Heather M.A. Prince

Penetration of Tenofovir and Emtricitabine in Mucosal Tissues: Implications for Prevention of HIV-1 Transmission

Antiretroviral drugs are found at different concentrations in human cervical, vaginal, and rectal mucosal tissues with implications for designing PrEP trials to prevent HIV transmission. HAARTening News for HIV Prevention Antiretroviral drug therapy (ART) has been used successfully for treating HIV-1. However, HIV transmission, principally through sexual intercourse, is still a major problem, with 7000 new infections reported daily worldwide. ART is now being investigated as a way not only to treat individuals with HIV but also to prevent the transmission of HIV infection. This strategy, called PrEP, or PrEP, requires that the concentrations of the antiretroviral drugs under investigation are at high enough concentrations in the target mucosal tissues, predominantly genital and colorectal, to prevent HIV infection. Such knowledge will be essential for designing new HIV prevention trials and also for interpreting the data from several prevention trials that have yielded conflicting results. In a new study, Patterson et al. set out to obtain this information for two antiretroviral drugs that have shown promise for HIV prevention: tenofovir (TFV) disoproxil fumarate (TDF) and emtricitabine (FTC). The researchers gave 12 healthy men and women a single oral dose of the fixed-dose combination pill TDF/FTC (Truvada) and then took samples of blood plasma, genital secretions, and vaginal, cervical, and rectal tissue biopsies over the next 14 days. The pro-drug forms TFV and FTC were measured in blood plasma and genital secretions, whereas the active intracellular phosphorylated metabolites of these drugs (TFV-DP and FTC-TP) were measured in vaginal, cervical, and rectal tissues. After a single oral dose, TFV and FTC were detected in blood plasma for the 14-day duration of the study. Encouragingly, the concentration of FTC was 27-fold greater in genital secretions than in blood plasma. However, the concentration of TFV was only 2.5-fold greater in genital secretions than in blood plasma. Both TFV and its active phosphorylated form TFV-DP were detected in rectal tissue for 14 days after dosing, and the cumulative exposure of rectal tissue to TFV-DP was 100-fold greater than that in vaginal and cervical tissues. In contrast, the cumulative exposure of vaginal and cervical tissues to FTC was only 10- to 15-fold greater than that for rectal tissue. Despite high concentrations of the pro-drug FTC in vaginal and cervical tissues, its active form FTC-TP was detected for ≤2 days in these tissues. This study demonstrates that the penetration of different human mucosal tissues and their exposure to the pro-drugs TFV and FTC and their active metabolites TFV-DP and FTC-TP is wide ranging and dependent on the tissue type. These findings reinforce the fact that the success of PrEP will depend on selecting the correct ART that achieves a critical concentration and duration of exposure in vulnerable mucosal tissues. Continuing pharmacological investigations will be crucial for ensuring the success of PrEP and ultimately for preventing HIV transmission. A mainstay of strategies to prevent HIV-1 transmission is to use antiretroviral therapy (ART) for pre-exposure prophylaxis (PrEP). Critical to the design and interpretation of PrEP prevention trials is the ability to make accurate pharmacological measurements of ART drugs in human genital and colorectal mucosal tissues, the principal route of HIV transmission. Here, we evaluated two drugs that are preferentially used for PrEP: tenofovir (TFV) disoproxil fumarate (TDF) and emtricitabine (FTC). A single oral dose of TDF/FTC (Truvada) was administered to 15 healthy individuals. Over the next 14 days, TFV and FTC were measured in blood plasma and genital secretions using a sensitive assay (lower level of quantification, 0.1 ng/ml). The active intracellular phosphorylated metabolites of these drugs [TFV diphospate (TFV-DP) and FTC triphosphate (FTC-TP)] were measured in homogenates prepared from rectal, vaginal, and cervical tissues. TFV and FTC were detected in blood plasma 14 days after administration of a single dose. The area under the concentration-time curve from 24 hours to 14 days (AUC1–14d) for FTC in genital secretions was 27-fold greater than in blood plasma, whereas the AUC1–14d for TFV was only 2.5-fold greater in genital secretions than in blood plasma. In rectal tissue, TFV and TFV-DP concentrations were detectable for 14 days and were 100-fold higher than the concentrations in vaginal and cervical tissues. Vaginal and cervical tissue concentrations of FTC were 10- to 15-fold higher than in rectal tissue. Despite high concentrations of FTC in vaginal and cervical tissue, FTC-TP concentrations in all tissue types were detected for only 2 days after dose. The exposure to TFV, TFV-DP, FTC, and FTC-TP was wide ranging depending on the type of mucosal tissue. These results demonstrate the need for detailed pharmacological studies to improve the application of ART for PrEP to prevent transmission of HIV.

