CONRAD provided regulatory support for the tenofovir gel IND given their ongoing management of tenofvoir gel development. However, NIH held the IND for the tenofovir gel in this study, not CONRAD, not Gilead. CONRAD did not provide financial support. Gilead had no role in this study - neither the design, drug supply, data collection and analysis, decision to publish, or preparation of the manuscript. MTN paid for the tenofovir gel. Finally, there are no restrictions to data from MTN-001 which is controlled by CONRAD or Gilead Sciences. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: CWH BAC VG JJ CN RS LST AMM KG BAR. Performed the experiments: CWH BAC VG CH JJ CN RS LST KP AMM SG KG BAR NNB. Analyzed the data: CWH KP AMM SG BAR NNB. Wrote the paper: CWH BAC VG CH JJ CN RS LST KP AMM SG KG BAR NNB.
Oral and vaginal preparations of tenofovir as pre-exposure prophylaxis (PrEP) for human immunodeficiency virus (HIV) infection have demonstrated variable efficacy in men and women prompting assessment of variation in drug concentration as an explanation. Knowledge of tenofovir concentration and its active form, tenofovir diphosphate, at the putative vaginal and rectal site of action and its relationship to concentrations at multiple other anatomic locations may provide key information for both interpreting PrEP study outcomes and planning future PrEP drug development.
MTN-001 was designed to directly compare oral to vaginal steady-state tenofovir pharmacokinetics in blood, vaginal tissue, and vaginal and rectal fluid in a paired cross-over design.
We enrolled 144 HIV-uninfected women at 4 US and 3 African clinical research sites in an open label, 3-period crossover study of three different daily tenofovir regimens, each for 6 weeks (oral 300 mg tenofovir disoproxil fumarate, vaginal 1% tenofovir gel [40 mg], or both). Serum concentrations after vaginal dosing were 56-fold lower than after oral dosing (p<0.001). Vaginal tissue tenofovir diphosphate was quantifiable in ≥90% of women with vaginal dosing and only 19% of women with oral dosing. Vaginal tissue tenofovir diphosphate was ≥130-fold higher with vaginal compared to oral dosing (p<0.001). Rectal fluid tenofovir concentrations in vaginal dosing periods were higher than concentrations measured in the oral only dosing period (p<0.03).
Compared to oral dosing, vaginal dosing achieved much lower serum concentrations and much higher vaginal tissue concentrations. Even allowing for 100-fold concentration differences due to poor adherence or less frequent prescribed dosing, vaginal dosing of tenofovir should provide higher active site concentrations and theoretically greater PrEP efficacy than oral dosing; randomized topical dosing PrEP trials to the contrary indicates that factors beyond tenofovir’s antiviral effect substantially influence PrEP efficacy.
ClinicalTrials.gov
Four recently completed clinical trials demonstrated the effectiveness of both vaginal and oral tenofovir (TFV)-containing regimens as pre-exposure prophylaxis (PrEP) to prevent HIV infection in susceptible men, women, and partners of HIV-infected individuals
Knowledge of active drug at the site of action, arguably the vaginal tissue in women, linked with seroconversion events in these clinical trials could provide critical information for interpreting outcomes and guiding dose and frequency decisions for future clinical trials by indicating the critical concentration needed to prevent infection. To date, none of these clinical trials have reported the concentration of active drug in the site of action due largely to logistical constraints in these large clinical studies.
Drug concentration at anatomic sites more distant from the rectal or vaginal mucosal tissue, however, was associated with HIV seroconversion events in all trials where pharmacokinetic (PK) results have been reported. Detecting drug in plasma or peripheral blood mononuclear cells (PBMCs) was associated with significantly higher relative risk reduction when compared to the primary modified intent-to-treat analysis in iPrEX (92% vs. 44%) and Partner’s PrEP (86% vs. 67% for TDF, 90% vs. 75% for TDF/FTC)(all p values <0.05)
We report the results of such a PK bridging study, MTN-001, which directly compares vaginal TFV gel and oral tenofovir disoproxil fumarate (TDF) tablets in a cross-over design thus removing inter-individual variation to provide more precise paired comparisons. We measured steady-state pharmacokinetics of TFV and its active form, TFV diphosphate (TFV-DP), in blood, vaginal tissue, vaginal lumen, and rectal lumen to better understand concentrations in a wide range of anatomic locations after different routes of dosing. The substantially different TFV concentrations in blood and tissue after oral compared to vaginal dosing was successfully described.
