Nonoxynol-9 Research Articles

Increased COX-2 Expression in Human Vaginal Epithelial Cells Exposed to Nonoxynol-9, a Vaginal Contraceptive Microbicide that Failed to Protect Women from HIV-1 Infection

Despite displaying virucidal activity in vitro, nonoxynol-9 (N-9), a vaginal contraceptive microbicide candidate, failed to reduce the rate of human immunodeficiency virus (HIV) transmission in clinical trials. With frequent use, it even increased the risk of HIV acquisition. Such outcome was postulated to be because of N-9-induced mucosal inflammation, which resulted in recruitment of HIV-target immune cells to the sites of virus entry. Understanding the mechanism underlying the response of the vaginal epithelium to N-9 is critical to properly evaluate the safety of prospective vaginal microbicides and contraceptives. Using DNA microarray and quantitative RT-PCR techniques, we observed that N-9 initiated a strong transcriptional upregulation of cyclooxygenase-2 (COX-2) in immortalized human vaginal epithelial cells (VK2/E6E7 cell line). Increased COX-2 protein expression evaluated by immunoblotting was dose- and time-dependent. The level of prostaglandin E(2) (PGE(2) ) increased subsequently to COX-2 elevation. This upregulation was in part because of NF-kB activation. Expression of COX-2, a potent inflammation-related enzyme, as well as increased secretion of PGE(2) , an important local mediator of mucosal immunoinflammatory responses, by human vaginal epithelial cells exposed to vaginal microbicide and contraceptive candidates may be used as a biomarker of undesirable compound properties.

Design and synthesis of 3-(azol-1-yl)phenylpropanes as microbicidal spermicides for prophylactic contraception

We designed a series of 25 3-(azol-1-yl)phenylpropanes which yielded 10 compounds (3, 4, 7, 8, 13, 14, 19, 21, 23, 26) that irreversibly immobilized 100% human sperm at 1% (w/v) concentration in 60s; 12 compounds (8, 9, 15, 16, 19–21, 23–25, 27, 28) that showed potent microbicidal activity at 12.5–50μg/mL against Trichomonas vaginalis; and 17 compounds (3–11, 13, 15, 19, 21, 23, 26, 28, 30) that exhibited potent anticandida activity with minimum inhibitory concentration (MIC) of 12.5–50μg/mL. Almost all the compounds exhibited high level of safety towards normal vaginal flora (Lactobacillus) and human cervical (HeLa) cells in comparison to the marketed spermicide nonoxynol-9 (N-9). All the biological activities were evaluated in vitro. Two compounds (4, 8) with good safety profile exhibited multiple (spermicidal, antitrichomonas and anticandida) activities, warranting further lead optimization for furnishing a prophylactic vaginal contraceptive.

742 MicroRNAs in the differential diagnosis of aggressive B-cell lymphomas

Heterosexual transmission of human immunodeficiency virus (HIV-1) is the predominant mode of infection worldwide. However, the early steps of transepithelial infection still need to be clarified. Using epithelial cells, originating from the female genital tract, and peripheral blood mononuclear cells as subepithelial target cells, an in vitro dual-chamber model of the female genital tract was developed. Remarkably, an intact layer of some cell types (HEC-1A, CaSki and Ect1) served as a protective barrier against cell-free but not against cell-associated HIV-1 that crossed the epithelial barrier through transmigration. Furthermore, dysfunctions of the epithelial layers were assessed by monitoring transepithelial electric resistance and transepithelial passage of FluoSpheres® and HIV-1 after treatment with nonoxynol-9 (N-9). Most of the functional assays showed dysfunction of the epithelial barrier at lower concentrations compared to a widely used colorimetric toxicity assay (WST-1). Finally, N-9 treatment caused a significant increase in the production of interleukin-8 (IL-8) and macrophage inflammatory protein-3α (MIP-3α) and a decrease of Secretory Leukocyte Protease Inhibitor (SLPI) and Monocyte Chemotactic Protein-1 (MCP-1) in this model. In conclusion, this model is a useful tool to (1) study HIV-1 transmission mechanisms and (2) evaluate epithelial toxicity of candidate microbicides.