Single and Multiple Dose Pharmacokinetics of Maraviroc in Saliva, Semen, and Rectal Tissue of Healthy HIV-Negative Men

BACKGROUND Antiretroviral pharmacology in seminal plasma (SP) and rectal tissue (RT) may provide insight into antiretroviral resistance and the prevention of sexual transmission of human immunodeficiency virus (HIV). Saliva may be of utility for noninvasively measuring adherence. METHODS A pharmacokinetic study was performed in 12 HIV-negative men receiving maraviroc 300 mg twice daily for 8 days. Seven time-matched pairs of blood plasma (BP) and saliva samples were collected over 12 h on day 1 (PK1) and days 7 and 8 (PK2). One RT sample from each subject was collected during PK1 and PK2. Two SP samples were collected from each subject during PK1, and 6 SP samples were collected from each subject during PK2. RESULTS SP AUCs were ∼50% lower than BP. However, protein binding in SP ranged from 4% to 25%, resulting in protein-free concentrations >2-fold higher than BP. RT AUCs were 7.5- to 26-fold higher than BP. Maraviroc saliva AUCs were ∼70% lower than BP, but saliva concentrations correlated with BP (r(2) = 0.58). CONCLUSIONS More pharmacologically available maraviroc was found in SP than BP. High RT concentrations are promising for preventing rectal HIV acquisition. Saliva correlation with BP suggests that this may be useful for monitoring adherence. CLINICAL TRIALS REGISTRATION NCT00775294.

A Translational Pharmacology Approach to Predicting Outcomes of Preexposure Prophylaxis Against HIV in Men and Women Using Tenofovir Disoproxil Fumarate With or Without Emtricitabine.

BACKGROUND A novel translational pharmacology investigation was conducted by combining an in vitro efficacy target with mucosal tissue pharmacokinetic (PK) data and mathematical modeling to determine the number of doses required for effective human immunodeficiency virus (HIV) preexposure prophylaxis (PrEP). METHODS A PK/pharmacodynamic (PD) model was developed by measuring mucosal tissue concentrations of tenofovir, emtricitabine, their active metabolites (tenofovir diphosphate [TFVdp] and emtricitabine triphosphate [FTCtp], respectively), and competing endogenous nucleotides (dATP and dCTP) in 47 healthy women. TZM-bl and CD4(+) T cells were used to identify 90% effective concentration (EC90) ratios of TFVdp to dATP and FTCtp to dCTP (alone and in combination) for protection against HIV. Monte-Carlo simulations were then performed to identify minimally effective dosing strategies to protect lower female genital tract and colorectal tissues. RESULTS The colorectal TFVdp concentration was 10 times higher than that in the lower female genital tract, whereas concentrations of endogenous nucleotides were 7-11 times lower. Our model predicted that ≥98% of the population achieved protective mucosal tissue exposure by the third daily dose of tenofovir disoproxil fumarate plus emtricitabine. However, a minimum adherence to 6 of 7 doses/week (85%) was required to protect lower female genital tract tissue from HIV, while adherence to 2 of 7 doses/week (28%) was required to protect colorectal tissue. CONCLUSIONS This model is predictive of recent PrEP trial results in which 2-3 doses/week was 75%-90% effective in men but ineffective in women. These data provide a novel approach for future PrEP investigations that can optimize clinical trial dosing strategies.

Differential penetration of raltegravir throughout gastrointestinal tissue: implications for eradication and cure.