The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1.
As this research involved human subjects, written informed consent was obtained from all research participants and the clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki. The study was conducted following US National Institutes of Health and local IRB approval at seven clinical sites: Umkomaas and Botha’s Hill, Durban, South Africa; Makerere University-Johns Hopkins University Research Collaboration, Kampala, Uganda; Case Western Reserve University in Cleveland, OH, USA; University of Pittsburgh, Pittsburgh, PA, USA; University of Alabama at Birmingham, Birmingham, AL, USA; Bronx-Lebanon Hospital Center, New York City, NY, USA. The study is registered at ClinicalTrials.gov (Identifier NCT00592124). The full protocol is available online:
MTN-001 was a 21-week, phase 2, open-label, crossover study in which all participants were assigned to a randomized sequence of daily tenofovir orally, vaginally, or both orally and vaginally in three 6-week study periods, separated by one-week washouts between study periods (
N | Period 1 | Washout | Period 2 | Washout | Period 3 | |
6 weeks | 1 week | 6 weeks | 1 week | 6 week | ||
Sequence A | 24 | Oral | – | Vaginal | – | Oral+Vaginal |
Sequence B | 24 | Vaginal | – | Oral | – | Oral+Vaginal |
Sequence C | 24 | Oral+Vaginal | – | Oral | – | Vaginal |
Sequence D | 24 | Oral+Vaginal | – | Vaginal | – | Oral |
Sequence E | 24 | Oral | – | Oral+Vaginal | – | Vaginal |
Sequence F | 24 | Vaginal | – | Oral+Vaginal | – | Oral |
Formulations: Oral, 300 mg tenofovir disoproxil fumarate; Vaginal, 1% tenofovir gel.
Sampling occurs at the 3-week mid-point (blood only) and 6-week end of period visit (blood, PBMC, vaginal biopsy [intensive sites], vaginal fluid, and rectal fluid [Bronx-Lebanon site]).
Number of samples at each visit varied between intensive (US) and non-intensive (African) clinical sites.
Women were recruited through clinical research sites and community outreach. After the nature and possible consequences of the study was explained, all participants provided written informed consent prior to screening and study enrollment. Eligible participants were women aged 18–45 years, HIV-uninfected, sexually active, non-pregnant, and using an effective method of contraception. Participant self-identified race/ethnicity was recorded. Major exclusion criteria included: pregnancy, significant blood chemistry or hematology laboratory abnormalities, hepatitis B surface antigen positivity, clinically apparent gynecological abnormality, sexually transmitted or urinary tract infections requiring treatment, recent use or intended use of specific forms of contraception (diaphragm, vaginal ring, spermicide) or use of non-study vaginal products.
Participants evaluable for the final analysis included women who were dispensed study product and returned to report on product use at least once in each of the three study periods.
Eligible research participants were randomized equally to one of 6 sequences of oral, vaginal, and combination dosing of study drug for each of the three 6-week periods. Randomization, conducted by the data coordinating center, was stratified by site. Blocks of size 6 and 12 were chosen randomly to distribute the six treatment sequences. The uniform distribution was used to generate the random assignments. Sites received sealed, numbered randomization envelopes that were assigned in sequential order to each participant at the time of enrollment. The randomized dosing sequence was revealed to the study team and research participant after receipt of randomization envelope. No drug was taken during a one week washout between study periods (
Study visits took place at enrollment, at the 3-week midpoint, and again at the end of each six-week study period, and after the final one-week washout period for a total duration of 21 weeks. The PK sampling took place at the 3-week mid-period and 6-week end-of-period visits. Blood for serum and PBMCs was collected at the mid-period visits. More intensive end-of-period sampling differed by clinical research site. At the African sites, blood (serum, PBMCs) was collected prior to a dose in clinic and during one specified randomized time interval (1–3, 3–5, or 5–7 hours post dose) and repeated at the same time in each study period. Cervicovaginal lavage (CVL) fluid was collected in the same specified time interval as the blood collection. At the US sites, more intensive sampling was performed, with blood (serum, PBMCs) collected pre-dose and 1, 2, 4, 6, and 8 hours after dosing in clinic for each study period. In addition, CVL, endocervical cytobrush sampling (ECC), and 2 vaginal tissue biopsies were collected from each participant in each study period at one randomly assigned time (pre-dose, 2, 4, and 6 hours after dosing). US sites collected PBMCs for flow cytometry assessment at end-of-period visits. At the Bronx-Lebanon site, rectal sponges were collected to coincide with scheduled vaginal sampling.