Evaluation of Spermicidal Activity of Mi-Saponin A

The seed extracts of Madhuca latifolia were reported to have spermicidal activity. The current investigation identified the spermicidal component of the extracts and evaluated its spermicidal potential in vitro. As characterized by infrared, mass, and nuclear magnetic resonance (NMR) spectral analyses, Mi-saponin A (MSA) was found to be the most potent component among a mixture of saponins. The mean effective concentrations of MSA that induced irreversible immobilization were 320 microg/mL for rat and 500 microg/mL for human sperm, as against the respective concentrations of 350 and 550 microg/mL of nonoxynol 9 (N-9). The mode of spermicidal action was evaluated by a battery of tests including (a) double fluoroprobe staining for sperm viability, (b) hypoosmotic swelling test and, assays for 5' nucleotidase and acrosin for physiological integrity of sperm plasma membrane, (c) scanning and transmission electron microscopy for sperm membrane ultrastructure, and (d) plasma membrane lipid peroxidation (LPO). The observations, taken together, were interpreted to mean that the spermicidal effect of MSA involved increased membrane LPO leading to structural and functional disintegration of sperm plasma membrane and acrosomal vesicle. A comparative in vitro cytotoxicity study in human vaginal keratocyte (Vk2/E6E7) and endocervical (End/E6E7) cell lines demonstrated that the 50% cell cytotoxicity (CC(50)) values, and consequently the safety indices, for MSA were >or= 8-fold higher as compared to those of N-9. In conclusion, MSA is a potent spermicidal molecule that may be explored further for its suitability as an effective component of vaginal contraceptive.

Functional attenuation of human sperm by novel, non-surfactant spermicides: precise targeting of membrane physiology without affecting structure

We have attempted to identify structural, physiological and other targets on human sperm vulnerable to the spermicidal action of two novel series of non-detergent molecules, reported to irreversibly immobilize human sperm in <30 s, apparently without disrupting plasma membrane. Three sperm samples were studied. Scanning and transmission electron microscopy were used to assess structural aberrations of sperm membrane; plasma membrane potential and intracellular pH measurements (fluorometric) were used to detect changes in sperm physiology; reactive oxygen species (ROS, fluorometric) and superoxide dismutase activity (colorimetric) were indicators of oxidative stress; and sperm dynein ATPase activity demonstrated alterations in motor energy potential, in response to spermicide treatment. Post-ejaculation tyrosine phosphorylation of human sperm proteins (immunoblotting) was a marker for functional integrity. Disulfide esters of carbothioic acid (DSE compounds) caused complete sperm attenuation at > or =0.002% concentration with hyper-polarization of sperm membrane potential (P < 0.001), intracellular alkalinization (P < 0.01), ROS generation (P < 0.05) and no apparent effect on sperm (n = 150) membrane structure. Isoxazolecarbaldehyde compounds required > or =0.03% for spermicidal action and caused disrupted outer acrosomal membrane structure, depolarization of membrane potential (P < 0.001), intracellular acidification (P < 0.01) and ROS generation (P < 0.01). Detergent [nonoxynol-9 (N-9)] action was sustainable at > or =0.05% and involved complete breakdown of structural and physiological membrane integrity with ROS generation (P < 0.001). All spermicides caused functional attenuation of sperm without inhibiting motor energetics. Unlike N-9, DSE-37 (vaginal dose, 200 microg) completely inhibited pregnancy in rats and vaginal epithelium was unchanged (24 h,10 mg). The study reveals a unique mechanism of action for DSE spermicides. DSE-37 holds promise as a safe vaginal contraceptive. CDRI Communication No. 7545.

Development of an in vitro dual-chamber model of the female genital tract as a screening tool for epithelial toxicity

Heterosexual transmission of human immunodeficiency virus (HIV-1) is the predominant mode of infection worldwide. However, the early steps of transepithelial infection still need to be clarified. Using epithelial cells, originating from the female genital tract, and peripheral blood mononuclear cells as subepithelial target cells, an in vitro dual-chamber model of the female genital tract was developed. Remarkably, an intact layer of some cell types (HEC-1A, CaSki and Ect1) served as a protective barrier against cell-free but not against cell-associated HIV-1 that crossed the epithelial barrier through transmigration. Furthermore, dysfunctions of the epithelial layers were assessed by monitoring transepithelial electric resistance and transepithelial passage of FluoSpheres ® and HIV-1 after treatment with nonoxynol-9 (N-9). Most of the functional assays showed dysfunction of the epithelial barrier at lower concentrations compared to a widely used colorimetric toxicity assay (WST-1). Finally, N-9 treatment caused a significant increase in the production of interleukin-8 (IL-8) and macrophage inflammatory protein-3α (MIP-3α) and a decrease of Secretory Leukocyte Protease Inhibitor (SLPI) and Monocyte Chemotactic Protein-1 (MCP-1) in this model. In conclusion, this model is a useful tool to (1) study HIV-1 transmission mechanisms and (2) evaluate epithelial toxicity of candidate microbicides.

