PRO 2000 elicits a decline in genital tract immune mediators without compromising intrinsic antimicrobial activity.

OBJECTIVE: Vaginal microbicides should protect against infection without disrupting the mucosal environment or its mediators of host defense. The objective of this study was to examine the effect of 14 daily applications of 0.5% PRO 2000 or placebo gel on mediators of mucosal immunity and intrinsic antimicrobial activity. DESIGN AND METHODS: A randomized, prospective, double-blind, placebo-controlled study was conducted among 24 healthy, abstinent women. Levels of cytokines, chemokines, defensins, and other protective factors and intrinsic antimicrobial activity were determined in cervicovaginal lavage samples collected on study days 0, 7, 14, and 21. RESULTS: No increase in pro-inflammatory cytokines was observed. Rather cytokines and protective factors including interleukin (IL)-1 receptor antagonist, immunoglobulins and human beta-defensin 2 were lower in the drug compared with the placebo group. All of the mediators returned towards baseline on day 21. Women who were cycling had lower levels of most proteins on study days 7 and/or 14 compared with women on oral contraceptives; however, the magnitude of decline was greater in women who received PRO 2000 compared with placebo gel. The reduction in protective factors was not associated with a loss in the intrinsic anti-viral (HIV or herpes simplex virus) activity or anti-bacterial activity (Escherichia coli or Staphylococcus aureus). CONCLUSION: In contrast to experience with nonoxynol-9, PRO 2000 did not trigger an inflammatory response in cervicovaginal secretions. There was a modest reduction in mucosal immune mediators, but this loss was not associated with a reduction in intrinsic antimicrobial activity.

Amphipathic DNA polymers are candidate vaginal microbicides and block herpes simplex virus binding, entry and viral gene expression.

BACKGROUND: Amphipathic DNA polymers are promising therapies for the prevention of HIV and genital herpes infections. Recent studies on a panel of such compounds indicated potent activity against HIV binding and entry. This current study was conducted to explore the anti-herpes simplex virus (HSV) activity of the same panel of compounds and to determine their mechanism of activity. METHODS: The anti-HSV activity of a 40-nucleotide degenerate polymer (REP 9), a 40-nucleotide polycytidine amphipathic DNA polymer (REP 9C) and an analogue lacking amphipathic activity (Randomer 3) were compared in plaque reduction assays in the absence or presence of human genital tract secretions; the mechanisms of anti-HSV activity were explored. RESULTS: REP 9 inhibited HSV infection 10,000-fold, whereas Randomer 3 displayed no anti-HSV activity. The antiviral activity was independent of sequence but was dependent on size: the most potent activity was observed for analogues of 40 nucleotides in length. Mechanistic studies indicated that REP 9 and REP 9C blocked HSV-2 binding and entry, were active when added post-entry, inhibited viral gene expression and blocked HSV-induced apoptosis. Confocal microscopy studies showed rapid delivery of fluorescently tagged REP 9 and REP 9C into human epithelial cells, and delivery was significantly greater in infected cells as compared with uninfected cells. REP 9 exhibited no cytotoxicity and retained anti-HSV activity in the presence of cervicovaginal secretions and when virus was introduced in seminal plasma. CONCLUSIONS: REP 9 and REP 9C represent a novel class of antiviral agents that act by multiple mechanisms. These compounds warrant further development for systemic or topical delivery for the prevention and treatment of HIV and HSV.

Bridging the gap between preclinical and clinical microbicide trials: blind evaluation of candidate gels in murine models of efficacy and safety.