Objective:To investigate the concentration of the integrase strand inhibitor raltegravir (RAL) throughout gastrointestinal (GI) tissue, especially gut-associated lymphoid tissue (GALT), as an adjunct to current prevention and cure strategies. Design:Open-label pharmacokinetic (PK) study. Methods:HIV-negative men received RAL 400 mg twice daily for 7 days. Seven blood plasma specimens were collected over 12-h intervals; timed tissue specimens from terminal ileum, splenic flexure, and rectum were also obtained by colonoscopy following the first dose and on day 7 [multiple dose (MD)]. RAL concentrations were measured by validated LC–MS assay with 1 ng/ml lower limit of detection. Data were analyzed by noncompartmental methods (WinNonlin 6). Tissue exposures are reported as composite medians and tissue density of 1.04 g/ml is assumed for comparisons. Results:Fourteen men completed evaluations. Median (range) age was 24 (19–49) years and BMI 25 (19–31) kg/m2. After the first dose, area under the time-concentration curve (AUC)0-12h was highest in the terminal ileum (594 &mgr;g*h/ml). Exposures were 160, 68 and 39-fold greater than blood plasma at the terminal ileum, splenic flexure and rectum, respectively. After multiple doses, exposure was highest at the splenic flexure (2240 &mgr;g*h/ml); exposure at the terminal ileum and rectum were equivalent (both 788 &mgr;g*h/ml). Following multiple doses, exposures were 160 to 650-fold greater than blood plasma throughout the colon. Conclusion:RAL rapidly disseminates into GI tissue and concentrations remain significantly higher than blood plasma. RAL exposure in GI tissue remains higher than any antiretroviral investigated to date. These data suggest that RAL should result in full suppression of viral replication in GI tissue and GALT.

Expression of six drug transporters in vaginal, cervical, and colorectal tissues: Implications for drug disposition in HIV prevention.

Effective antiretroviral (ARV)‐based HIV prevention strategies require optimizing drug exposure in mucosal tissues; yet factors influencing mucosal tissue disposition remain unknown. We hypothesized drug transporter expression in vaginal, cervical, and colorectal tissues is a contributing factor and selected 3 efflux (ABCB1/MDR1, ABCC2/MRP2, ABCC4/MRP4) and 3 uptake (SLC22A6/OAT1, SLC22A8/OAT3, SLCO1B1/OATP1B1) transporters to further investigate based on their affinity for 2 ARVs central to prevention (tenofovir, maraviroc). Tissue was collected from 98 donors. mRNA and protein expression were quantified using qPCR and immunohistochemistry (IHC). Hundred percent of tissues expressed efflux transporter mRNA. IHC localized them to the epithelium and/or submucosa. Multivariable analysis adjusted for age, smoking, and co‐medications revealed significant (P < 0.05) differences in efflux transporter mRNA between tissue types (vaginal ABCB1 3.9‐fold > colorectal; vaginal ABCC2 2.9‐fold > colorectal; colorectal ABCC4 2.0‐fold > cervical). In contrast, uptake transporter mRNA was expressed in <25% of tissues. OAT1 protein was detected in 0% of female genital tissues and in 100% of colorectal tissues, but only in rare epithelial cells. These data support clinical findings of higher maraviroc and tenofovir concentrations in rectal tissue compared to vaginal or cervical tissue after oral dosing. Quantifying mucosal transporter expression and localization can facilitate ARV selection to target these tissues.

Dolutegravir pharmacokinetics in the genital tract and colorectum of HIV-negative men after single and multiple dosing

Objectives:To describe first-dose and steady state pharmacokinetics (PKs) of dolutegravir (DTG) in blood plasma (BP), seminal fluid (SF), colorectal tissue (RT), and rectal mucosal fluid (RF) of healthy HIV-negative men. Design:A phase 1, open-label, PK study that enrolled 12 healthy men taking 50 mg DTG daily for 8 days. Methods:Eleven paired BP samples and 3 SF and RF samples were collected over 24 hours after first (PK1) and multiple (PK2) dosing. RT biopsies were collected at 1 of 6 time points at PK1 and PK2 to generate composite PK profiles. DTG concentrations were analyzed by validated liquid chromatography-tandem mass spectrometry (LC-MS/MS). Noncompartmental PK analysis was conducted with Phoenix WinNonlin v6.3, and Spearman rank correlations were determined using SAS v9.3. Results:BP area under the concentration–time curves (AUCs) were similar to previous reports, and concentrations at 24 hours (C24 h) were 6- to 34-fold greater than the protein-adjusted concentration required for 90% viral inhibition (PA-IC90) of 64 ng/mL. SF exposures were <7% of BP and below the PA-IC90. RT exposures were 17% of BP and ∼2-fold greater than the PA-IC90. RF AUCs were ∼2%–5% of RT and did not correlate with RT (rho = 0.43, P = 0.17). Accumulation of DTG with multiple dosing was observed in BP, SF, and RT. Conclusions:DTG BP PKs were consistent with previously published values. SF concentrations were <7% BP, with SF C24 h below the PA-IC90. However, SF protein binding was not measured. Although the AUC of DTG in RT was <20% BP, RT C24 h remained ∼2-fold higher than the PA-IC90. RF was not a strong surrogate for RT concentrations.

