Intravaginal ring eluting tenofovir disoproxil fumarate completely protects macaques from multiple vaginal simian-HIV challenges

Abstract

Topical preexposure prophylaxis interrupts HIV transmission at the site of mucosal exposure. Intermittently dosed vaginal gels containing the HIV-1 reverse transcriptase inhibitor tenofovir protected pigtailed macaques depending on the timing of viral challenge relative to gel application. However, modest or no protection was observed in clinical trials. Intravaginal rings (IVRs) may improve efficacy by providing long-term sustained drug delivery leading to constant mucosal antiretroviral concentrations and enhancing adherence. Although a few IVRs have entered the clinical pipeline, 100% efficacy in a repeated macaque vaginal challenge model has not been achieved. Here we describe a reservoir IVR technology that delivers the tenofovir prodrug tenofovir disoproxil fumarate (TDF) continuously over 28 d. With four monthly ring changes in this repeated challenge model, TDF IVRs generated reproducible and protective drug levels. All TDF IVR-treated macaques (n = 6) remained seronegative and simian-HIV RNA negative after 16 weekly vaginal exposures to 50 tissue culture infectious dose SHIV162p3. In contrast, 11/12 control macaques became infected, with a median of four exposures assuming an eclipse of 7 d from infection to virus RNA detection. Protection was associated with tenofovir levels in vaginal fluid [mean 1.8 × 10(5) ng/mL (range 1.1 × 10(4) to 6.6 × 10(5) ng/mL)] and ex vivo antiviral activity of cervicovaginal lavage samples. These observations support further advancement of TDF IVRs as well as the concept that extended duration drug delivery devices delivering topical antiretrovirals could be effective tools in preventing the sexual transmission of HIV in humans.

Keywords: PrEP; controlled drug delivery; nonhuman primate; pharmacokinetics.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

TDF IVR design and in vitro release. (A) Schema depicting mechanism of drug release from TDF reservoir IVR. Vaginal fluid hydrates the swellable HPEU tubing (Left) and water is driven into the osmotically active drug–NaCl core along a gradient, resulting in TDF dissolution and elution from the IVR (Right). NaCl aids in establishing soluble drug in the core and achieving equilibrium drug release (Fig. 2B). (B) Photograph of macaque (left) and human (right) TDF IVR. (Scale bar: 1 cm.) (C) Comparison of 28-d in vitro TDF release rates from matrix (n = 3) and reservoir (n = 6) IVRs under simulated vaginal conditions (2). Data represented as mean ± SD. The SDs of some of the collected data are too small to be visualized.

Fig. 2.

Matrix release and design evaluation of reservoir TDF IVR. (A) Comparison of TDF released from matrix PEU IVR in vitro on days 3 and 21 to drug concentrations in vaginal fluid of Chinese rhesus macaques (n = 3) in a 28 + 3-d study. Each data point represents a single sample and the bar corresponds to the mean for that dataset. (B) In vitro TDF release rate from HPEU reservoir IVR filled with TDF and TDF-NaCl formulation under simulated vaginal conditions (n = 3). Data represented as mean ± SD. (C) In vitro TDF and NaCl release rates from HPEU reservoir IVR under simulated vaginal conditions (n = 3). Data represented as mean ± SD. (D) Comparison of initial in vitro TDF release (up to day 3) from heat-treated and control-unheated TDF IVR (n = 3). Heat treatment of the TDF reservoir IVR at 65 °C for 5 d increased TDF release in the first 2 d. Data represented as mean ± SD.

Fig. 3.

Drug PK in pigtailed (28 + 2-d) and rhesus (14-d) macaques. Each data point represents a single sample and the bar corresponds to the mean for that dataset. (A) TFV (Upper) and TDF (Lower) concentrations in pigtailed macaque vaginal fluid with 28-d TDF IVR administration (n = 6). Samples were collected proximal (open symbols) and distal (closed symbols) to IVR placement for the indicated time points. (B) TFV concentrations in vaginal biopsies from 28-d TDF IVR administration. Samples were collected proximal (open symbols) and distal (closed symbols) to IVR placement for the indicated time points. (C) TFV-DP levels in lymphocytes isolated from the indicated tissues of rhesus macaques after 14-d IVR administration (n = 3).

Fig. 4.

TDF IVR protects macaques from repeated vaginal viral challenge. (A) Six TDF IVR-treated cycling female macaques received weekly 50 TCID50 SHIV162p3 inoculations starting 6 d after the first IVR insertion. Control macaques (n = 6 real time and n = 6 historical controls) were challenged similarly. The ring was replaced as shown in red (every 28 d starting 2 d after the fourth virus exposure). Macaques were monitored weekly (until week 20) for presence of SHIV by RT-PCR and confirmed by Western blot. Macaques were defined as infected and exposures discontinued if vRNA was detected in plasma for 2 consecutive weeks. (B) Kaplan-Meier plot showing time to infection for TDF IVR (n = 6; red) and control (n = 6 real time and 6 historical naïve; black) groups (nonparametric log-rank test; P = 0.0007). The median number of exposures to infection in the untreated group was four. (C) Plasma viral load kinetics in infected macaques aligned at peak. The red line is the median for all infected macaques (11/12). vRNA, viral RNA.

Fig. 5.

Drug PK from efficacy study and PK/PD correlation. (A) Monthly TFV (open symbols) and TDF (closed symbols) concentrations in vaginal fluid of pigtailed macaques in the efficacy study with four TDF IVR changes (n = 6 macaques). Each data point represents a single sample (proximal or distal to IVR placement) and the bar corresponds to the mean for that dataset (n = 12; two samples per animal). (B) To monitor drug PK/PD during the efficacy challenge study, two macaques were treated with TDF IVR (closed symbol) or placebo IVR control (open symbol) and CVL samples were collected at the indicated times in the absence of viral challenge. CVL samples (1:10 dilution) were assayed for drug levels and ability to inhibit HIV-1BaL infection in TZM-bl cells. Results are presented as percentage inhibition of infection relative to control wells; each data point represents the average of two experiments conducted in triplicate (n = 2 macaques, mean ± SEM). (C) Correlation of CVL (diluted 1:10) antiviral activity against HIV-1BaL infection in TZM-bl cells to TDF and TFV concentrations (Spearman PK/PD correlation; TDF, r = 0.57; TFV, r = 0.61). Samples with TDF or TFV levels below the LLOQ were attributed the value of 0.1 ng/mL so that data could be plotted on a log scale.