Mackenzie L. Cottrell

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.

Pharmacokinetics of antiretrovirals in mucosal tissue

Introduction: In the absence of an HIV vaccine or cure, antiretroviral (ARV)-based prevention strategies are being investigated to reduce HIV incidence. These prevention strategies depend on achieving effective drug concentrations at the site of HIV exposure, which is most commonly the mucosal tissue of the lower gastrointestinal tract and the female genital tract. Areas covered: This article collates all known data regarding drug exposure in these vulnerable mucosal tissues and reviews important mechanisms of ARV drug distribution. Research papers and abstracts describing ARV pharmacokinetics (PK) in the female genital tract and lower gastrointestinal mucosal tissues available in MEDLINE® or presented at scientific conferences prior to December 2014 are reviewed in detail. Important influences on ARV mucosal tissue distribution, including protein binding, active drug transport and endogenous hormones are also reviewed. Expert opinion: ARVs exhibit highly variable PK in mucosal tissues. In general, ARV exposure is higher in the lower gastrointestinal tract compared with the female genital tract, but concentrations required for protective efficacy are largely unknown. The expected site of HIV exposure represents an important consideration when designing and optimizing ARV-based prevention strategies.

Topical microbicides and HIV prevention in the female genital tract

Worldwide, HIV disproportionately affects women who are often unable to negotiate traditional HIV preventive strategies such as condoms. In the absence of an effective vaccine or cure, chemoprophylaxis may be a valuable self‐initiated alternative. Topical microbicides have been investigated as one such option. The first generation topical microbicides were non‐specific, broad‐spectrum antimicrobial agents, including surfactants, polyanions, and acid buffering gels, that generally exhibited contraceptive properties. After extensive clinical study, none prevented HIV infection, and their development was abandoned. Second generation topical microbicides include agents with selective mechanisms of antiviral activity. Most are currently being used for, or have previously been explored as, drugs for treatment of HIV. The most advanced of these is tenofovir 1% gel: the first topical agent shown to significantly reduce HIV infection by 39% compared to placebo. This review summarizes the evolution of topical microbicides for HIV chemoprophylaxis, highlights important concepts learned, and offers current and future considerations for this area of research.Worldwide, HIV disproportionately affects women who are often unable to negotiate traditional HIV preventive strategies such as condoms. In the absence of an effective vaccine or cure, chemoprophylaxis may be a valuable self-initiated alternative. Topical microbicides have been investigated as one such option. The first generation topical microbicides were non-specific, broad-spectrum antimicrobial agents, including surfactants, polyanions, and acid buffering gels, that generally exhibited contraceptive properties. After extensive clinical study, none prevented HIV infection, and their development was abandoned. Second generation topical microbicides include agents with selective mechanisms of antiviral activity. Most are currently being used for, or have previously been explored as, drugs for treatment of HIV. The most advanced of these is tenofovir 1% gel: the first topical agent shown to significantly reduce HIV infection by 39% compared to placebo. This review summarizes the evolution of topical microbicides for HIV chemoprophylaxis, highlights important concepts learned, and offers current and future considerations for this area of research.

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 r2 values were used to select the final predictive models. Results:CVF exposure increased linearly with dose for all antiretrovirals (r2 ≥ 0.23, P ⩽ 0.02) except raltegravir (r2 = 0.08, P = 0.19). In RF, only emtricitabine increased linearly with dose (r2 = 0.27, P = 0.01). For all antiretrovirals, CVF and RF concentrations significantly correlated with mucosal tissue concentrations (female genital tract r2 ≥ 0.37, rectal tissue r2 ≥ 0.50, P ⩽ 0.001). In the final multivariate models, plasma and fluid concentrations were both associated with FGT concentrations for all antiretrovirals (r2 ≥ 0.81, P < 0.001). The same was noted for rectal tissue (r2 ≥ 0.58, P < 0.001) except for tenofovir, for which RF alone was predictive of tissue concentration (r2 = 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.

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 alafenamides 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 alafenamides pharmacology.