Blood was collected in clot tubes and PBMCs were isolated from whole blood using cell preparation tubes (CPTs). Serum was processed by centrifugation and aliquoting into cryovials. Cells were isolated from the buffy coat after centrifugation of the CPTs, washed twice in phosphate buffered saline (PBS), counted, and then lysed in 70% methanol before storage in cryovials. All samples were frozen at −80°C until analysis.
Lavage fluid from a 10 mL saline CVL was collected into a syringe, transferred into a 15 mL conical tube, centrifuged, and the supernatant was aliquoted into cryovials. For the ECC sample, a cervical cytobrush was inserted into the cervical os, rotated twice through 360 degrees and removed. The brushes were immersed in PBS with gentle agitation to remove cells. Cells were washed twice in PBS, counted, lysed using 70% cold methanol and aliquoted into cryovials. Two vaginal wall biopsies were taken using 3×5 mm vaginal biopsy forceps and placed in a cryovial. All vaginal samples were flash frozen and stored in a −80°C freezer. At the time of sample analysis, the biopsies were weighed and homogenized with an electric mortar and pestle in a 1.5 mL cryovial with 500 µL ice-cold 70% methanol. Cervicovaginal lavage results were corrected for estimated average 20× dilution of 0.5 mL cervicovaginal fluid in 10 mL lavage fluid
Sponges (Ultracell® Medical Technologies, North Stonington, CT) pre-wetted with PBS were applied to the rectal mucosa through an anoscope for 5 minutes to adsorb rectal fluid. Sponges were centrifuged and the rectal fluid removed was stored at −80°C until analysis. TFV concentrations were not corrected for adsorbed volume, due to variable estimated sample weights due to evaporative differences across samples.
TFV and TFV diphosphate (TFV-DP) concentrations were determined by previously described LC-MS/MS methods
Thawed aliquots of serum, and tissue homogenate, with 13C5-TFV internal standard, were protein precipitated with methanol. CVL and rectal fluid aliquots with13C5-TFV underwent solid phase extraction using HLB oasis cartridges. The supernatants and eluants were collected and dried and reconstituted in 0.5% acetic acid for analysis. Samples underwent chromatographic separation using gradient elution with a Zorbax Eclipse XDB-C18 column, with positive electrospray ionization (ESI), and detection via multiple reaction monitoring using an LC-MS/MS system (Waters Acquity UPLC and Agilent 1100 HPLC Applied Biosystem API4000 mass spectrometer). Calibration standards for assay ranged from 0.31–1280 ng/ml. (0.25–50 ng/sample for tissue).
Cell lysates and tissue homogenates were analyzed using an indirect assay, which measures TFV in the sample after isolation of TFV-DP and enzymatic conversion to TFV essentially. TFV-DP was isolated from cell lysates on a Waters QMA cartridge (Waters Corporation, Milford MA) over a salt (KCl) gradient. TFV and TFV monophosphate (TFV-MP) were eluted from the cartridge under lower salt concentrations followed by elution of TFV-DP with application of 1 M KCl to the cartridge. Isolated TFV-DP was then enzymatically dephosphorylated to TFV via phosphatase digestion with sweet potato phosphatase with 13C5-TFV internal standard. Samples were desalted using trifluoroacetic acid and eluted with 1 mL methanol. The effluent was dried and reconstituted in 0.5% acetic acid for analysis. Processed samples were then chromatographically separated using a reverse phase Waters BEH C18 column and TFV and IS were ionized using negative ion mode in electrospray ionization and detected via multiple reaction monitoring (Waters Acquity UPLC, Applied Biosystem API5000 mass spectrometer) The assay is linear over the range of 50.0–1,500 fmol TFV-DP/sample.
