Next-Generation Contraceptive Intravaginal Ring: Comparison of Etonogestrel and Ethinyl Estradiol In Vitro and In Vivo Release from 3D-Printed Intravaginal Ring and NuvaRing.

Intravaginal rings (IVRs) represent a well-established, woman-controlled and sustained vaginal drug delivery system suitable for a wide range of applications. Here, we sought to investigate the differences in etonogestrel (ENG) and ethinyl estradiol (EE) release from a 3D-printed IVR utilizing continuous liquid interface production (CLIP™) (referred to as CLIPLOW for low drug loading and CLIPHIGH IVRs for high drug loading) and NuvaRing, a commercially available injection molded IVR. We conducted in vitro release studies in simulated vaginal fluid to compare the release of ENG and EE from CLIPLOW IVRs and NuvaRing. CLIPLOW IVRs had a similar hormone dose to NuvaRing and exhibited slightly slower ENG release and greater EE release in vitro compared to NuvaRing. When administered to female sheep, NuvaRing demonstrated greater ENG/EE levels in plasma, vaginal tissue and vaginal fluids compared to CLIPLOW IVR despite similar drug loadings. Leveraging observed hormones levels in sheep from NuvaRing as an effective contraceptive benchmark, we developed a long-acting CLIPHIGH IVR with increased ENG and EE doses that demonstrated systemic and local hormone levels greater than the NuvaRing for 90 days in sheep. No signs of toxicity were noted regarding general health, colposcopy, or histological analysis in sheep after CLIPHIGH IVR administration. Our results provided (1) a comparison of ENG and EE release between a 3D-printed IVR and NuvaRing in vitro and in vivo, (2) a preclinical pharmacokinetic benchmark for vaginally delivered ENG and EE and (3) the generation of a 90-day CLIP IVR that will be utilized in future work to support the development of a long-acting ENG/EE IVR combined with an antiretroviral for the prevention of HIV and unplanned pregnancy.

EFdA efficiently suppresses HIV replication in the male genital tract and prevents penile HIV acquisition.

Sexually transmitted HIV infections in heterosexual men are acquired through the penis. Low adherence to condom usage and the fact that 40% of circumcised men are not protected indicate the need for additional prevention strategies. Here, we describe a new approach to evaluate the prevention of penile HIV transmission. We demonstrated that the entire male genital tract (MGT) of bone marrow/liver/thymus (BLT) humanized mice is repopulated with human T and myeloid cells. The majority of the human T cells in the MGT express CD4 and CCR5. Direct penile exposure to HIV leads to systemic infection including all tissues of the MGT. HIV replication throughout the MGT was reduced 100-1,000-fold by treatment with 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), resulting in the restoration of CD4+ T cell levels. Importantly, systemic preexposure prophylaxis with EFdA effectively protects from penile HIV acquisition. IMPORTANCE Over 84.2 million people have been infected by the human immunodeficiency virus type 1 (HIV-1) during the past 40 years, most through sexual transmission. Men comprise approximately half of the HIV-infected population worldwide. Sexually transmitted HIV infections in exclusively heterosexual men are acquired through the penis. However, direct evaluation of HIV infection throughout the human male genital tract (MGT) is not possible. Here, we developed a new in vivo model that permits, for the first time, the detail analysis of HIV infection. Using BLT humanized mice, we showed that productive HIV infection occurs throughout the entire MGT and induces a dramatic reduction in human CD4 T cells compromising immune responses in this organ. Antiretroviral treatment with novel drug EFdA suppresses HIV replication in all tissues of the MGT, restores normal levels of CD4 T cells and is highly efficient at preventing penile transmission.

Immune reconstitution inflammatory syndrome drives emergence of HIV drug resistance from multiple anatomic compartments in a person living with HIV.

Reservoirs of HIV maintained in anatomic compartments during antiretroviral therapy prevent HIV eradication. However, mechanisms driving their persistence and interventions to control them remain elusive. Here we report the presence of an inducible HIV reservoir within antigen-specific CD4+T cells in the central nervous system of a 59-year-old male with progressive multifocal leukoencephalopathy immune reconstitution inflammatory syndrome (PML-IRIS). HIV production during PML-IRIS was suppressed by modulating inflammation with corticosteroids; selection of HIV drug resistance caused subsequent breakthrough viremia. Therefore, inflammation can influence the composition, distribution and induction of HIV reservoirs, warranting it as a key consideration for developing effective HIV remission strategies.

A quantitative LC-MS/MS method for the determination of tissue brincidofovir and cidofovir diphosphate in a MuPyV-infected mouse model.

