Intravaginal ring delivery of tenofovir disoproxil fumarate for prevention of HIV and herpes simplex virus infection

Abstract

Objectives: A safe and effective topical prevention strategy will likely require sustained delivery of potent antiviral drugs and a delivery system that simultaneously maximizes drug distribution and overcomes the behavioural challenges related to adherence. Activity against HIV and herpes simplex virus (HSV) would be advantageous, given the epidemiological link between the two pathogens. We hypothesize that tenofovir disoproxil fumarate (tenofovir DF), a prodrug of tenofovir, may be more potent than tenofovir and ideal for sustained intravaginal ring (IVR) delivery.

Methods: The anti-HIV and anti-HSV activity of tenofovir and tenofovir DF were assessed in cell and explant models. Cumulative tenofovir DF release and stability from polyether urethane (PEU), ethylene-co-vinyl acetate (EVA) and silicone IVRs were compared, and the activity and safety of drug released were evaluated in cervical explants and in a polarized dual-chamber model.

Results: Tenofovir DF inhibited HIV and HSV at ≈ 100-fold lower concentrations than tenofovir and retained activity in the presence of semen. PEU rings delivered >1 mg/day of tenofovir DF for 30 days. Pre-treatment of cervical explants with 10 μg/mL tenofovir DF or eluants from PEU minirings resulted in >90% inhibition of HIV and reduced HSV-2 yields by 2.5 log. Tenofovir DF and eluants did not prevent cell growth or polarization, or have any deleterious effects on an epithelial barrier.

Conclusions: The findings support the development of a PEU tenofovir DF ring, which may provide potent and sustained protection against HIV and HSV.

Figures

Figure 1.

(a) Tenofovir disoproxil hydrolysis to form mPTFV with the loss of one molecule each of isopropanol, formaldehyde and carbon dioxide. Further breakdown of the mPTFV yields tenofovir. (b) Proposed pharmacokinetic model for vaginal delivery of tenofovir DF from an IVR. Tenofovir DF is released from the IVR compartment because of a large concentration gradient into vaginal fluid, where the drug may hydrolyse to form mPTFV and tenofovir. Tenofovir DF more readily diffuses into the cellular compartment due to its increased hydrophobicity and therefore partitions into the cell membrane. In cells, tenofovir DF is converted into TFV-DP after several enzyme-catalysed reactions. Ultimately, the drug partitions into the bloodstream and is eliminated. TDF, tenofovir DF; TFV, tenofovir; mPTFV, monoprotected tenofovir.

Figure 2.

Antiviral activity of tenofovir and tenofovir DF in vitro. (a) JT-CCR5 cells were pre-treated with indicated concentrations of tenofovir or tenofovir DF for 24 h prior to challenge with 103 TCID50 of HIV-1BaL in the presence or absence of 25% SP. Following 2 h of incubation at 37°C, cells were washed to remove drug and inocula, and cultured. Infection was monitored by determining the p24 level in culture supernatants 5 days post-infection. Results are means ± SEM from two experiments conducted in triplicate. (b) CaSki cells were pre-treated with the indicated doses of tenofovir, tenofovir DF or aciclovir for 12–16 h and then challenged with HSV-2 (4674) in the presence or absence of 25% semen (SE). Following 1 h of incubation, the inoculum was removed and cells were overlaid with medium. Plaques were counted 48 h post-infection. Results are means ± SEM obtained from at least two experiments conducted in duplicate. TDF, tenofovir DF; TFV, tenofovir; ACV, aciclovir.

Figure 3.

(a) Photograph showing 10 wt% tenofovir DF-loaded human-sized PEU IVR (55 × 5.3 mm) and miniring (6.7 × 1.4 mm). (b) 30 day cumulative tenofovir DF release in vitro from different polymers (PEU, EVA and silicone) under simulated conditions. (c) In vitro daily and cumulative tenofovir DF release from PEU segments with and without 0.5 wt% PAA (pH modifier). (d) mPTFV recovery from PEU segments with and without 0.5 wt% PAA subjected to in vitro release studies for 30 days in simulated vaginal fluid. mPTFV levels were calculated from tenofovir DF calibration curves assuming similar molar absorption coefficients at 260 nm. The concentration of mPTFV was below the limit of quantification (LOQtenofovir DF = 0.06 μg/mL) on day 10 in PEU segments with PAA. Results are means ± SD from three independent experiments. TDF, tenofovir DF; PAA, poly(acrylic acid).

Figure 4.

Antiviral activity of tenofovir DF released from ring formulation. (a) TZM-bl cells were infected with 103 TCID50 of HIV-1BaL in the presence or absence of diluted eluants from minirings (10 wt% and 5 wt% tenofovir DF or placebo). Virus and eluant were left in culture for 48 h at 37°C, and infectivity was monitored by a luciferase assay. (b) Cell viability was determined by MTS assay following exposure to eluant for 48 h. Results are means ± SEM obtained from two experiments conducted in triplicate. (c) Ectocervical explants were exposed to miniring eluants [10 wt% (10), 5 wt% (5) or placebo (P)] or the indicated concentrations of tenofovir DF for 24 h prior to viral challenge with 105 TCID50 HIV-1BaL. Virus and drug were removed by extensive washing after 2 h and explants were cultured for 14 days. 50% of the culture medium was replaced every 2–3 days and HIV provirus integration was assessed by RTQ-PCR. LTR relative copy numbers are expressed as the percentage of control explants challenged in the absence of inhibitor and are representative of three experiments, where each condition was tested in triplicate. (d) Ectocervical explant cultures were exposed to minirings (5 wt% tenofovir DF and placebo) or 1 mg/mL tenofovir DF or aciclovir for 24 h prior to challenge with 106 pfu/explant HSV-2 (G); input virus and drug were removed as above. Supernatants were collected 5 days post-infection and virus yields determined by plaque assays on Vero cells. Results are presented as virus yields and are means ± SEM obtained from at least three experiments conducted in triplicate. RLUs, relative luciferase units; TDF, tenofovir DF; ACV, aciclovir; LTR, long terminal repeat; API, active pharmaceutical ingredient; RCN, relative copy number.

Figure 5.

Exposure to tenofovir DF has no deleterious effect on the epithelial barrier. HEC-1-A cells were cultured in Transwell® inserts (0.5–1 × 105 cells/insert) and TER was monitored daily. After the TER reached a plateau (4–5 days), cells were exposed to the indicated microbicides (0.1% v/v N-9 and 10–100 µg/mL tenofovir DF) for 18 h. After removal of microbicides by washing three times, HIV-1BaL (40 ng p24) and JT-CCR5 cells (1 × 105/well) were added to the upper and lower chambers, respectively. (a) The TER was monitored 24 h after drug removal and then daily. (b) Supernatants were collected from the baso-lateral chambers at indicated times post-apical addition of HIV and tested for p24 content by ELISA. Results are means ± SD of two independent experiments, where each condition was tested in duplicate. (c) Alternatively, HEC-1-A cells were cultured in Transwell® inserts in the presence of tenofovir DF (unformulated or miniring eluant), culture medium or after treatment with mitomycin C (toxic control) and TER monitored daily. Results are means ± SD of three independent experiments, where each condition was tested in duplicate except miniring eluant and mitomycin C (two experiments). TDF, tenofovir DF.