Hypo-osmolar rectal douche tenofovir formulation prevents simian/human immunodeficiency virus acquisition in macaques

Abstract

In spite of the rollout of oral pre-exposure prophylaxis (PrEP), the rate of new HIV infections remains a major health crisis. In the United States, new infections occur predominantly in men having sex with men (MSM) in rural settings where access to PrEP can be limited. As an alternative congruent with MSM sexual behavior, we have optimized and tested tenofovir (TFV) and analog-based iso-osmolar and hypo-osmolar (HOsm) rectal douches for efficacy against rectal simian/human immunodeficiency virus (SHIV) infection of macaques. Single TFV HOsm high-dose douches achieved peak plasma TFV levels similar to daily oral PrEP, while other formulations yielded lower concentrations. Rectal tissue TFV-diphosphate (TFV-DP) concentrations at the portal of virus entry, however, were markedly higher after HOsm douching than daily oral PrEP. Repeated douches led to significantly higher plasma TFV and higher TFV-DP concentrations in rectal tissue at 24 hours compared with single douches, without detectable mucosal or systemic toxicity. Using stringent repeated intrarectal SHIV exposures, single HOsm high-dose douches delivered greater protection from virus acquisition for more than 24 hours compared with oral PrEP. Our results demonstrate a rapid delivery of protective TFV doses to the rectal portal of virus entry as a potential low-cost and safe PrEP alternative.

Keywords: AIDS/HIV; Pharmacology; Virology.

Figure 1. PK profile of TFV/TFV prodrug IOsm and HOsm douche formulations and oral TDF and TDF/FTC regimens in rhesus macaques.

Median values with 95% CI of (A) plasma TFV, (B) colorectal tissue TFV, and (C) tissue TFV-DP concentrations after administration in groups of 6 monkeys each. (D) Median plasma FTC, colorectal tissue FTC, or tissue FTC-TP after 1-week daily oral PrEP (22 mg/kg TDF combined with 20 mg/kg FTC). One-way ANOVA was used to compare TFV 5.28 mg/mL HOsm to all other douche formulations. The level of significance is indicated by P values as follows: *P < 0.05; **P < 0.01; ***P < 0.001. PK, pharmacokinetic; HOsm, hypo-osmolar; IOsm, iso-osmolar; TFV, tenofovir; TFV-DP, tenofovir diphosphate; TDF, tenofovir disoproxil fumarate; FTC, emtricitabine; FTC-TP, emtricitabine triphosphate.

Figure 2. PK evaluation of 5 repeated short-term intrarectal douches of TFV HOsm high-dose (5.28 mg/mL) formulation.

Expelled fluid volumes after 5 consecutive intrarectal administrations of (A) 30 mL and (B) 60 mL of HOsm high-dose formulation. (C) Comparison of plasma TFV between single-dose PK and 5 repeated doses PK. (D) Comparison of colorectal tissue TFV between single dose PK and 5 repeated doses PK. (E) Comparison of tissue TFV-DP between single-dose PK and 5 repeated doses PK. Differences between the 2 groups (6 monkeys each) were evaluated by the Wilcoxon rank-sum test. (F) PK correlation of retained volumes and TFV-DP concentration in rectal tissues. The correlation coefficients (r) and P values were derived using Spearman rank analysis. The level of significance is indicated by P values as follows: *P < 0.05; **P < 0.01; ***P < 0.001. PK, pharmacokinetic; HOsm, hypo-osmolar; TFV, tenofovir; TFV-DP, tenofovir diphosphate.

Figure 3. Mucosal toxicity testing of 5 repeated short-term intrarectal douches of TFV HOsm high-dose (5.28 mg/mL) formulation.

Representative colorectal biopsy histomorphological photomicrographs from 3 animals at postdose (0 hour), 1 hour, and 24 hours after 5 consecutive intrarectal administrations of (A) 30 mL and (B) 60 mL of HOsm high-dose formulation. There was no evidence of mucosal damage at any time point in any animal; however, microhemorrhages (black arrows) and mild edema (green arrows) are indicated. Scale bar: 200 μm. HOsm, hypo-osmolar; TFV, tenofovir.

Figure 4. Outcome of efficacy study of weekly repeated intrarectal SHIV challenges.

Plasma peak acute viral loads illustrate infection results following weekly repeated intrarectal challenges with simian/human immunodeficiency virus 162p3 (SHIV162p3) for (A) group 1, TDF oral daily; (B) group 2, TDF/FTC oral daily; (C) group 3, HOsm high; (D) group 4, IOsm high; and (E) group 5, mock vehicle control. Red arrow indicates time of infection and black arrows mark sequential weekly rectal SHIV challenges. # indicates Mamu A01⁺ macaque distribution among the groups. HOsm, hypo-osmolar; IOsm, iso-osmolar; TDF, tenofovir disoproxil fumarate; TFV, tenofovir; FTC, emtricitabine.

Figure 5. Protective efficacy of each intervention group against repeated intrarectal SHIV162p3 challenges.

Number of challenges required for acquisition of infection in each group. Each Kaplan-Meier survival curve represents the cumulative percentage of uninfected macaques as a function of the number of weekly rectal virus exposures. Animals in the HOsm and IOsm groups were administered a single douche 1, 24, or 48 hours prior to challenge, while oral treatments were continuous. The exact log-rank test was used to ascertain statistical difference between groups. Statistical analyses were considered significant of P < 0.05. Exp, exposure to SHIV162p3; HOsm, hypo-osmolar; IOsm, iso-osmolar; TDF, tenofovir disoproxil fumarate; FTC, emtricitabine.

Figure 6. No correlations between virus acquisition and plasma drug levels in group 1 (TDF oral daily) or 2 (TDF/FTC oral daily).

(A) Group 1 individual macaque plasma TFV value at time of infection (red-filled symbol) during oral TDF high-dose and low-dose treatment. # indicates Mamu A01⁺ macaques. (B) No correlation between virus acquisition and plasma TFV levels during high-dose TDF treatment in group 1. (C) Comparison of peak acute viremia of animals infected in high-dose or low-dose TDF treatment in group 1. (D) Group 2 individual macaque plasma TFV value at time of infection (red-filled symbol) during oral TDF/FTC high-dose and low-dose treatment. (E) No correlation between virus acquisition and plasma TFV levels during high-dose TDF/FTC treatment in group 2. (F) Comparison of peak acute viremia of animals infected in high-dose or low-dose and no TDF/FTC treatment in group 2. (G) Group 2 individual plasma FTC value at time of infection (red-filled symbol) during oral TDF/FTC high dose and low dose treatment. (H) No correlation between virus acquisition and plasma FTC levels during high-dose TDF/FTC treatment in group 2. The red number indicates time of infection. The correlation coefficients (r) and P values are from Spearman rank analysis. Differences between the 2 groups were evaluated by the Wilcoxon rank-sum test. Statistical analyses were considered significant for P values of < 0.05. TFV, tenofovir; TDF, tenofovir disoproxil fumarate; FTC, emtricitabine.