Prevalence of Cardiovascular Disease and Risk Factors Among Former National Football League Players.

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of death worldwide, but prevalence estimates in former professional athletes are limited. OBJECTIVES: HUDDLE (Heart Health: Understanding and Diagnosing Disease by Leveraging Echocardiograms) aimed to raise awareness and estimate the prevalence of CVD and associated risk factors among members of the National Football League (NFL) Alumni Association and their families through education and screening events. METHODS: HUDDLE was a multicity, cross-sectional study of NFL alumni and family members aged 50 years and older. Subjects reported their health history and participated in CVD education and screening (blood pressure, electrocardiogram, and transthoracic echocardiogram [TTE] assessments). Phone follow-up by investigators occurred 30 days postscreening to review results and recommendations. This analysis focuses on former NFL athletes. RESULTS: Of 498 participants screened, 57.2% (N = 285) were former NFL players, the majority of whom were African American (67.6%). The prevalence of hypertension among NFL alumni was estimated to be 89.8%, though only 37.5% reported a history of hypertension. Of 285 evaluable participants, 61.8% had structural cardiac abnormalities by TTE. Multivariable analysis showed that hypertension was a significant predictor of clinically relevant structural abnormalities on TTE. CONCLUSIONS: HUDDLE identified a large discrepancy between participant self-awareness and actual prevalence of CVD and risk factors, highlighting a significant opportunity for population health interventions. Structural cardiac abnormalities were observed in most participants and were independently predicted by hypertension, affirming the role of TTE for CVD screening in this population aged older than 50 years. (Heart Health: Understanding and Diagnosing Disease by Leveraging Echocardiograms [HUDDLE]; NCT05009589).

Design and rationale of the evaluation of transcatheter aortic valve replacement compared to surveillance for patients with asymptomatic severe aortic stenosis: The EARLY TAVR trial.

BACKGROUND: For patients with asymptomatic, severe aortic stenosis (AS) and preserved left ventricular ejection fraction, current guidelines recommend clinical surveillance every 6 to 12 months. To date, no randomized trials have examined whether an early intervention with transcatheter aortic valve replacement (TAVR) will improve outcomes among these patients. STUDY DESIGN AND OBJECTIVES: EARLY TAVR is a prospective, randomized, controlled, and multicenter trial, with an event-based design. Asymptomatic severe AS patients (n = 900) are randomized 1:1 to either clinical surveillance or TAVR with the Edwards SAPIEN 3/SAPIEN 3 Ultra transcatheter heart valve. Patients are stratified by whether they are able to perform a treadmill stress test. The primary end point is death, stroke, or unplanned cardiovascular hospitalization. Patients who are asymptomatic but have a positive stress test will be followed in a registry and undergo aortic valve replacement as per current guidelines. CONCLUSIONS: EARLY TAVR is the largest randomized trial to date assessing the role of early intervention among patients with asymptomatic severe AS compared to clinical surveillance and the first to study the role of TAVR. TRIAL REGISTRATION NUMBER: NCT03042104.

Cardiac Damage and Quality of Life After Aortic Valve Replacement in the PARTNER Trials.