Brincidofovir (BCV) is an investigational lipid conjugate of the nucleotide analog cidofovir (CDV), which is being developed as a medical countermeasure for the treatment of smallpox. BCV is active against double-stranded DNA viruses including BK and JC viruses. Here, we validated procedures for quantifying BCV and its pharmacologically active moiety cidofovir diphosphate (CDV-PP) in mouse kidney, brain and spleen tissue homogenates. Following homogenization, BCV and CDV-PP were extracted from the tissues by protein precipitation with their stable, isotopically labeled internal standards, BCV-d6 and 13 C3 15 N2 -CDV-PP. Then, samples were analyzed for BCV by reverse-phase chromatography on a Waters Xterra MS C18 (50 × 2.1 mm, 3.5 µm particle size) column while CDV-PP was analyzed on a Thermo BioBasic AX (50 × 2.1 mm, 5 µm particle size) column using anion exchange chromatography. Detection was achieved by electrospray ionization in positive ion mode on an AB Sciex API-5000 triple quadrupole mass spectrometer. The calibration curves were linear over a range of 1.00-1,000 ng/ml homogenate and 0.050-50.0 ng/ml homogenate for BCV and CDV-PP, respectively. These methods were validated according to US Food and Drug Administration guidance for industry and may be used to characterize the tissue pharmacology of both analytes to advance its preclinical development.

Pregnancy-Related Hormones Increase Nifedipine Metabolism in Human Hepatocytes by Inducing CYP3A4 Expression.

Pregnancy-related hormones (PRH) have emerged as key regulators of hepatic cytochrome P450 (CYP) enzyme expression and function. The impact of PRH on protein levels of CYP3A4 and other key CYP enzymes, and the metabolism of nifedipine (a CYP3A4 substrate commonly prescribed during pregnancy), was evaluated in primary human hepatocytes. Sandwich-cultured human hepatocytes (SCHH) from female donors were exposed to PRH (estradiol, estriol, estetrol, progesterone, and cortisol), individually or in combination as a cocktail. Absolute protein concentrations of twelve CYP isoforms in SCHH membrane fractions were quantified by nanoLC-MS/MS, and metabolism of nifedipine to dehydronifedipine in SCHH was evaluated. PRH significantly increased CYP3A4 protein concentrations and nifedipine metabolism to dehydronifedipine in a concentration-dependent manner. CYP3A4 mRNA levels in hepatocyte-derived exosomes positively correlated with CYP3A4 protein levels and dehydronifedipine formation in SCHH. PRH also increased CYP2B6, CYP2C8 and CYP2A6 levels. Our findings demonstrate that PRH increase nifedipine metabolism in SCHH by inducing CYP3A4 expression and alter expression of other key CYP proteins in an isoform-specific manner, and suggest that hepatocyte-derived exosomes warrant further investigation as biomarkers of hepatic CYP3A4 metabolism. Together, these results offer mechanistic insight into the increases in nifedipine metabolism and clearance observed in pregnant women.

Antiretroviral Penetration and Drug Transporter Concentrations in the Spleens of Three Preclinical Animal Models and Humans.

Adequate antiretroviral (ARV) concentrations in lymphoid tissues are critical for optimal antiretroviral therapy (ART). While the spleen contains 25% of the body's lymphocytes, there are minimal data on ARV penetration in this organ. This study quantified total and protein-unbound splenic ARV concentrations and determined whether drug transporters, sex, or infection status were modifiers of these concentrations in animal models and humans. Two humanized mice models (hu-HSC-Rag [n = 36; 18 HIV-positive (HIV+) and 18 HIV-negative (HIV-)] and bone marrow-liver-thymus [n = 13; 7 HIV+ and 6 HIV-]) and one nonhuman primate (NHP) model (rhesus macaque [n = 18; 10 SHIV+ and 8 SHIV-]) were dosed to steady state with ARV combinations. HIV+ human spleens (n = 14) from the National NeuroAIDS Tissue Consortium were analyzed postmortem (up to 24 h postdose). ARV concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS), drug transporter concentrations were measured with LC-MS proteomics, and protein binding in NHP spleens was determined by rapid equilibrium dialysis. Mice generally had the lowest splenic concentrations of the three species. Protein binding in splenic tissue was 6 to 96%, compared to 76 to 99% in blood plasma. NHPs had quantifiable Mrp4, Bcrp, and Ent1 concentrations, and humans had quantifiable ENT1 concentrations. None significantly correlated with tissue ARV concentrations. There was also no observable influence of infection status or sex. With these dosing strategies, NHP splenic penetration most closely resembled that of humans. These data can inform tissue pharmacokinetic scaling to humans to target HIV reservoirs by identifying important species-related differences.

Food Insecurity Is Associated With Lower Levels of Antiretroviral Drug Concentrations in Hair Among a Cohort of Women Living With Human Immunodeficiency Virus in the United States.

