of an additional 10 mL EDTA tube from a subject taking TDF/FTC.
The direct determination of TFV-DP and FTC-TP concentrations was performed in PBMC samples by protein precipitation followed by liquid chromatography/mass spectrometry analysis. Calibration standards and quality control samples were prepared in PBMC lysate (70:30 of methanol:water with 1 × 106 cells/mL of lysate). The stored PBMC samples were centrifuged and the methanolic extracts subjected to protein precipitation with 1:1 of methanol:1 mM ammonium phosphate solution containing the isotopically labeled internal standard 13C TFV-DP (Moravek Biochemicals, Brea, CA). Samples were evaporated to dryness under nitrogen and reconstituted with 1 mM of ammonium phosphate. Using a Shimadzu high performance liquid chromatography system (Shimadzu, Columbia, MD), the analytes were eluted from a Thermo Scientific BioBasic AX (50 × 2.1-mm 5-μm particle size) column (Thermo Fisher Scientific, Waltham, MA) with 70:30 of 10 mM ammonium acetate:acetonitrile (pH 5.55) and 75:25 of 10 mM ammonium acetate:acetonitrile (pH 9.45) as the mobile phases. An API-5000 triple quadrupole mass spectrometer (AB Sciex, Foster City, CA) operated in positive ion electrospray mode was used to detect analytes. Data were collected using AB Sciex Analyst Software, with m/z transitions of 448.0/270.0 (TFV-DP) and 488.0/130.1 (FTC-TP). The dynamic range of the assay was 1–2500 ng/mL lysate; raw concentration values were normalized for cells counts (cells per milliliter lysate) and molecular mass of the analyte, with final concentration values reported as femtomoles per 106 cells. All calibrators and quality control samples were within 15% of the nominal value for both within-day and between-day runs. Within-day and between-day precision was <15%. Recoveries of TFV-DP, FTC-TP, and 13C TFV-DP seen with this methodology were all approximately 100%.
The extraction of TFV-DP and FTC-TP from ULPC samples was performed in an ice bath to maintain analyte stability. Calibration standards and quality control samples were prepared in packed RBCs (Biological Specialty Corporation, Colmar, PA). Isotopically labeled 13C TFV-DP and lamivudine triphosphate (3TC-TP) were added as internal standards. Analytes were extracted with 70:30 of dichloromethane:methanol. The upper aqueous layer was removed, evaporated under nitrogen, and reconstituted with 1 mM of ammonium phosphate. Liquid chromatography/mass spectrometry analysis was performed using similar conditions to those described for the PBMC samples. The same anion exchange column (Thermo Scientific BioBasic AX) was used for ULPC sample analysis, but the mobile phases used were 750 mM of ammonium acetate and 75:25 of 5 mM ammonium actate:acetonitrile (pH 9.50). The use of high salt (750 mM of ammonium acetate) removed carryover from the assay, but a divert valve was required to redirect the flow to waste during the high salt portion of the gradient. In the ULPC analysis, m/z transitions of 448.0/350.0 (TFV-DP) and 488.0/130.1 (FTC-TP) were monitored. Using a 100 μL extraction volume, the dynamic range of the assay was 10,000–15,000,000 fmol/mL (TFV-DP) and 5,000–7,500,000 fmol/mL (FTC-TP). Cell counts were then used to calculate concentrations in femtomoles per 106 cells for comparison to PBMC values. The recoveries of TFV-DP and 13C TFV-DP were approximately 20%, whereas the recoveries of FTC-TP and 3TC-TP were approximately 60%. All calibrators and quality control samples were within 15% of the nominal value. Within-day and between-day precision was <15%.
Pharmacokinetic and Statistical Analysis
Noncompartmental analysis was performed using Phoenix Win Nonlin v6.1 (Pharsight, Inc, Cary, NC). Partial area-under-the-concentration-time curves over 6 hours (AUC0–6 h or AUC12–18 h) were determined using the trapezoidal rule (linear up/log down interpolation). Geometric Mean Ratios (GMRs) with 90% confidence intervals were calculated to compare ULPC and PBMC concentrations. Summary statistics and Spearman Rank Correlation were calculated using SAS 9.2 (SAS Institute Inc, Cary, NC). Data are presented as median (25th–75th percentile) unless otherwise noted.
Of the 10 subjects, 6 were women and 80% were African American. The median age was 49.5 years (range, 25–57 years). All participants had an undetectable viral load at their previous clinic visit (<40 copies/mL) and the median CD4 cell count at last draw was 911 cells/mL (range, 572–1380 cells/mL). The HIV ARV regimens varied, but all contained TDF and FTC. The other ARVs were as follows: atazanavir/ritonavir (3 subjects), lopinavir/ritonavir (2 subjects), darunavir/ritonavir (1 subject), efavirenz (2 subjects), raltegravir (1 subject), and a combination of raltegravir, maraviroc, and darunavir/ritonavir (1 subject).