BACKGROUND: The extent of extravalvular cardiac damage is associated with increased risk of adverse events among patients with severe aortic stenosis undergoing aortic valve replacement (AVR). OBJECTIVES: The goal was to describe the association of cardiac damage on health status before and after AVR. METHODS: Patients from the PARTNER (Placement of Aortic Transcatheter Valves) 2 and 3 trials were pooled and classified by echocardiographic cardiac damage stage at baseline and 1 year as previously described (stage 0-4). We examined the association between baseline cardiac damage and 1-year health status (assessed by the Kansas City Cardiomyopathy Questionnaire Overall Score [KCCQ-OS]). RESULTS: Among 1,974 patients (794 surgical AVR, 1,180 transcatheter AVR), the extent of cardiac damage at baseline was associated with lower KCCQ scores both at baseline and at 1 year after AVR (P < 0.0001) and with increased rates of a poor outcome (death, KCCQ-OS <60, or a decrease in KCCQ-OS of ≥10 points) at 1 year (stages 0-4: 10.6% vs 19.6% vs 29.0% vs 44.7% vs 39.8%; P < 0.0001). In a multivariable model, each 1-stage increase in baseline cardiac damage was associated with a 24% increase in the odds of a poor outcome (95% CI: 9%-41%; P = 0.001). Change in stage of cardiac damage at 1 year after AVR was associated with the extent of improvement in KCCQ-OS over the same period (mean change in 1-year KCCQ-OS: improvement of ≥1 stage +26.8 [95% CI: 24.2-29.4] vs no change +21.4 [95% CI: 20.0-22.7] vs deterioration of ≥1 stage +17.5 [95% CI: 15.4-19.5]; P < 0.0001). CONCLUSIONS: The extent of cardiac damage before AVR has an important impact on health status outcomes, both cross-sectionally and after AVR. (PARTNER II Trial: Placement of AoRTic TraNscathetER Valves II - XT Intermediate and High Risk (PII A), NCT01314313; The PARTNER II Trial: Placement of AoRTic TraNscathetER Valves - PII B [PARTNERII B], NCT02184442; PARTNER 3 Trial: Safety and Effectiveness of the SAPIEN 3 Transcatheter Heart Valve in Low Risk Patients With Aortic Stenosis [P3], NCT02675114).

Influence of hair treatments on detection of antiretrovirals by mass spectrometry imaging.

Analysis of drugs in hair by mass spectrometry imaging (MSI) has great potential as an objective, long-term measure of medication adherence. However, the fidelity of the chemical record in hair may be compromised by any cosmetic hair treatments. Here, we investigate infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) MSI response to multiple antiretrovirals (ARVs) in cosmetically treated hair. Hair strands from patients on different ARV regimens were mechanically treated with dye, bleach, and relaxer. The treatments had little or no effect relative to untreated controls for cobicistat, abacavir, dolutegravir, maraviroc, efavirenz, and darunavir, but all three treatments removed emtricitabine (FTC) to undetectable levels from patient hair strands. We also evaluated hair strands by IR-MALDESI MSI from 8 patients on FTC-based regimens who reported a range of hair treatments at varying recency prior to hair collection. While FTC was undetectable in the treated portion of these hair strands, ARVs coadministered with FTC remained detectable in hair strands after treatment. We conclude that IR-MALDESI MSI can be used when measuring adherence to ARV therapy, provided that ARVs other than FTC are targeted in people using hair treatments.

Using Real-Time Adherence Feedback to Enhance Communication About Adherence to Antiretroviral Therapy: Patient and Clinician Perspectives.

New technologies for real-time adherence monitoring hold the potential to enhance antiretroviral therapy adherence interventions by providing objective information about daily medication-taking behavior. To realize this potential, we need to understand how to integrate real-time adherence feedback into existing best practices to promote antiretroviral therapy adherence at the point of care. Using in-depth interviews with 30 HIV-infected patients and 29 HIV care clinicians, our primary aims were to understand patients' and clinicians' perceptions of anticipated benefits and preferred uses of objective feedback to enhance conversations about adherence and to identify concerns about the impact of objective monitoring on patient-clinician relationships and communication. Both patients and clinicians suggested that identifying patterns of nonadherence with real-time feedback could (a) facilitate collaborative adherence problem-solving, (b) motivate patient adherence, and (c) reinforce the importance of optimal adherence. Some clinicians worried that delivery of real-time feedback could imply mistrust of patient-reported adherence and suggested careful framing of monitoring results. A few patients and clinicians were concerned that negative reactions to monitoring could discourage retention in care and reduce adherence motivation. These results indicate the potential of real-time feedback to enhance existing evidence-based adherence interventions targeting the key adherence precursors of adherence information, motivation, and behavioral skills. Guidance for the delivery of real-time adherence feedback should focus on both optimizing adherence and mitigating negative perceptions of adherence monitoring.