Targeting Trojan Horse leukocytes for HIV prevention

…. is sure design'd, by fraud or force: trust not their presents, nor admit the horse. Virgil, Aeneid Human immunodeficiency virus type 1 (HIV-1), the lymphotropic virus that causes AIDS, has infected more than 60 million people worldwide since its clinical appearance in 1981. Despite intensive prevention efforts, the HIV/AIDS epidemic continues to spread, particularly in developing countries in sub-Saharan Africa, southeast Asia and the Caribbean, as well as the developed world [1]. Although HIV can be transmitted very efficiently parenterally, the advent of routine blood screening prior to transfusion and harm reduction programs for injection drug users, have made this mode of transmission much less common than mucosal transmission. Most new HIV infections are attributable to mucosal transmission: through genital and rectal mucosae in the case of sexual transmission and through oral or gastrointestinal mucosae in the case of mother-to-child transmission [2]. Much has been learned about HIV pathogenesis and infection mechanisms at the molecular level, but the scientific community has yet to develop an effective vaccine or microbicide for HIV prevention. Many unanswered questions remain concerning HIV-1 sexual transmission. In 1983, barely 2 years into the AIDS epidemic, we hypothesized that the agent that was subsequently identified as HIV-1 may be sexually transmitted by infected ‘Trojan Horse’ leukocytes in semen [3]. This hypothesis was based on our knowledge at the time that human semen contains substantial numbers of T lymphocytes and macrophages, which could host a T-cell tropic virus, and the following assumptions: intracellular virus would be better protected than free virus from adverse effects of antiviral factors in the genital environment such as antiviral antibodies likely to be present in genital secretions of the virus-infected transmitter, as well as antimicrobial peptides that play an important role in genital innate immune defense; and virus-infected allogeneic cells could also escape early detection by major histocompatibility complex (MHC)-restricted cytotoxic T cells in a new host. Over the intervening 25+ years, others have also championed this cause [4,5], and convincing evidence has emerged from clinical research as well as in-vitro and animal studies that infected leukocytes indeed play a role in HIV transmission. Yet, most recent research on sexual HIV transmission has focused on cell-free HIV in genital secretions because of the wide availability of HIV RNA quantification assays. Furthermore, the majority of HIV vaccines and microbicides have been designed to block transmission of cell-free virus and have been tested in animal and in-vitro models that use cell-free virus as the only infectious inoculum. As the molecular events underlying cell-associated HIV transmission differ from those underlying cell-free virus transmission, many of the current vaccine and microbicide candidates might not be expected to protect against cell-associated HIV transmission. The failure of several recent vaccine and microbicide clinical trials may be due in part to this oversight. It should be possible to design strategies that block cell-associated HIV transmission as well as cell-free HIV transmission. In this article, we present an overview of research that has been conducted on cell-associated HIV mucosal transmission and recommendations for future research. We focus on sexual HIV transmission, but this review also has relevance for mother-to-child HIV transmission, which may occur through mucosal transmission of cell-associated HIV from maternal genital or mammary gland secretions [6–8]. We review published reports that describe and enumerate HIV-infected cells in genital secretions, and compelling evidence from clinical, animal and in-vitro studies demonstrating that such cells can transmit HIV across genital tract epithelial surfaces; potential molecular mechanisms underlying cell-associated HIV transmission that could be specifically targeted by future HIV prevention strategies; and in-vitro and animal cell-associated HIV transmission models currently used for studies on cell-associated HIV transmission mechanisms and for testing vaccine and microbicide candidates. Using this information as a foundation, we discuss the evidence and probability that various current microbicide and vaccine approaches prevent cell-associated HIV transmission, and suggest additional microbicide and vaccine concepts and experiments that will move this field forward.