The median [interquartile range (IQR)] Cmax in plasma was 349 (314–433) ng/mL for TFV and 2970 (2020–3310) ng/mL for FTC in those subjects sampled from 0 to 6 hours postdose. The median (IQR) Tmax in plasma was 1 (1–1.5) hours for TFV and 2 (1–2) hours for FTC. For those subjects who were sampled from 12 to 18 hours postdose, the median (IQR) plasma concentration at C12 was 93.1 (77.1–102) ng/mL for TFV and 223 (184–584) for FTC.
ULPC Concentrations Compared With PBMC Concentrations
The median (IQR) concentration of TFV-DP in PBMCs was 143 (103–248) fmol/106 cells and in ULPCs was 227 (160–394) fmol/106 cells. The ULPC concentrations of TFV-DP were 64% higher on average than the PBMC concentrations with a GMR (90% confidence interval) of 1.64 (1.39–1.94). The concentrations of TFV-DP in PBMCs and ULPCs were significantly correlated (rho = 0.65; P < 0.0001) (Fig. 1A).
For FTC-TP, the median (IQR) concentration in PBMCs was 6660 (5650–10,000) fmol/106 cells and in ULPCs was 19.0 (12.0–27.8) fmol/106 cells. The ULPC concentration of FTC-TP was 99.7% lower than the PBMC concentrations (Fig. 1B). The GMR (90% confidence interval) between ULPC and PBMC concentrations was 0.0026 (0.0022–0.0030). The FTC-TP PBMC and ULPC concentrations were significantly correlated (rho = 0.55; P < 0.0001). The ULPC concentrations of TFV-DP and FTC-TP were also highly correlated (rho = 0.73; P < 0.0001) (Fig. 1C).
Individual subject pharmacokinetic profiles for TFV-DP and FTC-TP are shown in Figure 2, and demonstrate the differences in exposure between PBMCs and ULPCs. The intersubject variability [coefficient of variance, CV% (range)] of TFV-DP and FTC-TP in ULPC was 56.7% (34.5%–69.3%) and 49.3% (41.2%–73.2%), respectively, when calculated in femtomoles per 106 cells. Calculated from 5 samples collected over the 6-hour sampling interval, the intrasubject variability [CV% (range) for femtomoles per 106 cells] for TFV-DP and FTC-TP was 25.8% (14.2%–63.1%) and 28.0% (13.3%–61.5%), respectively. Comparisons to PBMC variability can be found in Table 1, as can average concentration and AUC comparisons. Concentrations are also reported in femtomoles per mL, as it is unlikely that clinical research sites will be able to perform cell counting on ULPC specimens.
The geometric mean (CV%) of the partial AUCs calculated over the 6-hour sampling period for TFV-DP in PBMCs was 966 (50.4) fmol × h/106 cells and in ULPC was 1590 (59.6) fmol × h/106 cells. These were significantly correlated (rho = 0.88;P = 0.0008). The geometric mean (CV%) of the partial AUCs for FTC-TP in PBMCs was 47700 (36.4) fmol × h/106 cells and in ULPC was 122 (45.2) fmol × h/106 cells. These were also significantly correlated (rho = 0.78; P = 0.0075).
Discrimination Between Single and Multiple Dosing
One additional subject prescribed TFV and FTC in combination with didanosine, darunavir/ritonavir, and raltegravir had undetectable concentrations of TFV-DP and FTC-TP in samples obtained just before, and 1 hour after, a witnessed dose of TDF/FTC. At 6 hours postdose, the TFV-DP concentrations in ULPC were 100-fold lower, and FTC-TP concentrations were 5-fold lower, than in the other 10 subjects (Fig. 3). In PBMCs, this subject had TFV-DP concentrations below the limit of detection at all time points. FTC-TP concentrations were undetectable at time zero and increased by 4 hours to concentrations similar to other subjects following a witnessed dose. In plasma, the concentrations of both TFV and FTC for this subject were below the limit of detection at t = 0 but similar to the other 10 subjects after a witnessed dose. These pharmacokinetic profiles are suggestive of exposure after a single dose and demonstrate that ULPC samples may provide discrimination of this dosing pattern.
Stability and Uniformity of ULPC Concentrations
When the ULPC samples that had been immediately frozen at −80°C were compared to those that were refrigerated for 14 hours, TFV-DP concentrations decreased from a median (IQR) of 205 (123–334) fmol/106 cells to 192 (109–313) fmol/106 cells corresponding with a median (range) change of 7% (−19% to 9%). The FTC-TP concentrations increased from a median (IQR) of 14.3 (10.5–24.1) fmol/106 cells to 26.9 (14.9–35.5) fmol/106 cells corresponding with a median (range) change of 52% (−18% to 123%) (Fig. 4).
Samples taken from the top layer of the ULPC had 5% lower TFV-DP concentrations and 15% higher FTC-TP concentrations, compared with those taken from the bottom of the tube. This is biologically plausible, as there is a greater concentration of PBMCs in the top layer, and FTC-TP is preferentially phosphorylated in PBMCs. Conversely, there is a greater concentration of RBCs in the bottom layer, and TFV-DP is preferentially phosphorylated in RBCs.