Translational Approach to Predicting the Efficacy of Maraviroc-Based Regimens as HIV Preexposure Prophylaxis.

Maraviroc-based regimens have been explored as preexposure prophylaxis (PrEP) against human immunodeficiency virus (HIV). In this study, we utilized mucosal tissue drug exposure data, combined with target concentrations generated in vitro, in a pharmacokinetic-pharmacodynamic analysis to predict the effects of drug combinations and adherence on PrEP efficacy. Mucosal tissue concentrations of maraviroc were measured in 24 healthy women. The 90% effective concentrations (EC90) of maraviroc (alone and combined with tenofovir and emtricitabine) for protection against HIV were identified in CD4+ T cells. Monte Carlo simulations were performed to identify dosing strategies to protect colorectal and female genital tract (FGT) tissues from HIV infection. Colorectal maraviroc concentrations were 350-fold higher than in the FGT. Under steady-state conditions, our model predicted that one 300-mg dose/week was sufficient to protect colorectal tissue from HIV in 99% of the population, while 300 mg daily would protect the FGT in only 63% of the population. FGT protection increased to >90% when maraviroc was used in combination with tenofovir (5 doses/week) or emtricitabine (3 doses/week). Poor adherence resulted in a drastic decrease in efficacy in the FGT but not colorectal tissue. However, greater forgiveness was seen when maraviroc was combined with tenofovir or emtricitabine, suggesting that maraviroc should not be used alone as PrEP.

Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry Imaging of Human Hair to Characterize Longitudinal Profiles of the Antiretroviral Maraviroc for Adherence Monitoring.

Here, we assess infrared matrix assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging (MSI) analysis of hair as a clinical tool for monitoring patient adherence to the antiretroviral maraviroc (MVC). A custom MATLAB-based algorithm has been developed to streamline data analysis and generate longitudinal profiles of drug incorporation along the length of hair strands. Hair strands from volunteers enrolled in a directly observed therapy study were analyzed by IR-MALDESI MSI and processed using this tool to characterize the profiles of single doses and a daily dose regimen of MVC. Single dose responses were 1.7 [1.1, 2.5] mm (median [range]) wide along the length of the hair and were detected in 8 out of 12 volunteers. Daily dose profiles capturing 28 days of continuous dosing were approximately 5 times the intensity of single dose profiles and 10.5 [7.0, 13] mm wide, corresponding to 1 month of hair growth. MVC ion abundance was observed in all 12 volunteers for the daily dosing period. Daily dosing profiles were consistent with a model of MVC accumulation in hair based on linear superposition of a single dose response, indicating the potential for prediction of daily drug-taking behavior based on deconvolution of a complex longitudinal profile in hair.

Heterogeneous antiretroviral drug distribution and HIV/SHIV detection in the gut of three species.

HIV replication within tissues may increase in response to a reduced exposure to antiretroviral drugs. Traditional approaches to measuring drug concentrations in tissues are unable to characterize a heterogeneous drug distribution. Here, we used mass spectrometry imaging (MSI) to visualize the distribution of six HIV antiretroviral drugs in gut tissue sections from three species (two strains of humanized mice, macaques, and humans). We measured drug concentrations in proximity to CD3+ T cells that are targeted by HIV, as well as expression of HIV or SHIV RNA and expression of the MDR1 drug efflux transporter in gut tissue from HIV-infected humanized mice, SHIV-infected macaques, and HIV-infected humans treated with combination antiretroviral drug therapy. Serial 10-µm sections of snap-frozen ileal and rectal tissue were analyzed by MSI for CD3+ T cells and MDR1 efflux transporter expression by immunofluorescence and immunohistochemistry, respectively. The tissue slices were analyzed for HIV/SHIV RNA expression by in situ hybridization and for antiretroviral drug concentrations by liquid chromatography-mass spectrometry. The gastrointestinal tissue distribution of the six drugs was heterogeneous. Fifty percent to 60% of CD3+ T cells did not colocalize with detectable drug concentrations in the gut tissue. In all three species, up to 90% of HIV/SHIV RNA was found to be expressed in gut tissue with no exposure to drug. These data suggest that there may be gut regions with little to no exposure to antiretroviral drugs, which may result in low-level HIV replication contributing to HIV persistence.

