Postcoital Bioavailability and Antiviral Activity of 0.5% PRO 2000 Gel: Implications for Future Microbicide Clinical Trials

The pharmacokinetics and pharmacodynamics of vaginal microbicides are typically assessed among sexually abstinent women. However, the physical act of sex may modulate gel distribution, and preclinical studies demonstrate seminal plasma interferes with the antiviral activity of several microbicides. This study compared the biological activity and concentration of PRO 2000 in cervicovaginal lavage (CVL) collected in the absence or following coitus.CVL samples were collected from ten heterosexual couples at baseline, after sex, after a single dose of 0.5% PRO 2000 gel and sex, and after gel application without sex. The impact of CVL on HIV-1 infection of TZM-bl cells and HSV-2 infection of CaSki cells was monitored by luciferase and plaque assay, respectively. PRO 2000 concentrations were measured by fluorescence.CVL collected after PRO 2000 application significantly inhibited HIV-1 and HSV-2 (p = 0.01). However, the antiviral activity was reduced following sex and no significant protective effect was observed in postcoital CVL obtained in the presence compared to the absence of PRO 2000 for HIV (p = 0.45) or HSV-2 (p = 0.56). Less PRO 2000 was recovered in postcoital CVL, which, in conjunction with interference by seminal plasma, may have contributed to lower antiviral activity.Postcoital responses to PRO 2000 differ from precoital measures and the results obtained may provide insights into the clinical trial findings in which there was no significant protection against HIV-1 or HSV-2. Postcoital studies should be incorporated into clinical studies before embarking on large-scale efficacy trials

A Randomized Trial to Assess Anti-HIV Activity in Female Genital Tract Secretions and Soluble Mucosal Immunity Following Application of 1% Tenofovir Gel

Preclinical and early phase clinical microbicide studies have not consistently predicted the outcome of efficacy trials. To address this gap, candidate biomarkers of microbicide pharmacodynamics and safety were evaluated in a double-blind, placebo-controlled trial of tenofovir gel, the first microbicide to demonstrate significant protection against HIV acquisition.30 women were randomized to apply a single daily dose of tenofovir or placebo gel for 14 consecutive days. Anti-HIV activity was measured in cervicovaginal lavage (CVL) on Days 0, 3, 7, 14 and 21 by luciferase assay as a surrogate marker of pharmacodynamics. Endogenous activity against E. coli and HSV-2 and concentrations of immune mediators were quantified in CVL as candidate biomarkers of safety. Tenofovir levels were measured in CVL and blood.A significant increase in anti-HIV activity was detected in CVL from women who applied tenofovir gel compared to their endogenous anti-HIV activity in genital tract secretions on Day 0 and compared to activity in CVL from women in the placebo group. The activity correlated significantly with CVL concentration of tenofovir (r = 0.6, p<0.001) and fit a sigmoid E(max) pharmacodynamic model. Anti-HIV activity in CVL from women who applied tenofovir persisted when virus was introduced in semen, whereas endogenous anti-HIV activity decreased. Tenofovir did not trigger an inflammatory response or induce sustained loss in endogenous antimicrobial activity or immune mediators.Tenofovir gel had no deleterious impact on soluble mucosal immunity. The increased anti-HIV activity in CVL, which persisted in the presence of semen and correlated with tenofovir concentration, is consistent with the efficacy observed in a recent clinical trial. These results promote quantified CVL anti-HIV activity as a surrogate of tissue pharmacodynamics and as a potential biomarker of adherence to product. This simple, feasible and inexpensive bioassay may promote the development of models more predictive of microbicide efficacy.ClinicalTrials.gov NCT00594373

Endogenous anti-HIV activity in CVL collected in the absence or following coitus.

<p>TZM-bl cells were infected with 10³ TCID₅₀ HIV-1_BaL in the presence of Visit 1 (baseline) or Visit 2 (postcoital) CVL or control buffer (saline with 1.5 mg/ml bovine serum albumin). Mock infected cells were included as controls. Results are mean RLU obtained from 2 independent experiments, each conducted in triplicate.</p

Loss in the anti-HSV activity in CVL collected following PRO 2000 gel application in the absence or following coitus.

<p>CaSki cells were infected with serial 10-fold dilutions of HSV-2(G) mixed with each CVL sample. After incubation for 2 h, the inoculum was removed by washing and the cells were overlaid with fresh media. Infection was monitored by counting plaques after 48 h. Results are viral titer (pfu/ml) calculated from 2 independent experiments where each sample was tested in duplicate. The upper panel shows results for each subject for Visit 1 (baseline) versus Visit 4 (post-gel), middle panel for Visit 4 (post-gel) versus Visit 3 (post-gel and postcoital) and lower panel for Visit 2 (postcoital) versus Visit 3 (post-gel and postcoital).</p

Seminal plasma interferes with anti-HIV activity of PRO 2000 in cell culture.

<p>TZM-bl cells were infected with HIV-1_BaL that had been diluted in media (no SP) or media containing 5% pooled SP and mixed 1∶1 with serial 2-fold dilutions of unformulated PRO 2000 diluted in pooled CVL obtained from healthy controls. Results are mean ± sd obtained from triplicate wells and are representative of 3 independent experiments (upper panel). Cell proliferation was assessed in parallel in uninfected plates treated with the CVL and pooled SP for 48 h (lower panel). Results are expressed as % viability (relative to mock-treated controls) and are means ± SD of 2 independent experiments where each condition was tested in triplicate.</p

Loss in the anti-HIV activity in CVL collected following a single application of PRO 2000 gel in the absence or following coitus.

<p>TZM-bl indicator cells were exposed to 10³ TCID₅₀ HIV-1_BaL in the presence of each CVL sample and after 48 h incubation, the cells were lysed and infection monitored by luciferase assay. Results are mean RLU obtained from 2 independent experiments, each conducted in triplicate. The upper panel shows results for each subject for Visit 1 (baseline) versus Visit 4 (post-gel), middle panel for Visit 4 (post-gel) versus Visit 3 (post-gel and postcoital) and lower panel for Visit 2 (postcoital) versus Visit 3 (post-gel and postcoital).</p

Demographic and clinical data from 10 couples.

<p>F, female; M, male; min, minutes.</p>*<p>Time (minutes) between intercourse and CVL collection for Visit 2 and time after gel application of CVL collection for Visits 3 and 4.</p

Endogenous anti-HSV activity in CVL collected in the absence or following coitus.

<p>CaSki cells were infected with HSV-2(G) in the presence of Visit 1 (baseline) or Visit 2 (postcoital) CVL or control buffer. Results are presented as viral titer (pfu/ml) obtained from 2 independent experiments, each conducted in duplicate.</p

Viral infection in presence of each CVL or control buffer.

*<p>IQR interquartile range.</p