vitro and ex vivo testing of tenofovir shows it is effective as an HIV-1 microbicide

Abstract Background: Tenofovir 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

Tenofovir releases from the formulated gel.

<p>The <i>in vitr</i>o release data show the release profile of tenofovir from the gel; the slope of the line represents the release rate of the product. The data shown represent the mean ± standard deviation of 13 replicates.</p

Microbicide pre-clinical testing algorithm.

<p>Tenofovir and vehicle control gels were evaluated through a comprehensive pre-clinical algorithm. The algorithm focuses on testing the formulation's physiochemical properties and the ability to release the drug; <i>in vitro</i> testing that includes safety of vaginal flora, epithelial and immune cells, and efficacy against multiple HIV-1 clades; and <i>ex vivo</i> testing using ectocervical and colorectal explants to evaluate drug absorption and formulation safety and efficacy against HIV-1. The data obtained from this algorithm along with data supplied by the manufacturer aid in the decision to continue testing the product.</p

Efficacy of tenofovir against primary isolates of HIV-1.

<p>Peripheral blood mononuclear cells were activated and cultured with HIV-1 (BaL, laboratory-adapted CCR5-using clade B isolate; A103, primary CCR5/CXCR4-using clade A isolate; C012, primary CCR5-using clade C isolate; and C959, primary CCR5-using clade C isolate) with or without tenofovir or vehicle control gel. After 4 hours, the cultures were washed and fresh medium was added. Supernatant was collected every 3 to 4 days and stored at -80°C. HIV-1 infection was followed using a p24gag ELISA. The data shown represent the log₁₀-transformed (pg/mL) ±95% confidence interval of 4 (BaL) or 5 (A103, C012, and C959) independent experiments.</p

Tenofovir permeates through ectocervical tissue.

<p>Tissue permeability data is shown as the cumulative amount of tenofovir which permeated through excised human ectocervical tissue into the receptor chamber of a Franz cell over time. The permeability data represent five separate tissue donors. Tissue permeability data illustrate the intra- and inter-patient variability.</p

Viability of explant cultures after a 24 hour exposure to tenofovir or vehicle control gels.

<p>Ectocervical and colorectal explants were polarized and tenofovir or vehicle control gels were diluted 1:5 in the appropriate culture medium and applied to the apical surface. A 1:5 dilution of nonoxynol-9 (N9) was applied apically to the explants at the same time as the tenofovir and vehicle control gels as a toxicity control. Untreated explants were the negative control tissues. The explants were cultured for 24 h, washed five times, and placed in either medium containing 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) to assess tissue viability by measuring mitochondrial activity (A) or formalin to fix the tissue for hematoxylin-eosin staining for histology (original magnification 20×; bar length 0.05 mm) (B). Explants from five tissue donors were evaluated for viability after exposure to topical gels. The % viability was determined by dividing the corrected optical density of the treated explant by the corrected optical density of the control explant. Histology shown is representative of the five tissues evaluated.</p

Tenofovir applied topically or systemically protect ectocervical and colorectal explants from HIV-1 infection.

<p>Tenofovir or vehicle control gels were mixed with HIV-1_BaL and applied to the apical side of ectocervical (A) or colorectal (B) explants. In a separate study to simulate systemic dosing, tenofovir (1 mg/ml) was added to the basolateral side of the explants 15 min prior to the addition of HIV-1_BaL to the apical side of the tissue. After an overnight incubation, the explants were washed and cultured for 21 days with medium sampled and replaced every 3 to 4 days. HIV-1 replication was monitored in the supernatants using a p24gag ELISA. The data shown represent the median ±95% confidence interval of a minimum of 3 independent tissues performed in duplicate. The wide confidence intervals reflect the p24 variability between tissue donors. For ectocervical tissue, immunohistochemistry for p24gag positive cells of the day 21 explants was done and representative pictures are shown. Arrows indicate p24gag positive cells.</p