Decreased Tenofovir Diphosphate Concentrations in a Transgender Female Cohort: Implications for Human Immunodeficiency Virus Preexposure Prophylaxis.

Feminizing hormone therapy (FHT) may interact with human immunodeficiency virus preexposure prophylaxis (PrEP). We found that transgender women who took FHT exhibited a 7-fold lower rectal tissue ratio of PrEP's active metabolites vs competing deoxynucleotides compared to cisgender women and men (P = .03) that inversely correlated with estradiol (ρ = -0.79; P < .05). Thus, FHT may negatively impact PrEP efficacy. Clinical Trials Registration . NCT02983110.

Population Modeling Highlights Drug Disposition Differences Between Tenofovir Alafenamide and Tenofovir Disoproxil Fumarate in the Blood and Semen.

Understanding antiretroviral disposition in the male genital tract, a distinct viral compartment, can provide insight for the eradication of HIV. Population pharmacokinetic modeling was conducted to investigate the disposition of tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF), and emtricitabine and their metabolites in blood and semen. Blood plasma and seminal plasma (SP) concentrations of tenofovir and emtricitabine were measured, as were tenofovir-diphosphate and emtricitabine-triphosphate concentrations in peripheral blood mononuclear cells (PBMCs) and seminal mononuclear cells. Sequential compartmental modeling described drug disposition in blood and semen. Our modeling suggests slower elimination of apparent tenofovir-diphosphate PBMC and faster elimination of tenofovir SP after administration of TAF compared with TDF, likely reflecting flip-flop kinetics. Additionally, TAF metabolism to tenofovir appeared slower in semen compared with blood; however, SP elimination of TAF-derived tenofovir appeared faster than its blood plasma elimination. These findings provide valuable insight for further mechanistic study of cellular entry and drug metabolism in the male genital tract.

Patient and clinician perspectives on optimizing graphical displays of longitudinal medication adherence data.

OBJECTIVE: New pharmacological measures assessing medication adherence, including longitudinal drug levels in hair, are emerging. Little is known, however, about how best to present results from such measures to patients and clinicians in comprehensive, easy-to-understand, acceptable formats. We, therefore, developed three graphical display prototypes of hypothetical daily drug concentrations measured in hair, and assessed their acceptability among participants. METHODS: We interviewed 30 HIV-positive patients and 29 clinicians to examine perceived acceptability for each graphical display prototype. RESULTS: Patients and clinicians generally found the prototypes acceptable for facilitating understanding of patient adherence; however, areas for optimization were identified. For patients with lower health literacy, prototypes did not provide sufficient understanding of the link between medication-taking and drug concentrations in hair. These patients also preferred pictographs over bar or line graphs. Clinicians largely preferred daily drug concentration data in bar graphs with information included about the measure's accuracy. Participants questioned the utility of showing drug concentrations above a therapeutic range, though they found color-coding results acceptable. CONCLUSIONS: Assessing prototype versions of graphical displays of hypothetical longitudinal adherence data indicated ways to optimize their acceptability. PRACTICE IMPLICATIONS: Acceptable prototype-tested graphical displays of longitudinal patient-specific drug concentrations may enhance adherence monitoring in clinical settings.

Differential extracellular, but similar intracellular, disposition of two tenofovir formulations in the male genital tract.