BACKGROUND: Despite significant protection in preclinical studies, cellulose sulfate (CS) failed to protect women against HIV-1/2 and was associated with a trend toward increased HIV-1 acquisition in one of the clinical trials. These results highlight the need for preclinical tests more predictive of clinical outcomes. The objective of this study was to test coded vaginal gels, including CS, in murine models of safety and efficacy to determine the models' utility for evaluating future products. METHODS: Four coded formulations, including 6% CS, 2% PRO 2000 and two placebo gels, were administered intravaginally to medroxyprogesterone-treated mice and their ability to prevent genital herpes (efficacy) or to alter the susceptibility to low dose HSV challenge (safety) was determined. Nonoyxnol-9 served as a positive toxicity control. RESULTS: CS and PRO 2000 significantly protected mice from genital herpes following infection with a laboratory or clinical isolate of HSV-2 introduced in buffer (p<0.001). However, protection was reduced when virus was introduced in seminal plasma. Moreover, mice were significantly more susceptible to infection with low doses of HSV-2 when challenged 12 h after the 7th daily dose of CS or nonoxynol-9 (p<0.05). The increased susceptibility was associated with alterations in epithelial architecture. CONCLUSIONS: CS prevented genital herpes when present at the time of viral challenge, but increased the rate of infection when gel was applied daily for 7 days with a vaginal wash prior to viral inoculation. The findings presumably reflect altered epithelial architecture, which may have contributed to the trend towards increased HIV observed clinically.

Susceptibility to genital herpes as a biomarker predictive of increased HIV risk: expansion of a murine model of microbicide safety.

BACKGROUND: A crucial gap in the development of microbicides for HIV prevention is the absence of models predictive of safety. Previous studies have demonstrated an increased susceptibility to genital herpes in mice following repeated applications of nonoxynol-9 (N-9). This study was designed to explore the underlying mechanisms, focusing on the effects that N-9 has on genital tract epithelium and to apply this expanded model to evaluate the safety of microbicides that have been advanced to clinical trials. METHODS: Mice were treated intravaginally with formulated 3.5% N-9, 1% tenofovir, 0.5% or 2% PRO 2000, hydroxyethylcellulose (HEC) placebo or no treatment and the effect on herpes simplex virus 2 (HSV-2) susceptibility, epithelial cell architecture, junctional proteins and inflammation were assessed. RESULTS: Mice treated with seven daily doses of N-9, but not tenofovir, PRO 2000 or HEC, were significantly more susceptible to challenge with low doses of HSV-2; confocal microscopy demonstrated increased numbers of viral particles deep within the genital tract. N-9 disrupted the epithelium with loss of tight and adherens junctional proteins. By contrast, the epithelium was relatively preserved following tenofovir, PRO 2000 and HEC exposure. Additionally, N-9, but not the other microbicides, triggered a significant inflammatory response relative to untreated mice. CONCLUSIONS: These findings indicate that disruption of the epithelium contributes to increased HSV-2 susceptibility and might provide a biomarker predictive of increased risk for HIV acquisition. The results are consistent with the safety outcomes of the recently completed Phase IIb clinical trial with 0.5% PRO 2000 gel, and predict that tenofovir gel will not adversely affect the genital tract.

Disruption of tight junctions by cellulose sulfate facilitates HIV infection: model of microbicide safety.

BACKGROUND: The lack of biomarkers that are predictive of safety is a critical gap in the development of microbicides. The present experiments were designed to evaluate the predictive value of in vitro models of microbicide safety. METHODS: Changes in the epithelial barrier were evaluated by measuring transepithelial electrical resistance (TER) after exposure of human epithelial cells to candidate microbicides in a dual-chamber system. The significance of observed changes was addressed by challenging cultures with human immunodeficiency virus (HIV) and measuring the ability of virus to cross the epithelium and infect target T cells cultured in the lower chamber. RESULTS: Exposure to nonoxynol-9 (N-9) or cellulose sulfate (CS), but not 9-[2-(phosphonomethoxy)propyl]adenine (also referred to as tenofovir) or PRO2000, resulted in a rapid and sustained reduction in TER and a marked increase in HIV infection of T cells cultured in the lower chamber. Moreover, CS triggered nuclear factor kappaB activation in peripheral blood mononuclear cells and increased HIV replication in chronically infected U1 cells. CONCLUSIONS: Epithelial barrier disruption and enhanced viral replication may have contributed to the increased risk of HIV acquisition observed in phase 3 trials of N-9 and CS. Expansion of in vitro safety testing to include these models would provide a more stringent preclinical assessment of microbicide safety and may prove to be more predictive of clinical outcomes.