BACKGROUND: Food insecurity is a well-established determinant of suboptimal, self-reported antiretroviral therapy (ART) adherence, but few studies have investigated this association using objective adherence measures. We examined the association of food insecurity with levels of ART concentrations in hair among women living with human immunodeficiency virus (WLHIV) in the United States. METHODS: We analyzed longitudinal data collected semiannually from 2013 through 2015 from the Women's Interagency HIV Study, a multisite, prospective, cohort study of WLHIV and controls not living with HIV. Our sample comprised 1944 person-visits from 677 WLHIV. Food insecurity was measured using the US Household Food Security Survey Module. ART concentrations in hair, an objective and validated measure of drug adherence and exposure, were measured using high-performance liquid chromatography with mass spectrometry detection for regimens that included darunavir, atazanavir, raltegravir, or dolutegravir. We conducted multiple 3-level linear regressions that accounted for repeated measures and the ART medication(s) taken at each visit, adjusting for sociodemographic and clinical characteristics. RESULTS: At baseline, 67% of participants were virally suppressed and 35% reported food insecurity. In the base multivariable model, each 3-point increase in food insecurity was associated with 0.94-fold lower ART concentration in hair (95% confidence interval, 0.89 to 0.99). This effect remained unchanged after adjusting for self-reported adherence. CONCLUSIONS: Food insecurity was associated with lower ART concentrations in hair, suggesting that food insecurity may be associated with suboptimal ART adherence and/or drug absorption. Interventions seeking to improve ART adherence among WLHIV should consider and address the role of food insecurity.

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.

Antiretroviral Drug Concentrations in Lymph Nodes: A Cross-Species Comparison of the Effect of Drug Transporter Expression, Viral Infection, and Sex in Humanized Mice, Nonhuman Primates, and Humans.

In a "kick and kill" strategy for human immunodeficiency virus (HIV) eradication, protective concentrations of antiretrovirals (ARVs) in the lymph node are important to prevent vulnerable cells from further HIV infection. However, the factors responsible for drug distribution and concentration into these tissues are largely unknown. Although humanized mice and nonhuman primates (NHPs) are crucial to HIV research, ARV tissue pharmacology has not been well characterized across species. This study investigated the influence of drug transporter expression, viral infection, and sex on ARV penetration within lymph nodes of animal models and humans. Six ARVs were dosed for 10 days in humanized mice and NHPs. Plasma and lymph nodes were collected at necropsy, 24 hours after the last dose. Human lymph node tissue and plasma from deceased patients were collected from tissue banks. ARV, active metabolite, and endogenous nucleotide concentrations were measured by liquid chromatography-tandem mass spectrometry, and drug transporter expression was measured using quantitative polymerase chain reaction and quantitative targeted absolute proteomics. In NHPs and humans, lymph node ARV concentrations were greater than or equal to plasma, and tenofovir diphosphate/deoxyadenosine triphosphate concentration ratios achieved efficacy targets in lymph nodes from all three species. There was no effect of infection or sex on ARV concentrations. Low drug transporter expression existed in lymph nodes from all species, and no predictive relationships were found between transporter gene/protein expression and ARV penetration. Overall, common preclinical models of HIV infection were well suited to predict human ARV exposure in lymph nodes, and low transporter expression suggests primarily passive drug distribution in these tissues. SIGNIFICANCE STATEMENT: During human immunodeficiency virus (HIV) eradication strategies, protective concentrations of antiretrovirals (ARVs) in the lymph node prevent vulnerable cells from further HIV infection. However, ARV tissue pharmacology has not been well characterized across preclinical species used for HIV eradication research, and the influence of drug transporters, HIV infection, and sex on ARV distribution and concentration into the lymph node is largely unknown. Here we show that two animal models of HIV infection (humanized mice and nonhuman primates) were well suited to predict human ARV exposure in lymph nodes. Additionally, we found that drug transporter expression was minimal and-along with viral infection and sex-did not affect ARV penetration into lymph nodes from any species.

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.

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.

Differences in Local and Systemic TFV PK Among Premenopausal Versus Postmenopausal Women Exposed to TFV 1% Vaginal Gel.