Susceptibility to Genital Herpes as a Biomarker Predictive of Increased HIV Risk: Expansion of a Murine Model of Microbicide Safety

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. 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. 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. 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.

Inflammatory cytokines in the lower genital tract: analyses by absolute cytokine levels and normalized cytokine/total protein ratios result in different outcomes

OBJECTIVE: Cytokines are increasingly used to study the impact of microbicides on immune response in the lower female genital tract. The objective was to test if 2 methods of analyzing cytokine levels in the reproductive tract gave different results.DESIGN: Randomized, assessor-blinded crossover trial.MATERIALS AND METHODS: In a clinical trial assessing the impact of inert and pro-inflammatory topical gels on the vagina and endometrium, 14 subjects underwent baseline evaluation without gel exposure and at least one (and up to two) more set of vaginal lavage (VL) and endometrial lavage (EL) after exposure to two possible conditions: 3 days of nonoxynol-9 (N-9) or placebo gel. 8 pro-inflammatory cytokines (Il-1rα, Il-6, Il-8, MCP-1, MIP-1α, MIP-1β, RANTES, and TNF-α 3 anti-inflammatory cytokines (Il-1rα, Il-10, Slp-1) and total protein levels were assayed in all VL and EL specimens (RayBio Human Cytokines Array). Cytokines were analyzed as absolute values and cytokine/total protein ratios. These measures were compared by gel exposure using signrank tests.RESULTS: Compared to baseline and placebo, N-9 exposure changed both absolute cytokine levels and cytokine/total protein ratios in the vagina and the endometrium. However, both the magnitude and direction of change in cytokines differed between analyses using absolute values versus normalized ratios. For example, compared to placebo, N-9 was associated with significantly decreased Il-6 (p=0.01), MCP-1 (p=0.02), MIP-1α (p=0.02), TNF-α (p=0.05) Il-10 (p=0.02) and Slp-1 (p=0.03) cytokine/total protein ratios in the vagina. In contrast, N-9 was only associated with decreased Il-6 (p=0.05) and increased Il-8 (p=0.001)when analyzed by absolute levels.CONCLUSIONS: When analyzing the inflammatory impact of topical vaginal agents, results are highly impacted by the method of cytokine analyses. If the evaluation of microbicides candidates is to incorporate surrogates of markers of inflammation, a standardization of methodology is paramount. OBJECTIVE: Cytokines are increasingly used to study the impact of microbicides on immune response in the lower female genital tract. The objective was to test if 2 methods of analyzing cytokine levels in the reproductive tract gave different results. DESIGN: Randomized, assessor-blinded crossover trial. MATERIALS AND METHODS: In a clinical trial assessing the impact of inert and pro-inflammatory topical gels on the vagina and endometrium, 14 subjects underwent baseline evaluation without gel exposure and at least one (and up to two) more set of vaginal lavage (VL) and endometrial lavage (EL) after exposure to two possible conditions: 3 days of nonoxynol-9 (N-9) or placebo gel. 8 pro-inflammatory cytokines (Il-1rα, Il-6, Il-8, MCP-1, MIP-1α, MIP-1β, RANTES, and TNF-α 3 anti-inflammatory cytokines (Il-1rα, Il-10, Slp-1) and total protein levels were assayed in all VL and EL specimens (RayBio Human Cytokines Array). Cytokines were analyzed as absolute values and cytokine/total protein ratios. These measures were compared by gel exposure using signrank tests. RESULTS: Compared to baseline and placebo, N-9 exposure changed both absolute cytokine levels and cytokine/total protein ratios in the vagina and the endometrium. However, both the magnitude and direction of change in cytokines differed between analyses using absolute values versus normalized ratios. For example, compared to placebo, N-9 was associated with significantly decreased Il-6 (p=0.01), MCP-1 (p=0.02), MIP-1α (p=0.02), TNF-α (p=0.05) Il-10 (p=0.02) and Slp-1 (p=0.03) cytokine/total protein ratios in the vagina. In contrast, N-9 was only associated with decreased Il-6 (p=0.05) and increased Il-8 (p=0.001)when analyzed by absolute levels. CONCLUSIONS: When analyzing the inflammatory impact of topical vaginal agents, results are highly impacted by the method of cytokine analyses. If the evaluation of microbicides candidates is to incorporate surrogates of markers of inflammation, a standardization of methodology is paramount.

