Evaluation of the novel vaginal contraceptive agent PPCM in preclinical studies using sperm hyaluronan binding and acrosome status assays.

BACKGROUND: Polyphenylene carboxymethylene (PPCM) sodium salt is a promising multipurpose technology for prevention of both sexually transmitted infections (STIs) and pregnancy. In preclinical studies, PPCM has demonstrated significant (1) antimicrobial activity against several important viral and bacterial pathogens and (2) contraceptive activity associated with premature acrosome loss. OBJECTIVE: To further evaluate a vaginal antimicrobial compound as a contraceptive agent in preclinical studies utilizing a repurposed hyaluronan binding assay (HBA). MATERIALS AND METHODS: Semen samples containing either neat semen or washed spermatozoa were treated with increasing concentrations of PPCM or calcium ionophore A23187 (positive control). Sperm inactivation was measured by two methods: (1) double acrosome staining (AS), and (2) a hyaluronan binding assay (HBA® ). Percentage of inactivated sperm was compared between untreated control sperm and those treated with PPCM or A23187. RESULTS: PPCM had a significant (p < 0.05) and dose-dependent effect on sperm inactivation in both assays, with HBA detecting a higher proportion of inactivated sperm than AS. PPCM did not affect sperm motility and exhibited equivalent responses in the neat and washed samples. DISCUSSION: Both HBA and AS confirmed that spermatozoa were rapidly inactivated at PPCM concentrations likely present in the vagina under actual use conditions and in a time-frame comparable to in vivo migration of spermatozoa out of seminal plasma into cervical mucus. CONCLUSION: PPCM vaginal gel may provide contraceptive protection as well as help with STI prevention. HBA may be a sensitive and much needed biomarker for sperm activity in future contraceptive development.

Fish ingestion and congener specific polychlorinated biphenyl and p,p'-dichlorodiphenyldichloroethylene serum concentrations in a great lakes cohort of pregnant African American women.

A cohort of low income, city dwelling, pregnant African American Women (delivered from 1994-1999) was assembled to identify factors related to organochlorine exposure through consumption of Great Lakes resources. The cohort is known as the Great Lakes Cohort of Pregnant African American Women (GLCPAAW). Pregnant women from metropolitan Chicago, IL area clinics were administered a questionnaire on diet, demographics, and health history. Weight, height, and serum lipids were measured at delivery along with serum organochlorines such as PCBs and DDE. Congener specific concentrations of PCBs and p,p'-DDE found in the maternal serum are reported. Dominant PCB congeners found in the serum of the pregnant women at delivery included PCB 101, 118, 138, 153, and 180. The high prevalence and magnitude of PCB 101 (greater than the limit of detection in >80% of the women in the cohort) are unique characteristics of this cohort. Great Lakes fish has been identified as a source of exposure to organochlorines in several studies. Spearman correlations and robust regression models were utilized to identify the impact of Great Lakes fish ingestion on cohort serum organochlorine concentrations. Several potential confounders of the relationship between serum organochlorines and Great Lakes fish consumption were identified. Covariates related to organochlorines in correlations as well as regression models included age, body surface area, fish ingestion, lipids, parity, race and smoking. Lower chlorinated PCB congeners do not follow the same trends as the higher chlorinated congeners and DDE. The higher chlorinated PCB congeners (PCB 138, 153, and 180) and DDE were correlated with age while the lower chlorinated congeners were not. PCB 153 and 180 regression models included age as a significant covariate. None of the higher chlorinated congeners correlated to race, while both lower chlorinated congeners were correlated to race. Race was also significant in both lower chlorinated congeners' regression models. PCB 101, a lower chlorinated congener seldom found in human serum, is readily found in the cohort. Airborne PCB exposure as well as diminished metabolism of PCB 101 in African Americans may explain the increased presence of PCB 101 and it's correlation with race. High end sport fish consumers (> or =1 meal per week) carried elevated levels of DDE and higher chlorinated PCB congeners (138, 153, and 180) compared to non-sport fish eaters. Unexpectedly, DDE was correlated more consistently with fish ingestion and age (a marker of bioaccumulation) in comparison to PCBs. Small correlations were found between serum PCBs and fish ingestion (Spearman correlation=0.19 for total PCBs and fish meals per year). Additionally, Serum PCBs in low end Great Lakes sport fish consumers were not higher than non-consumers. These findings suggest the women of the cohort are being exposed to PCBs through other routes in addition to Great lakes sport fish. One major route of exposure may be Chicago air. The observed trends amongst individual PCB congeners has important ramifications because lower chlorinated congeners or their metabolites may be mediators of toxicity. Organochlorine exposure through Great Lakes fish ingestion was clearly identified in high end fish consumers while associations with race, metabolism, and possible airborne exposures pose new questions.

