Nonoxynol-9 Research Articles

Effects of nonoxynol-9 (N-9) on sperm functions: systematic review and meta-analysis.

ObjectiveTo summarize the currently available phase I and II clinical trials of the effects of nonoxynol-9 (N-9) on human sperm structure and functions.MethodsA systematic review and meta-analysis aiming to evaluate the spermicidal activity of N-9 on motility, was conducted in PubMed, EMBASE, and Cochrane databases by 10 March 2021. The counted numbers of progressive motile (PR) sperm in cervical mucus and the vanguard sperm penetration distances were analyzed. Other effects on sperm structures and physiological activities were reviewed as well.ResultsIn the pooled results, percentages or counted numbers of PR sperm decreased after the treatment of N-9. Vanguard sperm penetration distance was shortened in treated groups. N-9 has been confirmed to damage the structures of sperm, as well as other organelles like acrosome and mitochondria. The physiological activities such as generation of reactive oxygen species, superoxide dismutase activity, acrosin activity, and hemizona binding were all inhibited in the reviewed studies.ConclusionsN-9 has several impacts on sperm owing to its potency in reducing sperm motility and cervical mucus penetration, as well as other functional competencies.Lay summaryNonoxynol-9 (N-9) has been used worldwide as a spermicide to kill sperm for more than 60 years but can cause side effects including vaginal irritation and can increase the rate of contraceptive failure. A detailed analysis of published literature aiming to evaluate the spermicidal activity of N-9 on sperm was carried out. In the pooled results, N-9 reduced the number of active sperm and the distance they traveled. It also caused damage to the structures of sperm and to the way the sperm acted and interacted with the egg. In conclusion, N-9 impacts on sperm in a number of ways that lead to sperm death and dysfunction.

Development of Gram Stain Scoring System Based on Pro-Inflammatory Cytokines in the Sheep Model for Testing Toxicity of Vaginal Products.

Background: Development of safe, effective products to prevent the sexual transmission of HIV remains a priority. Prior to clinical testing, the products must undergo strict safety evaluations to avoid mucosal drug toxicity, inflammation, and vaginal microbiome (VMB) shifts. Based on the Food and Drug Administration (FDA) guidance, we designed a study to measure the inflammatory markers and VMB changes after intravaginal treatment with products that have been associated with toxicity, with the objective to develop a Gram stain slide scoring system, similar to Nugent scoring, correlated with the proinflammatory cytokines in sheep.Methods: Non-pregnant Dorset ewes (n = 34) were randomized to receive 5 ml intravaginal 4% nonoxynol-9 (N9) contraceptive gel, positive control (0.2% benzalkonium chloride), placebo control [hydroxethyl cellulose (HEC)], or no application daily for 10 days, with 11-day post-treatment follow-up. The vaginal swabs were collected for the cytokines, VMB, and Gram-stained slides. An enzyme-linked immunosorbent assay (ELISA) analysis of cytokines interleukin (IL)-1β, IL-8, CXCL10, and tumor necrosis factor-α (TNF-α) was used to determine inflammatory state of the sample. Vaginal microbiome community types (CT) were utilized to create five equivalent slide subsets for iterative development of a Gram-stained slide scoring system with comparisons with inflammatory state based on the cytokine levels.Results: Digital images of the Gram-stained slides were scored based on Gram staining and morphology of bacteria, presence of sheep epithelial cells, and immune cells. The scoring system was modified in an iterative fashion with weighting based on cytokine categorization of inflamed samples, with three of four cytokine values above the mean indicating that the sample was inflamed. The parameters in the final version of the scoring system included mature epithelial cells, Gram-negative rods, and Gram-positive diplococci indicating normal and immune cells indicating inflammation. The area under the receiver operator characteristic curve (ROC AUC) was 0.725 (ROC AUCs range between 0.5 and 1.0) with a greater area indicating higher diagnostic ability of a test with a binary outcome: inflamed or normal.Conclusion: The scoring system, derived from the advanced VMB and cytokine analyses, provides a validated, practical method for quantification of Gram-stained slides that can be performed in most laboratories, increasing the potential for standardization. The training plan can assist laboratories to determine the safety of intravaginal products in their sheep studies or the methodological approach can be applied to other animal models where such data are also needed.

High-Resolution Quantitative Mapping of Macaque Cervicovaginal Epithelial Thickness: Implications for Mucosal Vaccine Delivery.

Vaginal mucosal surfaces naturally offer some protection against sexually transmitted infections (STIs) including Human Immunodeficiency Virus-1, however topical preventative medications or vaccine designed to boost local immune responses can further enhance this protection. We previously developed a novel mucosal vaccine strategy using viral vectors integrated into mouse dermal epithelium to induce virus-specific humoral and cellular immune responses at the site of exposure. Since vaccine integration occurs at the site of cell replication (basal layer 100-400 micrometers below the surface), temporal epithelial thinning during vaccine application, confirmed with high resolution imaging, is desirable. In this study, strategies for vaginal mucosal thinning were evaluated noninvasively using optical coherence tomography (OCT) to map reproductive tract epithelial thickness (ET) in macaques to optimize basal layer access in preparation for future effective intravaginal mucosal vaccination studies. Twelve adolescent female rhesus macaques (5-7kg) were randomly assigned to interventions to induce vaginal mucosal thinning, including cytobrush mechanical abrasion, the chemical surfactant spermicide nonoxynol-9 (N9), the hormonal contraceptive depomedroxyprogesterone acetate (DMPA), or no intervention. Macaques were evaluated at baseline and after interventions using colposcopy, vaginal biopsies, and OCT imaging, which allowed for real-time in vivo visualization and measurement of ET of the mid-vagina, fornices, and cervix. P value ≤0.05 was considered significant. Colposcopy findings included pink, rugated tissue with variable degrees of white-tipped, thickened epithelium. Baseline ET of the fornices was thinner than the cervix and vagina (p<0.05), and mensing macaques had thinner ET at all sites (p<0.001). ET was decreased 1 month after DMPA (p<0.05) in all sites, immediately after mechanical abrasion (p<0.05) in the fornix and cervix, and after two doses of 4% N9 (1.25ml) applied over 14 hrs in the fornix only (p<0.001). Histological assessment of biopsied samples confirmed OCT findings. In summary, OCT imaging allowed for real time assessment of macaque vaginal ET. While varying degrees of thinning were observed after the interventions, limitations with each were noted. ET decreased naturally during menses, which may provide an ideal opportunity for accessing the targeted vaginal mucosal basal layers to achieve the optimum epithelial thickness for intravaginal mucosal vaccination.

