In Vitro and Ex Vivo Testing of Tenofovir Shows It Is Effective As an HIV-1 Microbicide

Abstract

BackgroundTenofovir gel has entered into clinical trials for use as a topical microbicide to prevent HIV-1 infection but has no published data regarding pre-clinical testing using in vitro and ex vivo models. To validate our findings with on-going clinical trial results, we evaluated topical tenofovir gel for safety and efficacy. We also modeled systemic application of tenofovir for efficacy.Methods and FindingsFormulation assessment of tenofovir gel included osmolality, viscosity, in vitro release, and permeability testing. Safety was evaluated by measuring the effect on the viability of vaginal flora, PBMCs, epithelial cells, and ectocervical and colorectal explant tissues. For efficacy testing, PBMCs were cultured with tenofovir or vehicle control gels and HIV-1 representing subtypes A, B, and C. Additionally, polarized ectocervical and colorectal explant cultures were treated apically with either gel. Tenofovir was added basolaterally to simulate systemic application. All tissues were challenged with HIV-1 applied apically. Infection was assessed by measuring p24 by ELISA on collected supernatants and immunohistochemistry for ectocervical explants. Formulation testing showed the tenofovir and vehicle control gels were >10 times isosmolar. Permeability through ectocervical tissue was variable but in all cases the receptor compartment drug concentration reached levels that inhibit HIV-1 infection in vitro. The gels were non-toxic toward vaginal flora, PBMCs, or epithelial cells. A transient reduction in epithelial monolayer integrity and epithelial fracture for ectocervical and colorectal explants was noted and likely due to the hyperosmolar nature of the formulation. Tenofovir gel prevented HIV-1 infection of PBMCs regardless of HIV-1 subtype. Topical and systemic tenofovir were effective at preventing HIV-1 infection of explant cultures.ConclusionsThese studies provide a mechanism for pre-clinical prediction of safety and efficacy of formulated microbicides. Tenofovir was effective against HIV-1 infection in our algorithm. These data support the use of tenofovir for pre-exposure prophylaxis.

Vaginal Microbicide Gel for Delivery of IQP-0528, a Pyrimidinedione Analog with a Dual Mechanism of Action against HIV-1

“…To confirm the results obtained with the VEC-100 tissue, human ectocervical tissue was obtained after surgery and placed in culture using a polarized system (7,26). The HEC IQP-0528 gel and HEC placebo gel preserved tissue viability similarly to control (untreated) explants, as measured by the MTT assay (Fig.…”

“…For the Carbopol formulation, we estimate approximately 0.003 M IQP-0528 permeated the ectocervical tissue after 6 h. After comparison of the drug release profiles of IQP-0528 from the HEC and the Carbopol gels, we observed that the HEC gel demonstrated a shorter lag time (4 h) and greater permeation across the tissue. However, for the IQP-0528 HEC formulation, the lag time, which is the time needed to attain inhibitory concentrations of drug in the basal compartment, was eight times longer than that of the 1% tenofovir gel (lag time Ͻ 30 min) (26). This delay can be attributed to the low permeability of IQP-0528.…”

“…Evaluations of the safety and antiviral activity of the lead formulation, IQP-0528, were performed as previously described (7,26). Briefly, the explant was placed in a transwell with the luminal side up.…”

“…To ensure an even spread of the gels and to allow them to be mixed with HIV-1 for the efficacy evaluation (below), a 1:5 dilution of IQP-0528 or HEC placebo gels was applied to the apical side of the explants for 18 h. Control explants were untreated or treated with an apically applied 1:5 dilution of 2% N-9 gel (Gynol II; purchased over the counter). The following day, the explants were washed and safety was evaluated by using an MTT [1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan] assay and histology methods (7,26).…”

“…We evaluated the safety and efficacy of the PYD gel formulation using reconstructed VEC-100 tissue (MatTek Corp.) and human polarized ectocervical tissue. The VEC-100 tissue recapitulates only the morphological characteristics of the stratified squamous epithelial layer, whereas the polarized ectocervical explants contains both epithelium and relevant immune cells (2,7,8,26). In an in vitro HIV-1 entry inhibition assay the 3.0% HEC gel with 0.25% IQP-0528 demonstrated a 50% effective concentration (EC 50 ) of 0.14 g/ml of gel in culture media, corresponding to ϳ0.001 M IQP-0528, and showed complete protection of the human polarized ectocervical tissue against HIV infection.…”