Conversions of concentrations to common molar units assume equivalent density for one gram tissue and one milliliter fluid with the following cell volume estimates: PBMC, 0.28 pL per cell, ECC cells, 2.8 pL, which assumed an arbitrary 50∶50 mix of stratified squamous, 4.8 pL, and columnar, 0.8 pL, epithelial cells
Flow cytometry was performed at the US sites using either FACSCalibur or Canto flow cytometers (Becton-Dickinson, Franklin Lakes, NJ) and the following reagents (Becton-Dickinson): CD38 PE (347687), HLA-DR FITC (347363 [L243]), CD3 PerCP (347334 [SK7]), CD4 APC (340443), IgG2a FITC (349051), and IgG1 PE (349043). Results were expressed as percent (using blood total lymphocyte count) and absolute counts for CD3, CD4 (CD3/CD4), CD38 (CD3/CD4/CD38), HLA-DR (CD3/CD4/HLA-DR), and CD38/HLA-DR. Mean fluorescence intensity (MFI) was assessed for CD38 and HLA-DR.
Primary TFV PK outcome measures were descriptive concentration-time curves for analytes, peak concentration (Cmax), time to peak concentration (Tmax), pre-dose concentration (Cpre-dose) based on individual data for intensive sites and composite concentration data from research participants at more sparsely sampled non-intensive sites. Sample size was based on adherence and acceptability outcomes, not PK outcomes, and is described elsewhere
In order to provide a rough estimate of very recent adherence, we estimated the time elapsed between the dose prior to the research clinic visit and the TFV serum sample collected during that upcoming visit. We used the observed peak serum TFV concentration as a crude estimate of the peak TFV concentration associated with the prior dose and estimated the time it would take for this peak concentration to fall to the observed pre-dose concentration in the research clinic. With only 8 hours of post-dose concentration data in our design, we were not confident of our half-life estimates using traditional PK analyses; so, we used a range of population-based half-life estimates, 12–17 hours
Adverse events were compared across treatment regimens using conditional logistic regression controlling for study period and randomization sequence. Drug concentrations and Tmax were compared across drug regimens using linear mixed effects models controlling for study period and sequence of randomization. Treatment regimen by period interactions were assessed and were not statistically significant for any of the outcomes. In addition, there were no statistically significant period or sequence effects for any of the outcomes. Correlations between different measures were assessed using Spearman rank correlation coefficient. Friedman’s test was used to test for differences in drug concentrations over time. All statistical tests were performed using IBM SPSS Statistics (v. 19, IBM Corporation, Somers, NY) with p value ≤0.05 indicating statistical significance.
In MTN-001, each research participant was prescribed open label daily TFV in a series of 3 different regimens, each for a 6-week period followed by a one-week washout between periods (
Characteristic | median (IQR) | # (%) |
Age (years) | 29 (25–37) | |
Weight (kg) | 73 (65–88) | |
CrCl (mL/min) | 122 (105–146) | |
Self-identified Race/Ethnicity | ||
Black | 97 (67) | |
White | 32 (22) | |
Asian | 8 (6) | |
Multi-racial | 6 (4) | |
Hispanic | 1 (1) | |
Contraceptive method | ||
Injectable | 62 (43) | |
Oral | 42 (29) | |
Surgical sterilization | 22 (15) | |
IUD | 14 (10) | |
Male partner sterilization | 4 (3) |
All 3 regimens were well tolerated with mild, transient symptoms with minimal differences among regimens. The adverse event profile among the 24 who did not complete the study did not differ from the study population as a whole. Transient and mild nausea was more frequent in the oral (15%) and combination periods (14%) when compared to the vaginal period (3%) (p<0.001). Headache was also more frequent during the combination dosing period (8%) compared to vaginal dosing periods (2%) (p<0.01) with intermediate frequency with oral only dosing (5%). Hypophosphatemia was the most commonly reported adverse event, but did not differ in frequency among regimens: 11% vaginal, 15% oral, 15% combination (p>0.05). Between screening and enrollment, prior to TFV dosing, phosphate was variable and differed as much as 2 mg/dL.