Adherence is critically important to interpreting the true effectiveness of PrEP strategies. In the iPrEX study in men, only 9% of those who became HIV infected had drug detected in plasma, whereas 51% of those who remained uninfected had detectable drug.1 Furthermore, adherence data from the iPrEX study indicated that men were >90% protected if their TFV-DP concentrations in cryopreserved PBMCs were >15.6 fmol/million cells. This was followed by modeling and simulation estimates of 76% (56%–96%) protection when 2 doses of TDF/FTC are taken per week, 96% (90%–99.9%) protection when 4 doses are taken per week and 99% (97%–99.9%) protection when 7 doses are taken per week.13 In Partners PrEP and TDF2, 81% and 80% of participants had detectable plasma concentrations of TFV indicating high levels of adherence and ultimately, high levels of protection.3,4 In contrast, plasma analysis of the FEM-PrEP samples found <35% of samples with detectable TFV concentrations indicating poor adherence and failure to detect a protective effect.5
As was evident in previous PrEP studies, measuring drug concentrations is a more accurate measure of adherence than is self-report.14,5 Yet TFV and FTC have plasma half-lives of 17 and 10 hours, respectively.8 Because of these short plasma half-lives and subsequently, minimal accumulation over time, it is difficult to differentiate consistent adherence from a participant who took doses only on the days of study visits (so-called “white coat” adherence). Indeed, we witnessed this in our subject with suspected nonadherence.
However, the intracellular phosphorylated metabolites of TFV and FTC, TFV-DP, and FTC-TP have documented intracellular half-lives of 6.25 days and 1.6 days, respectively.9,10 This long half-life results in significant intracellular accumulation over time, allowing for the assessment of adherence over the past 2–4 weeks. But intracellular TFV-DP and FTC-TP are traditionally measured in PBMCs, the isolation of which is complex and costly and not often done at rural international study sites.
Previous data suggest that TFV is phosphorylated in RBCs. A study published by Durand-Gasselin et al14 evaluated concentrations of the phosphorylated nucleoside reverse transcriptase inhibitors (NRTIs): zidovudine, lamivudine, and TFV in isolated PBMCs compared with samples in which contaminating RBCs had not been lysed. TFV-DP concentrations were 20% higher in the samples containing RBCs. Additionally, 3TC-TP concentrations were 99.3% lower in the samples containing RBCs. Because 3TC and FTC are both deoxycytidine analogues, they undergo very similar phosphorylation pathways.15 Therefore, the data found for 3TC-TP is similar to our findings of FTC-TP ULPC concentrations 99.5% lower than in PBMCs. Additionally, Rower et al11 recently demonstrated 70% higher TFV-DP exposure in RBCs compared with PBMCs, and Castillo-Mancilla et al16 demonstrated the RBC half-life of TFV-DP to be 17 (13–22) days.
We were able to measure both TFV-DP and FTC-TP in the ULPC samples because this layer contains both RBCs (in which TFV-DP is preferentially phosphorylated) and PBMCs (in which FTC-TP is preferentially phosphorylated). TFV-DP and FTC-TP correlated well with the PBMC concentrations. TFV-DP concentrations were 64% higher in ULPC samples than in PBMCs, and FTC-TP concentrations were 99.7% lower in ULPC samples. Low intrasubject variability across the sampling period suggests ULPC sampling could occur at any time during the dosing interval to measure adherence.
The additional subject, for whom the TFV-DP and FTC-TP concentrations were significantly lower than the other 10 subjects, is consistent with intermittent adherence. The plasma concentrations of TFV and FTC for this subject were below the limit of detection at t = 0 but similar to the other subjects at the time points after a witnessed dose, suggesting the potential ability to discriminate between “white coat adherence”: dosing just before a clinic visit. However, this will need confirmation in a larger group of subjects.
We also investigated the stability of ULPC intracellular concentrations when treated in a similar fashion to those collected in rural African FEM-PrEP study sites. We did not find significant drug degradation under refrigeration (4°C) for up to 14 hours, suggesting that samples can be kept cold for an extended period of time before freezing. Given the large concentrations of TFV-DP found in ULPC samples, the small percentage change in concentrations after refrigeration would not significantly alter interpretation of the results.
The small differences found in the ULPC concentrations taken from the top versus the bottom of the tube are consistent to what we would expect with slightly higher concentrations of TFV-DP in the bottom where there is a greater proportion of RBCs and slightly higher concentrations of FTC-TP at the top of the tube where there are greater proportion of PBMCs. These differences are not large enough to bias the clinical utility of results interpreted for adherence purposes. Therefore, it is possible to obtain samples for both virologic and pharmacologic measures from the same sample.
ULPC concentrations of both TFV-DP and FTC-TP were significantly correlated with PBMC concentrations with low inter- and intrasubject variability. Preliminary data suggest that these samples may discriminate between intermittent and consistent adherence, but more investigation is required to develop an adherence algorithm. Based on these results, TFV-DP and FTC-TP concentrations are being evaluated in ULPC samples from the FEM-PrEP clinical study to characterize adherence.
The authors would like to acknowledge Jill Palchinsky for her contributions to patient recruitment, scheduling, and study visit conduct. They would also like to acknowledge the study participants and the UNC Center for AIDS Research.
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