Seminal plasma reduces the effectiveness of topical polyanionic microbicides.

The objective of this study was to test the activity of microbicides against herpes simplex virus type 2 (HSV-2) introduced in seminal plasma. We found that seminal plasma interfered with the activity of PRO 2000 and of cellulose sulfate, increasing by 100-fold the concentration of drug required to inhibit 90% of viral plaque formation. Seminal plasma competitively inhibited binding of the microbicides to the HSV-2 envelope. Most of the interference was found in a high molecular-weight fraction; tandem mass spectrometry identified the proteins as fibronectin-1 and lactoferrin. In a murine model, the interference translated in vivo into a loss in protection. We found that 2% PRO 2000 gel protected 100% of mice challenged intravaginally with HSV-2 introduced in PBS, whereas only 55% of mice were protected if virus was introduced in seminal plasma (P=.0007, log rank test). If these findings are reflective of what occurs in humans, modifications to microbicides to ensure that they retain activity in the presence of seminal plasma are indicated.

PRO 2000 gel inhibits HIV and herpes simplex virus infection following vaginal application: a double-blind placebo-controlled trial.

BACKGROUND: Microbicides used to prevent the transmission of human immunodeficiency virus (HIV) are advancing to clinical trials on the basis of activity observed in vitro and in animal models. However, no data demonstrate activity of microbicides after application in humans. This study was designed to determine the antiviral activity in cervicovaginal lavage (CVL) samples collected after intravaginal application of 0.5% PRO 2000 gel (Indevus). METHODS: A randomized, double-blind study was conducted to assess the anti-HIV and anti-herpes simplex virus (HSV) activity of PRO 2000 in CVL samples obtained at screening (48 hours before) and 1 hour after application of study or placebo gel. HeLa cells or human macrophages were inoculated with CVL samples spiked with replication-defective HIV containing a luciferase indicator gene and pseudotyped with an R5 envelope. Human cervical epithelial cells were inoculated with CVL samples and challenged with HSV-2(G), and the virus titer was then determined. RESULTS: CVL samples obtained after application of PRO 2000 gel significantly inhibited HIV and HSV infection by at least 1000-fold, compared with CVL samples obtained at screening (P < .001). There were no differences in cytokine levels between the drug and placebo groups. CONCLUSIONS: PRO 2000 gel (0.5%) is sufficiently bioavailable and retains substantial antiviral activity after intravaginal application. This strategy provides a mechanism for testing the efficacy of a microbicide before embarking on large-scale clinical trials.

ACIDFORM inactivates herpes simplex virus and prevents genital herpes in a mouse model: optimal candidate for microbicide combinations.

The acidic vaginal milieu is presumed to inactivate pathogens but is neutralized by semen. This notion fostered the development of acid-buffering products, such as ACIDFORM (developed by Program for Topical Prevention of Conception and Disease, Rush University, and licensed by Instead), as microbicides. However, the extent and mechanism of protective activity provided by buffering gels is not known. Exposure of herpes simplex virus (HSV) to pH 4.5 or lower irreversibly inactivated HSV and reduced HSV yields by at least 90%; exposure to pH 5.0 had little or no effect. Pretreatment of HSV-2 with pH 3.5-4.5 triggered proteolysis, disrupting the HSV particle and resulting in a reduction in binding and invasion. ACIDFORM protected 21 (81%) of 26 mice from genital herpes, compared with 3 (12%) of 25 mice who received a placebo gel. ACIDFORM retained significant activity if mice were challenged with HSV delivered in seminal fluid. These findings suggest that ACIDFORM offers considerable protection against HSV and may be an optimal candidate for developing combination microbicides.