Serum TFV concentrations following observed dosing in clinic were quantifiable in all participants in all study periods (
Serum TFV (panel A) and PBMC TFV-DP (panel B) concentration versus time plots are shown for the observed 8 hour interval following a dose in clinic according to dosing regimen. Median with asymmetric upper and lower quartiles is shown. Values are only for the 70 US participants where all 6 PK samples were collected.
Oral | Combination | Vaginal | |||||
Matrix/Moiety/Parameter | Units | median (IQR) | %>LLOQ | median (IQR) | %>LLOQ | median (IQR) | %>LLOQ |
Serum TFV Cmax | ng/mL | 332 (257–406) | 100 | 337 (257–447) | 100 | 3.9 (2.2–7.9) | 100 |
Serum TFV Cpre-dose | ng/mL | 65 (14–103) | 80 | 57 (2–101) | 77 | 0.67 (<0.3–2.09) | 62 |
Serum TFV Tmax | hours | 1.0 (1.0–1.2) | – | 1.0 (1.0–1.4) | – | 2.1 (1.9–4.6) | – |
PBMC TFV-DP Cmax | fmol/106 cells | 51 (28–74) | 99 | 49 (31–70) | 100 | <6 (<6–<6) | 17 |
PBMC TFV-DP Cpre-dose | fmol/106 cells | 17 (3–36) | 66 | 16 (3–34) | 63 | <6 (<6–<6) | 7 |
PBMC TFV-DP Tmax | hours | 4.0 (2.0–6.1) | – | 4.0 (1.0–6.0) | – | 3.9 (1.1–6.1) | – |
Tissue TFV | ng/mg | 0.15 (<0.15–0.27) | 50 | 104 (40–268) | 96 | 113 (27–265) | 94 |
Tissue TFV-DP | fmol/mg | <25 (<25–<25) | 19 | 2,464 (917–6,112) | 96 | 1,807 (591–5,860) | 90 |
Cervicovaginal lavage | ng/mL | 5,380 (<6–201,560) | 59 | 1.6×106 (0.5×10–6.5×106) | 98 | 3.1×106 (0.6×106–8.1×106) | 97 |
Endocervical Cytobrush | fmol/106 cells | <130 (<130–<130) | 18 | 903 (159–4,283) | 62 | 1,181 (147–5,418) | 68 |
Rectal Sponge | ng/sponge | 20 (7–404) | 83 | 576 (140–2887) | 100 | 119 (53–2150) | 100 |
Data from end-of-period visit showing median (interquartile range) by dosing regimen in common concentration units. Serum TFV Cpre-dose, PBMC TFV-DP Cpre-dose, and cervicovaginal lavage include African clinical sites; other parameters are calculable only for US clinical sites. Rectal sponges are only from one US site. For values below the LLOQ,<[median LLOQ] is shown. Cervicovaginal lavage results were corrected for estimated average 20× dilution of 0.5 mL cervicovaginal fluid in 10 mL lavage fluid
In contrast, the median intracellular PBMC TFV-DP concentrations changed only between the pre-dose and 1-hour sample without further change through 8 hours (
For drug concentration in vaginal tissue homogenate, endocervical cytobrush cell lysate, and cervicovaginal lavage fluid, median concentration at each time was not different among the times from pre-dose to 8 hours post-dose (p>0.05). By contrast with blood, oral dosing achieved the lowest drug concentrations in vaginal tissue, endocervical cytobrush cells, and cervicovaginal lavage samples where the lower quartile was not quantifiable in any of these matrices. With vaginal or combination dosing, however, TFV and TFV-DP was quantifiable in 90 to 95% of tissue homogenates, 97 to 98% of CVL samples, and 62 to 68% of endocervical cytobrush cell samples.