OBJECTIVE: We describe and compare the local and systemic pharmacokinetics (PK) of tenofovir (TFV) and TFV-diphosphate (TFV-DP) in healthy premenopausal (PRE) and postmenopausal (POST) women using TFV 1% gel and correlate local PK with other mucosal end points. METHODS: PRE (n = 20) and POST (n = 17) women used 2 doses of TFV 1% vaginal gel, separated by 2 hours. Blood and cervicovaginal samples were obtained 3 and 23 hours after the second dose. PRE women used gel in the follicular and luteal phases of the menstrual cycle. POST women used gel at baseline and again after approximately 2 months of treatment with 0.01% vaginal estradiol (E2) cream. RESULTS: Median TFV concentrations in cervicovaginal aspirate (ng/mL) and vaginal tissue (ng/mg) were significantly higher in PRE (4.3E10, 49.8) versus POST women (2.6E10, 2.2). POST women had significantly higher median molecular ratios of TFV-DP to TFV (3.7%) compared with PRE (0.19%). After vaginal E2 treatment, the local and systemic PK end points in POST women were generally similar to PRE women (all P values > 0.05). Importantly, median vaginal tissue TFV-DP concentrations (fmol/mg) among PRE, POST, and POST women after E2 therapy were similar (292.5, 463.3, and 184.6, respectively). Vaginal tissue TFV concentrations were significantly positively correlated with vaginal epithelial thickness, whereas vaginal tissue TFV-DP concentrations were positively correlated with density of vaginal CD4 and CD8 immune cells. CONCLUSIONS: The state of the cervicovaginal mucosa has a significant impact on local and systemic PK of a topically applied microbicide.

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.

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.

Lack of viral control and development of combination antiretroviral therapy escape mutations in macaques after bone marrow transplantation.

OBJECTIVE: We have previously demonstrated robust control of simian/human immunodeficiency virus (SHIV1157-ipd3N4) viremia following administration of combination antiretroviral therapy (cART) in pigtailed macaques. Here, we sought to determine the safety of hematopoietic stem cell transplantation (HSCT) in cART-suppressed and unsuppressed animals. DESIGN: We compared disease progression in animals challenged with SHIV 100 days post-transplant, to controls that underwent transplant following SHIV challenge and stable cART-dependent viral suppression. METHODS: SHIV viral load, cART levels, and anti-SHIV antibodies were measured longitudinally from plasma/serum from each animal. Flow cytometry was used to assess T-cell subset frequencies in peripheral blood and the gastrointestinal tract. Deep sequencing was used to identify cART resistance mutations. RESULTS: In control animals, virus challenge induced transient peak viremia, viral set point, and durable suppression by cART. Subsequent HSCT was not associated with adverse events in these animals. Post-transplant animals were challenged during acute recovery following HSCT, and displayed sustained peak viremia and cART resistance. Although post-transplant animals had comparable plasma levels of antiretroviral drugs and showed no evidence of enhanced infection of myeloid subsets in the periphery, they exhibited a drastic reduction in virus-specific antibody production and decreased T-cell counts. CONCLUSIONS: These results suggest that virus challenge prior to complete transplant recovery impairs viral control and may promote drug resistance. These findings may also have implications for scheduled treatment interruption studies in patients on cART during post-HSCT recovery: premature scheduled treatment interruption could similarly result in lack of viral control and cART resistance.

Quantitative mass spectrometry imaging of emtricitabine in cervical tissue model using infrared matrix-assisted laser desorption electrospray ionization.

A quantitative mass spectrometry imaging (QMSI) technique using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is demonstrated for the antiretroviral (ARV) drug emtricitabine in incubated human cervical tissue. Method development of the QMSI technique leads to a gain in sensitivity and removal of interferences for several ARV drugs. Analyte response was significantly improved by a detailed evaluation of several cationization agents. Increased sensitivity and removal of an isobaric interference was demonstrated with sodium chloride in the electrospray solvent. Voxel-to-voxel variability was improved for the MSI experiments by normalizing analyte abundance to a uniformly applied compound with similar characteristics to the drug of interest. Finally, emtricitabine was quantified in tissue with a calibration curve generated from the stable isotope-labeled analog of emtricitabine followed by cross-validation using liquid chromatography tandem mass spectrometry (LC-MS/MS). The quantitative IR-MALDESI analysis proved to be reproducible with an emtricitabine concentration of 17.2 ± 1.8 µg/gtissue. This amount corresponds to the detection of 7 fmol/voxel in the IR-MALDESI QMSI experiment. Adjacent tissue slices were analyzed using LC-MS/MS which resulted in an emtricitabine concentration of 28.4 ± 2.8 µg/gtissue.

Immunologic, virologic, and pharmacologic characterization of the female upper genital tract in HIV-infected women.

: A comparative analysis of cellular and soluble markers of immune activation in HIV-infected women on combination antiretroviral therapy showed that the upper genital tract (UGT) compared to the lower female genital tract was characterized by higher frequencies of potential HIV target cells and increased inflammatory molecules. Despite the activated UGT milieu, HIV RNA could not be detected in paired samples of plasma, cervicovaginal or endometrial lavage. As antiretroviral concentrations were ≥3-fold higher in the endometrium than in the lower genital tract, high antiretroviral penetration and/or metabolism may limit viral replication in the UGT.