BACKGROUND: The male genital tract (MGT) is a viral sanctuary and likely HIV reservoir; understanding MGT pharmacokinetics (PK) of antiretrovirals (ARVs) used for curative strategies is critical to eradication and cure. Tenofovir alafenamide (TAF) is a tenofovir (TFV) formulation designed to maximize efficacy/minimize toxicity with unknown MGT PK. METHODS: HIV-positive and HIV-negative men receiving TFV-based regimens provided six paired blood plasma (BP) and semen samples. Extracellular (TFV, TAF, emtricitabine [FTC]) drug concentrations in BP and seminal plasma (SP), and intracellular metabolite (IM) and endogenous nucleotide (EN) concentrations were measured in peripheral blood mononuclear cells (PBMCs) and seminal mononuclear cells (SMCs). Exposure ratios for SP:BP, SMC:PBMC and IM:EN were calculated from PK parameters generated by noncompartmental analysis. HIV viral load was measured in BP and SP. RESULTS: Sixteen HIV-positive (n=8, TDF/FTC; n=8, TAF/FTC) and eight HIV-negative (TDF/FTC) men provided samples. Median TFV SP:BP ratios differed between TDF and TAF (1.5 versus 7.4), due to lower TFV BP concentrations with TAF coupled with TFV SP concentrations similar to TDF. FTC SP: BP ratios were approximately 3. SMC concentrations of IMs and ENs were a fraction of PBMC concentrations (1-22%), though IM:EN ratios exceed a suggested protective threshold. CONCLUSIONS: TAF SP PK was unexpected. IM SMC concentrations were low relative to PBMC, as were EN concentrations, suggesting differences in cell phenotype and lineage in the MGT; these differences in phenotype and pharmacology may have an impact on selecting and dosing ARVs used in cure strategies.

A Pharmacokinetic/Pharmacodynamic Model to Predict Effective HIV Prophylaxis Dosing Strategies for People Who Inject Drugs.

The goal of this work was to evaluate dosing strategies for tenofovir disoproxil fumarate (TDF), tenofovir alafenamide (TAF), and emtricitabine (FTC) for pre-exposure prophylaxis (PrEP) with injection drug use with a pharmacokinetic/pharmacodynamics analysis of concentration data generated from two single-dose clinical studies conducted in healthy women. Population pharmacokinetic models were developed using measured intracellular metabolite, endogenous nucleotide competitors, and extracellular parent drug concentrations. Intracellular metabolite concentrations were normalized to endogenous competitors and compared with an EC90 target for PrEP efficacy. Monte Carlo simulations were used to select effective dose strategies of single agents (TAF, TDF, and FTC) and combinations (TDF + FTC and TAF + FTC). Daily, intermittent, and event-driven dosing regimens at varying dosage amounts were explored. When combined, TDF + FTC and TAF + FTC both provided quick (0.5 hours) and durable (up to 84 and 108 hours, respectively) protection of ≥99% after a single dose. When dosed twice per week, protection remained at 100%. Single-agent regimens provided lower estimates of protection than either combination tested. Here, the application of pharmacokinetic modeling to in vitro target concentrations demonstrates the added utility of including FTC in a successful PrEP regimen. While no TAF-based PrEP data are currently available for comparison, this analysis suggests TAF + FTC could completely protect against percutaneous exposure with as little as two doses per week.

Virological and immunological responses to raltegravir and dolutegravir in the gut-associated lymphoid tissue of HIV-infected men and women.