Disruption of Tight Junctions by Cellulose Sulfate Facilitates HIV Infection: Model of Microbicide Safety

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. 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. 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. 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.

Use of High‐Resolution Confocal Imaging of the Vaginal Epithelial Microstructure to Detect Microbicide Toxicity

High-resolution optical imaging by confocal reflectance microscopy (CRM) was investigated for its ability to delineate the epithelial microstructure of the vaginal tract and detect alterations that may result from the use of vaginal microbicides. The vaginal tracts of Swiss Webster mice treated with medroxyprogesterone acetate were exposed in vivo to a 4% nonoxynol-9 (N-9)-containing gel or saline. The vaginal tract was removed 4 h, 16 h, or 48 h after treatment and imaged by CRM without staining, and biopsy specimens were obtained from the imaged regions and processed for histological analysis. In control mice, CRM revealed a columnar epithelium and lamina propria with features resembling those observed via histological analysis. CRM revealed an exfoliated epithelium 4 h and 16 h after N-9 treatment, and quantitative measurement of epithelial thickness revealed a mean thickness (+/- standard error of the mean) of approximately ~41.7 +/- 1.7 mum in control specimens, compared with 14.9 +/- 4.5 mum for specimens obtained 4 h after treatment and 24.4 +/- 2.1 mum for specimens obtained 16 h after treatment. Inflammation 4 h after treatment was indicated through detection of inflammatory infiltrates. In samples collected 48 h after treatment, the epithelium was regenerating. The time line of changes in the morphological structure and epithelial thickness detected by CRM closely resembled that of changes revealed by histological analysis. This study demonstrates that CRM can delineate the epithelial structure and detect indicators of inflammation after treatment with N-9 and that it may be a useful imaging tool for evaluating the effects of vaginal microbicides.

Nonoxynol-9 (N-9), After Repeated Applications, Does not Result in Cumulative Damage to the Murine Cervicovaginal Epithelium

Nonoxynol-9 (N-9), After Repeated Applications, Does not Result in Cumulative Damage to the Murine Cervicovaginal Epithelium

In vitro testing of rationally designed spermicides for selectively targeting human sperm in vagina to ensure safe contraception†

Rational synthesis of novel structures resulted in two unique molecules (DSE-36 and DSE-37, disulphide esters of carbothioic acid) that killed sperm 25 times more strongly and with a precisely targeted action than nonoxynol-9 (N-9). We examine the effects of DSE-36 and DSE-37 on human spermatozoa versus HeLa cells to establish specificity and safety compared with N-9. At spermicidal EC(100) (20 microg/ml) DSE-36 and DSE-37 killed 100% sperm in <30 s (Sander-Cramer assay) and at EC(50) induced apoptosis in sperm (Annexin-V-fluorescein isothiocyanate and JC-1 labelling and Flow Cytometry) in 3 h. However, at EC(100) these molecules had no effect on HeLa cells by 24 h or on cell viability [3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay], surface ultrastructure (scanning electron microscopy), Annexin-V and JC-1 labelling pattern and reactive oxygen species (ROS) generation. N-9, with a spermicidal EC(100) of 500 microg/ml, decreased HeLa cell viability at 20 microg/ml in 24 h (P < 0.001), accompanied by acute damage to cell surface ultrastructural topography, induction of apoptosis and ROS generation. Unlike DSE-36 and DSE-37, N-9 also significantly induced mRNA levels (RT-PCR) of pro-inflammatory biomarkers (interleukin (IL)-1 alpha, IL-6, IL-8, RANTES) in HeLa cells and increased IL-6 and IL-8 secretion (P < 0.001, enzyme-linked immunosorbent assay). Furthermore, DSE-36 and DSE-37 did not inhibit Lactobacillus growth at EC(100) and exhibited mild microbicidal activity against Trichomonas vaginalis, while N-9 inhibited Lactobacillus and Trichomonas growth but had a lower prophylactic index. The ability of these novel spermicides to kill sperm almost instantaneously at innocuously low concentration indicates their worth as improved active ingredients for vaginal contraceptive preparations compared with N-9.