Tenofovir Diphosphate and Emtricitabine Triphosphate Concentrations in Blood Cells Compared with Isolated Peripheral Blood Mononuclear Cells: A New Measure of Antiretroviral Adherence?

Background:The active metabolites of tenofovir (TFV) and emtricitabine (FTC) in peripheral blood mononuclear cells (PBMCs) have been used as markers of long-term antiretroviral (ARV) adherence. However, the process of isolating PBMCs is expensive, complex, and not feasible in many settings. We compared concentrations of TFV-diphosphate (TFV-DP) and FTC-triphosphate (FTC-TP) in the upper layer packed cells (ULPCs) obtained after whole blood centrifugation to isolated PBMCs as a possible alternative marker of adherence. Methods:Ten HIV+ adults with HIV RNA <50 copies/mL on a TDF/FTC-containing regimen provided 5 paired PBMC and ULPC samples over 6 hours. TFV-DP and FTC-TP concentrations were analyzed by liquid chromatography/mass spectrometry. Partial areas under the curve were calculated using noncompartmental methods and Spearman Rank Correlations (rho) between PBMC and ULPC were determined. Results:The median (25th–75th percentile) concentration of TFV-DP in PBMCs was 143 (103–248) fmol/106 cells and in ULPC was 227 (160–394) fmol/106 cells (rho = 0.65; P < 0.0001). The concentration of FTC-TP in PBMCs was 6660 (5650–10,000) fmol/106 cells and in ULPC was 19.0 (12.0–27.8) fmol/106 cells (rho = 0.55; P < 0.0001). Compared to PBMCs, ULPC TFV-DP was 64% higher and FTC-TP was 99.7% lower. ULPC concentrations of TFV-DP and FTC-TP in one additional subject receiving a single dose of TDF/FTC were only 0.05% and 25%, of the other 10 subjects, respectively. Conclusions:ULPC concentrations significantly correlated with PBMC concentrations. Preliminary single-dose data suggest some discrimination between intermittent versus consistent dosing. ULPC concentrations of TFV-DP and FTC-TP should be further investigated as a simply collected surrogate measure of ARV adherence.

Single and multiple dose pharmacokinetics of dolutegravir in the genital tract of HIV-negative women

BACKGROUND Antiretrovirals that achieve adequate concentrations in anatomical sites of transmission are of interest for HIV prevention. A Phase I open-label pharmacokinetic (PK) study was performed to describe first dose (PK1) and steady-state (PK2) PKs of the integrase inhibitor dolutegravir (DTG) in blood plasma (BP), cervicovaginal fluid (CVF), cervical tissue (CT) and vaginal tissue (VT) in HIV type-1-negative women. METHODS A total of 8 healthy females given DTG 50 mg daily for 5-7 days had 11 paired BP and CVF samples collected over 24 h following the first dose (PK1) and multiple dosing (PK2). Each woman underwent CT and VT biopsies at 1/4 time points at PK1 and PK2 to generate composite PK profiles. DTG concentrations were analysed by validated liquid chromatography-tandem mass spectrometry methods. Non-compartmental PK analysis was performed and Spearman rank correlations determined between matrices. RESULTS BP areas under the concentration-time curve (AUCs) were similar to previous reports and concentrations remained greater than the protein-adjusted (PA) 90% inhibitory concentration (IC90) for wild-type HIV (64 ng/ml). CVF exposures were approximately 6% of BP with low inter-individual variability. CT and VT exposures were 7% of BP at PK1, and 9-10% of BP at PK2 with 94% of samples >PA-IC90. CT and VT concentrations were correlated to each other (ρ=0.70, P=0.003), and to CVF at steady state (ρ=0.52, P=0.04). Accumulation of DTG from PK1 to PK2 occurred in BP, CT and VT, but only marginally in CVF. CONCLUSIONS DTG BP PK were consistent with previously published values. CVF, CT and VT exposures were highly correlated. At PK2, DTG accumulated to a greater extent in tissue than in BP or CVF, suggesting increased tissue affinity.