BACKGROUND: Raltegravir (RTG) and dolutegravir (DTG) have different pharmacokinetic patterns in the gastrointestinal tract. To determine if this results in pharmacodynamic differences, we compared HIV RNA, HIV DNA and immunological markers in gut-associated lymphoid tissue (GALT) of HIV-infected participants receiving RTG or DTG with tenofovir+emtricitabine (TDF/FTC). METHODS: GALT specimens from the terminal ileum, splenic flexure and rectum were obtained by colonoscopy at a single time point in 20 adults treated with RTG (n=10) or DTG (n=10) with HIV RNA <50 copies/ml. Flow cytometry, drug concentrations, and HIV RNA and DNA were analysed in tissue. CD4/8+ T-cells were tested for γδ TCR, and markers of T-cell activation and exhaustion. Data are reported as median (Q1-Q3). RESULTS: A total of 15 men and 5 women were enrolled. There was no difference in time since HIV diagnosis for those on RTG (9.5 [4-22] years) and DTG (17 [1-24] years; P=0.6), although time on RTG (5.4 [2.3-6.7] years) was greater than DTG (1.0 [0.1-1.5] years; P<0.001). Concentrations of RTG and DTG in rectal tissue were similar to previous reports: median tissue:plasma ratio was 11.25 for RTG and 0.44 for DTG. RNA:DNA ratios were 1.14 (0.18-5.10) for the RTG group and 0.90 (0.30-18.87) for the DTG group (P=0.95). No differences (P≥0.1) between CD4+ and CD8+ T-cell markers were found. CONCLUSIONS: RTG produced higher tissue exposures than DTG, but no significant differences in GALT HIV RNA, DNA or most immunological markers were observed. ClinicalTrials.gov NCT02218320.

Validation of an LC-MS/MS assay to simultaneously monitor the intracellular active metabolites of tenofovir, emtricitabine, and lamivudine in dried blood spots.

The ability to monitor adherence to antiretroviral therapy is critical for the interpretation of outcomes from clinical studies of HIV, and for optimizing patient care. The antiretrovirals tenofovir (TFV), emtricitabine (FTC), and lamivudine (3TC) are commonly included in drug regimens for HIV prevention and treatment. The active form of the drugs tenofovir diphosphate (TFVdp), emtricitabine triphosphate (FTCtp), and lamivudine triphosphate (3TCtp) are found intracellularly in erythrocytes and peripheral blood mononuclear cells (PBMCs). The ability to collect and analyze dried blood spot (DBS) samples is an attractive alternative to PBMC sampling in many resource limited settings. We developed and validated an assay to quantify all three intracellular metabolites over the range of 100-25000 fmol/sample. This assay utilizes a simple protein precipitation/liquid-liquid extraction of a single 3-mm DBS punch (from a Whatman 903 Protein Saver card) with isotopically labeled 13C5-TFVdp included as the internal standard. Following extraction, samples are analyzed by anion exchange chromatography on a Thermo Biobasic AX 5µm column with detection by electrospray ionization in the positive mode on a AB Sciex API-5000 triple quadrupole mass spectrometer with a total run time of 8min. The assay was linear over the entire range (R2>0.996). The assay was accurate (inter-assay%bias within ±3.0%) and precise (inter-assay % CV≤9.8%). The assay was also reproducible from multiple punches within a spot as well as punches from separate blood spots. Stability was established at room temperature for 3days, and at -80°C for up to 63days. Clinical samples were analyzed from subjects on Truvada®, Stribild®, Descovy®, and Triumeq® regimens and intracellular metabolites were detected in all samples as expected, indicating the assay performs well for all current formulations of TFV, FTC, and 3TC.

Single-dose pharmacokinetics of tenofovir alafenamide and its active metabolite in the mucosal tissues.