Spermicidal Activity of Bivittoside D from Bohadschia vitiensis

A recent revelation about increased susceptibility to HIV by use of nonoxynol-9 (N-9) has called for identification of novel molecules with potent sperm-attenuating activity and lower side-effect profile, as suitable alternatives. The present study was designed to investigate spermicidal activity in Bohadschia vitiensis whole-body extracts followed by isolation and characterization of bioactive molecule. Bohadschia vitiensis (Semper) was collected from the Southern Andaman coast of India. Freshly collected marine animals were extracted with methanol. A portion of the crude extract was fractionated into four fractions by macerating with hexane, chloroform, and n-butanol successively. All fractions were evaluated for spermicidal activity. Because maximum activity was localized in the n-butanol soluble fraction, it was chromatographed over a silica gel column, and elution with chloroform-methanol-water (35:10:2, v/v) yielded the major compound bivittoside D (400 mg). Bivittoside D [molecular weight (MW) 1426] is a lanostane triterpenoid with six monosaccharide units. The structure of the compound was established on the basis of physicochemical data, acid hydrolysis of saponin, identification of sugar units and aglycon, melting point, and by comparison with data reported in the literature. The aqueous methanol extract of the Bohadschia vitiensis caused 100% mortality of human sperm at 0.01% concentration in vitro, whereas N-9 (reference control) exhibited an equivalent activity at 0.05%. On further fractionation, activity was localized in n-butanol soluble fraction from which the major compound purified was a lanostane triterpenoid called bivittoside D. Bivittoside D was found to be a more potent spermicide (approximately 2.3 times) than N-9 and killed 100% human sperm at the concentration of 350 muM in approximately 20 sec in vitro. Supravital staining and hypoosmotic swelling test revealed sperm membrane permeabilization by bivittoside D as the major mode of spermicidal action. However, bivittoside D was much safer than N-9 towards normal vaginal flora (Lactobacillus) in vitro, although it affected the viability of HeLa cells like other surfactants. Bivittoside D from B. vitiensis can adequately replace N-9 in vaginal contraceptives to make them more vaginally safe and ecofriendly.

SILCS diaphragm: postcoital testing of a new single-size contraceptive device

Background This study was conducted to compare the effectiveness of a new, single-size silicone contraceptive diaphragm used with either spermicide [2% nonoxynol-9 (N-9)] or lubricant in preventing sperm from penetrating midcycle cervical mucus. Study design A crossover postcoital test (PCT) in healthy, sexually active women not at risk for pregnancy due to tubal occlusion was conducted. Couples had a baseline PCT without a device to verify normal fertility parameters. Qualified couples underwent up to two test cycles using the SILCS diaphragm with a metal spring. A subgroup of couples underwent a third test cycle with the SILCS polymer spring diaphragm used with N-9 gel. Results Fifteen couples completed a baseline cycle and were randomized to order of study gel. Of these, 14 couples completed a baseline cycle and at least one test cycle, 12 couples completed a baseline cycle and two test cycles and 8 couples completed a third test cycle with the polymer spring prototype. Sperm was detected in the vaginal pool in all completed test cycles. The SILCS metal spring diaphragms used with N-9 gel reduced the average number of progressively motile sperm per high power field in the cervical mucus from a baseline of 12.5 to 0, while use of this device with lubricant reduced the number to 0.5. The SILCS polymer spring diaphragm used with N-9 performed the same as the metal spring used with N-9. Conclusion The SILCS diaphragm used with N-9 gel performed well. It is likely that the SILCS diaphragm will give acceptable results in a contraceptive effectiveness study but that adjunctive use of a chemical barrier such as N-9 gel will be necessary for it to be most effective.

The effects of spermicides containing nonoxynol-9 on cervical cytology

Background This analysis was undertaken to compare the effect of the different dosages and formulations of spermicides containing nonoxynol-9 (N-9) on cervical cytology. Study Design A randomized trial was conducted at 14 sites in the United States to evaluate the effectiveness and safety of five spermicides containing N-9. This Papanicolaou smear analysis included the data from all participants who provided two Papanicolaou smear samples: at admission and after discontinuation of the product. The effects of the spermicides were evaluated by comparing the rates of alteration of cervical cytology between five study groups. Results A total of 640 women were included in this analysis. The majority of the study participants (>85%) had no change of their baseline Papanicolaou smear result. The rates of alteration of cervical cytology were similar among women using the three gels containing the different doses of N-9 and three different formulations containing the same dose of N-9. Our analysis found no association between alteration of cervical cytology and duration or frequency of use of the five study spermicides. Conclusions Exposure to different formulations and doses of spermicides containing N-9 is unlikely to influence cervical cytology.