Protein binding of lopinavir and ritonavir during 4 phases of pregnancy: Implications for treatment guidelines

Objective:To investigate the intraindividual pharmacokinetics (PKs) of total (protein bound plus unbound) and unbound lopinavir/ritonavir (LPV/RTV) and to assess whether the pediatric formulation (100 mg/25 mg) can overcome any pregnancy-associated changes. Design:Prospective longitudinal PK study. Methods:HIV-infected pregnant antiretroviral therapy–naive and experienced women receiving LPV/RTV 400 mg/100 mg tablets twice daily. Intensive PK evaluations were performed at 20–24 weeks (PK1), 30 weeks (PK2) followed by empiric dose increase using the pediatric formulation (100 mg/25 mg twice daily), 32 weeks (PK3), and 8 weeks postpartum (PK4). Results:Twelve women completed prespecified PK evaluations. Median (range) age was 28 (18–35) years and baseline BMI was 32 (19–41) kg/m2. During pregnancy, total area under the time concentration (AUC0–12h) for LPV was significantly lower than postpartum (PK1, PK2, or PK3 vs. PK4, P = 0.005). Protein-unbound LPV AUC0–12h remained unchanged during pregnancy [PK1: 1.6 (1.3–1.9) vs. PK2: 1.6 (1.3–1.9) &mgr;g·h/mL, P = 0.4] despite a 25% dose increase [PK2 vs. PK3: 1.8 (1.3–2.1) &mgr;g·h/mL, P = 0.5]. Protein-unbound LPV predose concentrations (C12h) did not significantly change despite dose increase [PK2: 0.10 (0.08–0.15) vs. PK3: 0.12 (0.10–0.15) &mgr;g/mL, P = 0.09]. Albumin and LPV AUC0–12h fraction unbound were correlated (rs = 0.3, P = 0.03). Conclusions:Total LPV exposure was significantly decreased throughout pregnancy despite the increased dose. However, the exposure of unbound LPV did not change significantly regardless of trimester or dose. Predose concentrations of unbound LPV were not affected by the additional dose and were 70-fold greater than the minimum efficacy concentration. These findings suggest dose adjustments may not be necessary in all HIV-infected pregnant women.

Single and Multiple Dose Pharmacokinetics of Darunavir plus Ritonavir and Etravirine in Semen and Rectal Tissue of HIV-Negative Men

Background:Antiretroviral therapy has become a central component of combination in HIV prevention efforts. Defining the individual exposure of commercially available antiretroviral therapy in genital secretions and vulnerable mucosal tissues is paramount to designing future prevention interventions. Methods:A pharmacokinetic (PK) study was performed in 12 HIV-negative men receiving 600 mg of darunavir, 100 mg of ritonavir, and 200 mg of etravirine orally, twice daily for 8 days. Seven blood plasma (BP) samples were collected over 12 hours on day 1 (PK1) and days 7 and 8 (PK2). One rectal tissue (RT) sample from each subject was collected during PK1 and PK2. During PK1, 2 seminal plasma (SP) samples were collected from each subject. During PK2, 6 SP samples were collected from each subject over 2 days. Results:Antiretrovirals were detected in SP and RT within 1 hour after a single dose. Over PK1 and PK2, SP exposures were lower than BP by 80%–92% (DRV), 89–95% (RTV), and 83–88% (ETR). However, protein binding in SP (14% for darunavir, 70% for ritonavir, and 97% for etravirine) was lower than in BP. Rectal tissue exposures were higher than BP by 39- to 155-fold for darunavir, 12- to 61-fold for ritonavir, and 20- to 40-fold for etravirine. Conclusions:Lower SP protein binding resulted in higher pharmacologically active darunavir and etravirine concentrations compared with BP. High RT concentrations may also be favorable for suppressing viral replication in the gastrointestinal mucosa. The high protein-unbound exposures in SP and total exposures in RT support further investigations of darunavir plus ritonavir and etravirine in secondary prevention.