Because there were no temporal trends in locations other than blood and each subject provided only one sample from these locations at each end-of-period visit, results from tissue and cervicovaginal lavage fluid are summarized as median of values from all times in
Side-by-side boxplots of end-of-period visit data for all participants by anatomic site and dosing regimen are shown. Each box indicates the interquartile range with center bar as median and whiskers 1.5 times the quartile. *Lower quartile (LQ) is below the limit of quantitation (LOQ), only median and above are shown. **Median is below LOQ, so the median of values above the LOQ are shown as a single bar. X-axis key:
Because each daily dose was to be taken at the hour of sleep and clinic visits usually occurred in the morning, Cpre-dose is not a true trough (Ctau), but is a sample between Cmax and Ctau. The Cpre-dose sample occurs 13 (10–14) hours (median [IQR]) following the prior dose according to subject self-report – an interval consistent with the protocol. Similarly, Cmax is also potentially different than a true steady-state Cmax since it follows a short dosing interval. Assuming excellent adherence and steady-state, both Cpre-dose and Cmax will be overestimates. Poor adherence, however, would have an opposite, depressing effect on concentrations. Pre-dose concentrations are influenced both by individual pharmacokinetics and by the timing of prior doses with progressively greater influence of the more recent doses. We estimate that the median interval of time between the oral dose taken prior to the research visit blood collection was 1.2 to 1.7 days; the interval for the prior vaginal dose was between 1.3 to 1.8 days.
Rectal fluid was collected at only one US site. Rectal sponge TFV concentrations were quantifiable in 10 of 12 participants (83%) in the oral dosing study period and in all participants in the combination and vaginal dosing study periods. Median concentration between combination and oral dosing study periods were statistically significantly different (p = 0.008), as were mean concentrations between the vaginal and oral study periods (p≤0.03) (
Using linear mixed effect models controlling for study period and randomization sequence after log10 transformations of the end-of-period concentration data, vaginal only dosing was different than oral only dosing for all matrices tested (all p<0.001, except rectal sponge p = 0.03). Combination dosing was only different compared to vaginal only dosing for serum and PBMC’s (p<0.001) and compared to oral only dosing for tissue, CVL, endocervical cytobrush, and rectal (all p<0.001, except rectal p = 0.008). The relative magnitude of the dose regimen relationships across matrices via paired individual ratios are provided in
Vaginal:Oral | Vaginal:Combination | Combination:Oral | |||||
PK Parameter | Maximum N | N | Median (IQR) | N | Median (IQR) | N | Median (IQR) |
Serum TFV | 560 | 450 | 0.017 (0.008–0.034) | 449 | 0.018 (0.007–0.035) | 462 | 1.0 (0.8–1.3) |
PBMC TFV-DP | 560 | 260 | 0.06 (0.02–0.21) | 251 | 0.07 (0.02–0.20) | 452 | 1.0 (0.6–1.7) |
Tissue TFV | 70 | 63 | 797 (162–1623) | 63 | 1.1 (0.3–3.3) | 65 | 635 (179–1,474) |
Tissue TFV-DP | 70 | 49 | 130 (19–425) | 64 | 0.8 (0.3–2.1) | 50 | 173 (37–424) |
CVL TFV | 140 | 52 | 51 (3–365) | 93 | 1.1 (0.6–2.8) | 54 | 79 (5–391) |
CB TFV-DP | 70 | 28 | 2.2 (0.8–16.6) | 54 | 0.9 (0.4–4.2) | 33 | 3.6 (0.4–11.8) |
Rectal TFV | 12 | 10 | 6.1 (0.4–15.9) | 12 | 0.3 (0.2–4.0) | 10 | 3.1 (1.9–52.8) |
Pairs are included if at least one is above the limit of assay quantitation and the other is above the limit of assay detection. Other pairs are excluded. Serum and PBMC ratios are based on pooled values for data pairs from all times with research participants contributing more than one pair.
The ratio of TFV to TFV-DP in blood and tissue sources were all greater than unity with the lowest ratio seen in the pre-dose serum TFV to PBMC TFV-DP ratio, with median values of 2.5 (oral only) and 2.9 (combination) (
Oral | Combination | Vaginal | |||||
Molar Ratio | Maximum N | N | Median (IQR) | N | Median (IQR) | N | Median (IQR) |
Serum TFV/PBMC TFV-DP Cmax | 70 | 65 | 5.9 (3.6, 10.1) | 69 | 6.2 (4.1, 9) | *10 | NA |
Serum TFV/PBMC TFV-DP Cpre-dose | 144 | 89 | 2.5 (1.6, 4.5) | 104 | 2.9 (1.7, 5.3) | *6 | NA |
Serum TFVall/PBMC TFV-DPall | 560 | 475 | 4.8 (2.6, 9.5) | 479 | 4.2 (2.6, 9) | 245 | 1.2 (0.4, 4.6) |
Tissue TFV/Tissue TFV-DP | 70 | 27 | 48 (22, 130) | 67 | 170 (63, 418) | 64 | 207 (84, 485) |
Pairs are included if at least one value in the pair is above the limit of assay quantitation and the other is above the limit of assay detection. “N” is the number of pairs available meeting the inclusion criteria above. “Maximum N” is the number of possible pairs if all subjects at all visits provided a sample. Cmax only includes US participants where multiple serum samples were available; the pair is defined by serum Cmax matched with the corresponding PBMC TFV-DP concentration which is not necessarily peak TFV-DP after the same dose. Subscript “all” indicates that all PBMC-Serum pairs from each sample times are included, with participants contributing multiple pairs. All p<0.001 Wilcoxon signed rank test for TFV fmol/gm vs. TFV-DP fmol/gm. *Insufficient samples above the limit of assay quantitation and detection to estimate reliable medians given the inevaluable excluded pairs.