A spatio-temporal assessment of simian/human immunodeficiency virus (SHIV) evolution reveals a highly dynamic process within the host

The process by which drug-resistant HIV-1 arises and spreads spatially within an infected individual is poorly understood. Studies have found variable results relating how HIV-1 in the blood differs from virus sampled in tissues, offering conflicting findings about whether HIV-1 throughout the body is homogeneously distributed. However, most of these studies sample only two compartments and few have data from multiple time points. To directly measure how drug resistance spreads within a host and to assess how spatial structure impacts its emergence, we examined serial sequences from four macaques infected with RT-SHIVmne027, a simian immunodeficiency virus encoding HIV-1 reverse transcriptase (RT), and treated with RT inhibitors. Both viral DNA and RNA (vDNA and vRNA) were isolated from the blood (including plasma and peripheral blood mononuclear cells), lymph nodes, gut, and vagina at a median of four time points and RT was characterized via single-genome sequencing. The resulting sequences reveal a dynamic system in which vRNA rapidly acquires drug resistance concomitantly across compartments through multiple independent mutations. Fast migration results in the same viral genotypes present across compartments, but not so fast as to equilibrate their frequencies immediately. The blood and lymph nodes were found to be compartmentalized rarely, while both the blood and lymph node were more frequently different from mucosal tissues. This study suggests that even oft-sampled blood does not fully capture the viral dynamics in other parts of the body, especially the gut where vRNA turnover was faster than the plasma and vDNA retained fewer wild-type viruses than other sampled compartments. Our findings of transient compartmentalization across multiple tissues may help explain the varied results of previous compartmentalization studies in HIV-1.

Tenofovir/emtricitabine metabolites and endogenous nucleotide exposures are associated with p16INK4a 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.

Effect of HIV infection and menopause status on raltegravir pharmacokinetics in the blood and genital tract

BACKGROUND This study describes first dose and steady state pharmacokinetics of raltegravir (RAL) in cervicovaginal fluid (CVF) and blood plasma (BP). METHODS Three cohorts of women were enrolled sequentially in a single-site, open-label pharmacokinetic study of oral raltegravir 400 mg twice daily: HIV-negative premenopausal, HIV-infected premenopausal and HIV-infected post-menopausal women. BP and CVF were collected over 12 h after a single observed dose and at steady state. RAL concentrations were measured by HPLC-MS methods. Data are expressed as median (IQR). The ANOVA rank-sum test was used to evaluate between-group differences in steady state raltegravir exposure (area under the concentration-time curve over the 12-h dosing interval [AUC0-12 h]). RESULTS First dose pharmacokinetics were obtained in HIV-negative premenopausal women and HIV-infected post-menopausal women only. The median (IQR) BP AUC0-12 h was 3,099 (985-5,959) and 4,239 (2,781-13,695) ng•h/ml and the median (IQR) CVF AUC0-12 h was 1,720 (305-5,288) and 13,797 (11,066-19,563) ng•h/ml for HIV-negative premenopausal and HIV-infected post-menopausal women, respectively. All cohorts contributed to steady-state pharmacokinetic profiles. Median (IQR) BP AUC0-12 h did not differ between the groups: 8,436 (3,080-10,111), 5,761 (1,801-10,095) and 6,180 (5,295-8,282) ng•h/ml in HIV-negative premenopausal, HIV-infected premenopausal and HIV-infected post-menopausal women, respectively. There was a trend for lower CVF AUC0-12 h among HIV-negative women 3,164 (1,156-9,540) compared to 11,465 (9,725-17,138) and 9,568 (4,271-24,306) ng•h/ml HIV-infected premenopausal and HIV-infected post-menopausal women, respectively, but this was not statistically significant (P=0.08). HIV-negative premenopausal women had a median (IQR) CVF:BP AUC0-12 h ratio of 0.46 (0.2-1.1), whereas HIV-infected premenopausal and post-menopausal women had median (IQR) CVF:BP AUC0-12 h ratio of 3.9 (1.2-6.7) and 1.4 (0.7-4.3), respectively. CONCLUSIONS This is the first study to investigate RAL exposure in BP and CVF in premenopausal HIV-negative and pre- and post-menopausal HIV-infected women. These data indicate HIV and menopausal status may influence antiretroviral distribution into the female genital tract.

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-1β, 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.

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

Abstract: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.