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.

Specific human CYP 450 isoform metabolism of a pentachlorobiphenyl (PCB-IUPAC# 101).

Polychlorinated biphenyl IUPAC# 101-PCB 101 (chlorination pattern-2,2',4',5,5') is a common, persistent non-coplanar PCB congener found in the ambient environment but information related to its metabolism in humans is lacking. Previous studies indicate PCB 101 is rapidly metabolized in mammals through CYP 2B and 3A family enzymes. Recently, PCB metabolism through a 2A family isoform in hamsters was also reported. To specifically identify the human CYP 450 isoforms responsible for PCB 101 metabolism, we compared human microsome metabolism to metabolism using several specific recombinant human CYP isoforms. These data characterized selective and extensive metabolism by human CYP 2A6. The product formed was the 4-hydroxy-PCB 101 metabolite (4-hydroxy-2,2',4',5,5') and was the only major metabolite observed in the recombinant and human microsome investigation. This is important information for predicting human specific toxicokinetics of PCBs.

Candidate sulfonated and sulfated topical microbicides: comparison of anti-human immunodeficiency virus activities and mechanisms of action.

Poly(styrene 4-sulfonate), cellulose sulfate, polymethylenehydroquinone, and PRO 2000 are sulfated or sulfonated polymers (SPs) under development as topical microbicides. They are presumed to work through similar mechanisms of action, although to date there has been no extensive comparison of their anti-human immunodeficiency virus activities. To determine whether any of these candidate microbicides offers a potential advantage, their in vitro activities, mechanisms of action, stabilities in biological secretions, and toxicities were compared. All four compounds were found to be active against X4, R5, and dualtropic primary isolates and against X4 and R5 laboratory-adapted strains in CD4+ T cells, macrophages, and single-coreceptor cell lines. Our single-cycle experiments using pseudotyped virus suggest that all four SPs function at the binding and entry stages of the viral life cycle but differ in degree of postentry effect. Surface plasmon resonance analyses demonstrate that SPs bind to X4 and R5 monomeric glycoprotein 120 with similar high binding affinities. When mixed with cervicovaginal lavage fluid, SPs maintain inhibitory activity at concentrations achievable in formulations.

Candidate topical microbicides bind herpes simplex virus glycoprotein B and prevent viral entry and cell-to-cell spread.

Topical microbicides designed to prevent acquisition of sexually transmitted infections are urgently needed. Nonoxynol-9, the only commercially available spermicide, damages epithelium and may enhance human immunodeficiency virus transmission. The observation that herpes simplex virus (HSV) and human immunodeficiency virus bind heparan sulfate provided the rationale for the development of sulfated or sulfonated polymers as topical agents. Although several of the polymers have advanced to clinical trials, the spectrum and mechanism of anti-HSV activity and the effects on soluble mediators of inflammation have not been evaluated. The present studies address these gaps. The results indicate that PRO 2000, polystyrene sulfonate, cellulose sulfate, and polymethylenehydroquinone sulfonate inhibit HSV infection 10,000-fold and are active against clinical isolates, including an acyclovir-resistant variant. The compounds formed stable complexes with glycoprotein B and inhibit viral binding, entry, and cell-to-cell spread. The effects may be long lasting due to the high affinity and stability of the sulfated compound-virus complex, as evidenced by surface plasmon resonance studies. The candidate microbicides retained their antiviral activities in the presence of cervical secretions and over a broad pH range. There was little reduction in cell viability following repeated exposure of human endocervical cells to these compounds, although a reduction in secretory leukocyte protease inhibitor levels was observed. These studies support further development and rigorous evaluation of these candidate microbicides.