Initial median (IQR) values for PBMC surface markers were absolute lymphocyte count 2209 (1827, 2722), %CD3 77 (74, 81), %CD4 49 (44, 54), %CD38 66 (51, 78), %HLA-DR 5 (3, 7), %CD38/HLA-DR 3 (2, 4). There were no differences among the 3 study periods for any cell surface marker (p>0.1). When assessed within each dosing regimen, mean fluorescence intensity values for both CD38 and HLA-DR on PBMCs were negatively correlated with TFV-DP tissue concentrations (all rs >0.34, all p<0.001), but these surface markers did not correlate with TFV-DP concentration in PBMC or endocervical cytobrush cells. There was no correlation of percent or absolute number of either CD4 or CD8 with TFV-DP in any anatomic site by any route of dosing.
Our cross-over design allows for the direct paired comparisons of TFV PK within individuals in multiple anatomic sites, avoiding inter-individual variability. The most significant finding is the greater than 100-fold higher concentration of the active TFV moiety, TFV-DP, in vaginal tissue homogenates with vaginal dosing when compared to an oral regimen alone. Conversely, systemic exposure (serum TFV) after vaginal dosing was at least 56-fold lower than after oral dosing regimens. This difference in systemic exposure based on route of dosing may have been the cause of the greater frequency of nausea, diarrhea, and headache with oral regimens, though the gastrointestinal symptoms may have been due to local gastrointestinal effects in oral dosing regimens. The large systemic exposure differences, yet with similar rates of hypophosphatemia with both oral and vaginal regimens, argues against a significant dose-dependent relationship between TFV and hypophosphatemia.
The temporal patterns of drug concentration we observed are consistent with expectations based on the relatively shorter 12–17 hour serum half-life of TFV
The amount of TFV extracted from rectal sponges was 3 times higher with combination dosing when compared to oral dosing, though vaginal dosing was not statistically different from oral or combination dosing. This may result from an additive effect of oral and vaginal dosing not seen in any other anatomic site, but may also have resulted from assay variability due to the collection method and should be cautiously interpreted. Use of an internal standard in the rectal sponge would greatly improve the accuracy of this sampling method. Without this, heterogeneity of both fluid volume adsorbed onto the sponge and subsequent evaporative losses prior to sample freezing may introduce significant and unmeasured variation which prevents accurate estimates of concentration. Regardless, the finding of greater or similar rectal fluid concentrations after vaginal dosing alone compared to oral dosing suggests that vaginal dosing may also provide some level of protection from receptive anal intercourse. A similar vaginal to rectal drug migration has been reported by Nuttall,
The most notable result of the study is the 100-fold greater TFV-DP concentrations in vaginal tissue associated with vaginal dosing. This more precise estimate from our cross-over design confirms what can be inferred from combining results of separate oral dosing studies
The modest (CAPRISA 004) or absent (VOICE) efficacy seen in the TFV gel studies, despite vaginal tissue levels expected (based on MTN-001) to be far higher than in the very successful Partner’s PrEP and TDF2 studies, suggests that efficacy is more complex than would be predicted by vaginal tissue concentration alone
Adherence is a powerful explanatory variable among oral studies given the enhanced relative risk reduction when drug can be measured in the blood (Partner’s PrEP, iPrEX) and absence of efficacy when concentrations are lower (FEM-PrEP)
To put this adherence difference in temporal terms, consider the half-life of elimination of TFV-DP from vaginal tissue. The terminal half-life of TFV-DP after a single oral dose as measured in vaginal tissue homogenates over two weeks of sampling is estimated at 53 hours