Objectives: Tenofovir alafenamide, a prodrug of tenofovir, produces higher PBMC concentrations of tenofovir diphosphate (tenofovir-dp) than tenofovir disoproxil fumarate. To understand tenofovir alafenamide's mucosal tissue distribution and its implications for pre-exposure prophylaxis, we characterized tenofovir-dp in female genital tract (FGT) and lower gastrointestinal (GI) tissues. Methods: Healthy seronegative women were given 5, 10 or 25 mg of tenofovir alafenamide ( n = 8/group). Each participant provided plasma, PBMC and cervical, vaginal and rectal tissue samples over 14 days. Plasma, cell lysate and tissue homogenate concentrations were analysed by LC-MS/MS. Dose proportionality was declared in plasma and PBMCs if the natural log AUC versus natural log dose regression line 90% CI was within 0.57-1.43. In vitro tenofovir-dp formation was assessed in PBMCs and ectocervical (Ect1/E6E7) and vaginal (VK2/E6E7) cells incubated in 0.5 and 10 µM tenofovir alafenamide or tenofovir. clinicaltrials.gov: NCT02357602. Results: Following single doses of 5, 10 and 25 mg, median (IQR) tenofovir plasma AUC 0-14 days was 52.8 (49.5-59.6), 78.1 (68.2-86.9) and 169.7 (131.2-211.4) ng·h/mL and tenofovir-dp PBMC AUC 0-14 days was 2268 (1519-4090), 4584 (3113-5734) and 9306 (6891-10785) fmol·h/10 6 cells, respectively. Tenofovir was quantifiable in 52% and 92% of FGT and GI tissues, whereas tenofovir-dp was quantifiable in only 5% and 19% of FGT and GI tissues, respectively. Plasma tenofovir and PBMC tenofovir-dp were dose proportional (90% CI = 0.87-1.15 and 0.62-1.02, respectively). In vitro tenofovir-dp was 1.7-17-fold higher in epithelial cells than PBMCs. Conclusions: After tenofovir alafenamide dosing in vivo , tenofovir-dp was unquantifiable in most tissues (91%) although cervical and vaginal epithelial cells efficiently formed tenofovir-dp from tenofovir alafenamide in vitro . These findings warrant further investigation of tenofovir alafenamide's pharmacology.

Concentrations of Pro-Inflammatory Cytokines Are Not Associated with Senescence Marker p16INK4a or Predictive of Intracellular Emtricitabine/Tenofovir Metabolite and Endogenous Nucleotide Exposures in Adults with HIV Infection.

OBJECTIVES: As the HIV-infected population ages, the role of cellular senescence and inflammation on co-morbid conditions and pharmacotherapy is increasingly of interest. p16INK4a expression, a marker for aging and senescence in T-cells, is associated with lower intracellular concentrations of endogenous nucleotides (EN) and nucleos(t)ide reverse transcriptase inhibitors (NRTIs). This study expands on these findings by determining whether inflammation is contributing to the association of p16INK4a expression with intracellular metabolite (IM) exposure and endogenous nucleotide concentrations. METHODS: Samples from 73 HIV-infected adults receiving daily tenofovir/emtricitabine (TFV/FTC) with either efavirenz (EFV) or atazanavir/ritonavir (ATV/r) were tested for p16INK4a expression, and plasma cytokine and intracellular drug concentrations. Associations between p16INK4a expression and cytokine concentrations were assessed using maximum likelihood methods, and elastic net regression was applied to assess whether cytokines were predictive of intracellular metabolite/endogenous nucleotide exposures. RESULTS: Enrolled participants had a median age of 48 years (range 23-73). There were no significant associations between p16INK4a expression and cytokines. Results of the elastic net regression showed weak relationships between IL-1Ra and FTC-triphosphate and deoxyadenosine triphosphate exposures, and MIP-1ß, age and TFV-diphosphate exposures. CONCLUSIONS: In this clinical evaluation, we found no relationships between p16INK4a expression and cytokines, or cytokines and intracellular nucleotide concentrations. While inflammation is known to play a role in this population, it is not a major contributor to the p16INK4a association with decreased IM/EN exposures in these HIV-infected participants.

Cervicovaginal and Rectal Fluid as a Surrogate Marker of Antiretroviral Tissue Concentration: Implications for Clinical Trial Design.