Surfactants as Microbicidal Contraceptives: A Calorimetric Study of Partitioning and Translocation in Model Membrane Systems

Surfactant partitioning into lipid vesicles was studied using isothermal titration calorimetry (ITC), comparing the behavior of four surfactants with current or potential application in contraception and the prevention of sexually transmitted diseases: nonoxynol-9 (N-9), the amphoteric mixture known as C31G, benzalkonium chloride (BZK), and sodium dodecyl sulfate (SDS). Membranes varied in composition from a single-component system, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, to a complex lipid mixture that models the sperm plasma membrane. The partitioning of N-9 into the membranes was found to be endothermic in contrast to the other surfactants studied. For all four surfactants, the partition coefficient decreased as the membrane cholesterol content increased. Surfactant translocation across the membrane leaflets was also determined, with SDS being the only surfactant of the four not to exhibit “flip-flop” on experimental time scales. The results of these studies shed light on the process of surfactant-induced membrane permeabilization.

Update on nonoxynol-9 as vaginal spermicide

Nonoxynol-9 (N-9) is a typical surfactant. For more than 30 years that very property of N-9 has been successfully exploited for its spermicidal action. It is available as an over-the-counter, locally acting vaginal spermicide. The suitability of N-9 as a spermicide is elaborated in this article. The reasons why N-9 may fail as a contraceptive are discussed. In spite of many drawbacks, which are mentioned in the article, N-9 is still often resorted to as a locally acting contraceptive. The review ends with suggestions to alter the molecular structure of N-9 and to adjust the dosages.

Synthesis of disulfide esters of dialkylaminocarbothioic acid as potent, non-detergent spermicidal agents

S, S′-[disulfanediylbis(dialkylaminopropane-2,1-diyl)]bis- (dialkylaminothiocarbamate) ( 14– 31) were prepared and evaluated for the spermicidal activity and antifungal activity. Dialkyldithiocarbamates ( 1– 5) were reacted with epichlorohydrin to give 1-dialkylaminocarbothioic acid S-[(2,3-epithio)propyl]ester ( 7– 11), these on further reaction with a secondary amine gave S, S′-[disulfanediylbis(dialkylaminopropane-2,1-diyl)]bis- (dialkylaminothiocarbamate) ( 14– 31). Some of these compounds ( 16, 19– 21, 23, 30, 31) were found to be very potent spermicidal agents with marginal antifungal activity. Two compounds ( 20, 21) were 25 times more active than nonoxynol-9 (N-9), the spermicide currently in the market.

Genital transmission of HPV in a mouse model is potentiated by nonoxynol-9 and inhibited by carrageenan

Genital human papillomavirus (HPV) infection is the most common sexually transmitted infection, and virtually all cases of cervical cancer are attributable to infection by a subset of HPVs (reviewed in ref. 1). Despite the high incidence of HPV infection and the recent development of a prophylactic vaccine that confers protection against some HPV types, many features of HPV infection are poorly understood. It remains worthwhile to consider other interventions against genital HPVs, particularly those that target infections not prevented by the current vaccine. However, productive papillomavirus infection is species- and tissue-restricted, and traditional models use animal papillomaviruses that infect the skin or oral mucosa. Here we report the development of a mouse model of cervicovaginal infection with HPV16 that recapitulates the establishment phase of papillomavirus infection. Transduction of a reporter gene by an HPV16 pseudovirus was characterized by histology and quantified by whole-organ, multispectral imaging. Disruption of the integrity of the stratified or columnar genital epithelium was required for infection, which occurred after deposition of the virus on the basement membrane underlying basal keratinocytes. A widely used vaginal spermicide, nonoxynol-9 (N-9), greatly increased susceptibility to infection. In contrast, carrageenan, a polysaccharide present in some vaginal lubricants, prevented infection even in the presence of N-9, suggesting that carrageenan might serve as an effective topical HPV microbicide.