Therefore, there must be unmeasured variables at work with topical TFV that negate the vaginal concentration-dependent protection of tenofovir demonstrated among oral TFV PrEP studies. Plausible explanations for this protection-negating, possibly HIV-enhancing, effect include tissue toxicity from either concentration-dependent TFV or TFV-DP effects or dose frequency-dependent gel vehicle effects. Although TFV 1% gel has been found to be safe and well tolerated in women, our hypothesized negative effects could be either beneath the level of sensitivity or beyond the scope of the safety measures employed in large clinical trials. To impute non-drug related HIV enhancing effects as the cause of lower than expected efficacy in topical studies – increased frequency of anal intercourse, elevated partner viral load, noxious environmental or para-sexual behaviors, increased prevalence of sexually transmitted infections, higher systemic innate immune activation
Poor adherence in our study could reduce observed concentrations and increase variability. We had clear evidence of poor adherence in many subjects which we detailed in another report (Minnis,
It is unclear why we detected a correlation between PBMC surface marker with TFV-DP concentration in vaginal tissue, but not PBMC on whose surface the increased activation markers were detected. It is possible that (unmeasured) vaginal tissue activation markers had an even greater correlation with tissue TFV-DP concentration and a modest correlation with PBMC markers, but we did not extract cells from vaginal tissue to test these correlations directly. Three separate
Limitations to our study were several and point out the difficulties in sampling and comparing findings in these complex and varied anatomic spaces. Due to the destructive sampling (CVL, endocervical cytobrush) or logistical complexity (biopsies), we used a sparse sampling approach for some anatomic and geographic sites. Population PK modeling is underway to best incorporate this data to understand the temporal and spatial relationships of drug in multiple anatomic compartments. The short, variable, and uncertain (self-reported) dosing interval before the clinic dose introduced additional noise in drug concentration assessments, but the impact was minimal for all but serum concentration data due to the relatively long half-life and flat concentration-time profile in other sites. Our CVL samples were based on dilution-adjusted TFV concentrations which bring all of the concentration values closer to their original state. However, this crude adjustment doesn’t account for variations in native cervicovaginal fluid volume and results in significant underestimates in subjects sampled post-gel dose in vaginal dosing periods due to residual TFV gel volume. An internal standard, like lithium
In healthy women, daily dosing of tenofovir 1% gel (40 mg) achieves substantially lower (56-fold) systemic exposure of tenofovir compared to daily oral dosing of tenofovir disoproxil fumarate (Viread™). Rectal fluid concentrations of tenofovir were greater after vaginal dosing than oral dosing raising the potential that a vaginal dosing route might provide some level of protection from receptive anal intercourse, though this remains to be studied. When compared to daily oral dosing, vaginal dosing achieved more than 130 times greater vaginal tissue concentrations of active drug (tenofovir diphosphate), leading to the expectation that vaginal TFV dosing should achieve greater protective efficacy than oral TFV dosing, even allowing for very poor adherence. Because this concentration advantage has not been seen in the seroconversion outcomes of two randomized clinical trials (CAPRISA 004 and VOICE), it raises concern that factors beyond TFV’s antiviral effect may substantially reduce PrEP efficacy when dosed topically. This discordance between drug concentration and expected outcome warrants further investigation to better understand TFV and other antiretroviral approaches to topical HIV prevention.
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We wish to acknowledge the significant contribution to this research from the research participants without whom the work is not possible. In addition, the authors wish to specifically acknowledge the contributions of the MTN-001 Study Group with special note of: Nicole Anders, Dawn Antosh, Jane Baum, Katherine Bunge, Martha Cavallo, Jianmeng Chen, Mitchell D. Creinin, Brodie Daniels, Moira S. Flynn, Angelina Gangestad, Faye E. Howard, Sherri Johnson, Samuel Kabwigu, Betty Kamira, Flavia M. Kiweewa, Nicolette A. Louissaint, Teresa Parsons, Arendevi Panther, Carol Priest, Sharon Riddler, and Shay J. Warren.