BACKGROUND: Quantifying tissue drug concentrations can yield important information during drug development, but complicates pharmacokinetic study design. Mucosal fluids collected by direct aspiration (cervicovaginal fluid; CVF) or swab (rectal fluid; RF) might be used as tissue concentration surrogates, but these relationships are not well characterized. METHODS: Forty-nine healthy women, given a single oral dose of tenofovir, maraviroc, emtricitabine, or raltegravir at 50%-200% of the treatment dose, provided 13 plasma, 12 CVF, 12 RF and one cervical, vaginal and rectal tissue biopsy over 48 hours. Relationships between these paired samples were characterized by linear and multiple linear regression. Adjusted r values were used to select the final predictive models. RESULTS: CVF exposure increased linearly with dose for all antiretrovirals (r(2) ≥ 0.23, P ≤ 0.02) except raltegravir (r(2) = 0.08, P = 0.19). In RF, only emtricitabine increased linearly with dose (r(2) = 0.27, P = 0.01). For all antiretrovirals, CVF and RF concentrations significantly correlated with mucosal tissue concentrations (female genital tract r(2) ≥ 0.37, rectal tissue (2)r ≥ 0.50, P ≤ 0.001). In the final multivariate models, plasma and fluid concentrations were both associated with FGT concentrations for all antiretrovirals (r(2) ≥ 0.81, P < 0.001). The same was noted for rectal tissue (r(2) ≥ 0.58, P < 0.001) except for tenofovir, for which RF alone was predictive of tissue concentration (r(2) = 0.91, P < 0.001). CONCLUSIONS: Mucosal fluids were positively correlated with tissue concentrations and including plasma concentrations improved the regression models in most cases. Dose linearity in CVF, but not RF, suggests a saturation process in lower gastrointestinal tract tissue. These findings suggest that mucosal fluid and plasma concentrations may be used for qualitative inference of tissue concentrations for these antiretrovirals.

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.

Tenofovir/emtricitabine metabolites and endogenous nucleotide exposures are associated with p16(INK4a) expression in subjects on combination therapy.

BACKGROUND: HIV may amplify immunological, physiological and functional changes of ageing. We determined associations of frailty phenotype, a T-cell senescence marker (p16(INK4a) expression), age and demographics with exposures of the intracellular metabolites (IM) and endogenous nucleotides (EN) of tenofovir/emtricitabine (TFV/FTC), efavirenz (EFV), atazanavir (ATV) and ritonavir (RTV). METHODS: Plasma and peripheral blood mononuclear cell samples for drug, IM and EN concentrations were collected at four time points in HIV+ adults receiving TFV/FTC with EFV or ATV/RTV. Subjects underwent frailty phenotyping and p16(INK4a) expression analysis. Non-compartmental analysis generated an area under the curve (AUC) for each analyte. Spearman rank correlation and Kruskal-Wallis tests were used to assess associations between AUC, demographics and ageing markers, adjusting for multiple comparisons with the Holm procedure. RESULTS: Subjects (n=79) ranged in age from 22-73 years (median 48 years); 48 were African-American, 24 were female, 54 received EFV. Three subjects (range 51-60 years) demonstrated frailty, with 17 subjects (range 26-60 years) demonstrating pre-frailty. Negative associations were observed between p16(INK4a) expression and each of FTC-triphosphate (r=-0.45), deoxyadenosine triphosphate (dATP; r=-0.47) and deoxycytidine triphosphate (dCTP; r=-0.57) AUCs (P-values <0.02). TFV and FTC AUCs were larger among subjects with lower renal function or higher chronological age (P-values ≤0.05). No associations were observed for EFV, ATV or RTV AUCs. CONCLUSIONS: Associations of IM/EN exposure and p16(INK4a) expression observed here suggest that senescence may alter drug phosphorylation, metabolism or transport. This finding warrants further mechanistic study to ensure optimal treatment in the ageing HIV+ population. Clinicaltrials.gov NCT01180075.