Comparative in vitro study on the local tolerance and efficacy of benzalkonium chloride, myristalkonium chloride and nonoxynol-9 as active principles in vaginal contraceptives

Background Spermicides have been identified as a potentially attractive alternative to hormonal contraceptives and/or intrauterine devices. Thus, this study aimed evaluating the efficacy and local tolerance of benzalkonium chloride (BKC) and myristalkonium chloride (MKC) contained in Pharmatex® vaginal formulations and compare them with nonoxynol-9 (N-9), the most common active ingredient in topical vaginal contraceptives. Methods Human normozoospermic samples were assessed for motility, viability, acrosome status and penetration ability after exposure to control, N-9 or different BKC and MKC doses for 0 and 10 minutes. Local tolerance on HeLa cells was evaluated by the Trypan-blue and MTT assays. Results Exposure to BKC and MKC reduced acrosome integrity while promoting total immobilisation and complete loss of sperm viability (p < .001, n = 15). Both compounds also compromised sperm penetration ability upon exposure (p < .001, n = 15). N-9 induced the same outcomes (p < .001, n = 15); nevertheless, it was more toxic to HeLa cells than BKC and MKC (p < .05, n = 14). Conclusions BKC and MKC present strong in vitro spermicidal activity at lower doses than N-9 and were better tolerated after immediate exposure than N-9. Available Pharmatex® galenic formulations were as effective as products based on N-9.

A phase I randomized safety study of a single-size silicone rubber diaphragm used with or without a lactic-acid-containing diaphragm gel

ObjectivesTo evaluate a lactic-acid-containing diaphragm gel (Contragel®) approved outside the United States for use with a silicone rubber diaphragm (Caya®). The study gel is being evaluated as a safer alternative to nonoxynol-9 (N-9) gel, which has been associated with risk of increasing susceptibility to human immunodeficiency virus (HIV). Study designThis was a Phase I randomized, parallel study evaluating the safety of the novel diaphragm gel versus hydroxyethylcellulose (HEC) universal placebo gel delivered by the study diaphragm for two 7-day test cycles of daily use, without and with intercourse. The primary clinical safety endpoint was treatment emergent adverse events. Mucosal safety endpoints included colposcopic findings, anti-Escherichia coli activity of endocervical and vaginal fluid, immune mediators, Nugent score and ectocervical immune cell density. Endpoints were assessed prior to each test cycle and at day 7 of each test cycle. We compared the two independent groups and also evaluated paired changes from baseline in each gel cohort. ResultsTwenty-three participants used the study diaphragm with the novel gel (n=11) or with HEC (n=12). Use of either gel resulted in few genital AEs and no colposcopic findings. There were no differences in ectocervical histology and lymphocyte density or phenotype between the two cohorts at baseline or after each test cycle. We found no clinically important differences in the anti-microbial (anti Escherichia coli) activity of endocervical or vaginal fluid or concentrations of genital immune mediators (e.g. anti-inflammatory secretory leukocyte protease inhibitor (SLPI) or pro-inflammatory mediator RANTES) between the two gel cohorts at any visit. There were no important paired changes from baseline among participants using either gel in Nugent score, ectocervical histology or anti-microbial activity of genital secretions. ConclusionsWe found no clinically significant differences in clinical and mucosal safety endpoints between the two cohorts. The mucosal safety profiles of the study gel and HEC placebo gel were similar. ImplicationsOur data demonstrate no clinically important differences between the safety profiles of the lactic-acid-containing diaphragm gel versus HEC placebo gel when used with the study diaphragm. N-9 can no longer be used with contraceptive diaphragms in high HIV prevalence regions. Although larger studies are needed, the novel gel appears safe for use with the study diaphragm, which is the first over-the-counter, non-hormonal, diaphragm.

Tideglusib, a prospective alternative to nonoxynol-9 contraceptive

ObjectivesWe aimed to evaluate the antifertility activity and vaginal irritation effects of tideglusib in vivo using rabbit models and to evaluate the cytotoxical effects of tideglusib to sperm, vaginal cells and vaginal bacteria (L. acidophilus) in vitro. Study designWe treated female rabbits with vaginal tideglusib 1 mM, nonoxynol-9 (N-9) or vehicle control (Poloxamer 407). In experiment 1, we sacrificed females (n = 6 each) after 10 days of daily administration and assessed vaginal histological changes using Eckstein irritation score. In experiment 2, females (n = 9 each) received estradiol benzoate to induce ovulation 24 h prior to vaginal treatment followed by introduction of a fertile male. These females underwent necropsy at the 21st day to assess pregnancy status. In experiment 3, we used an HTM-TOX IVOS sperm motility analyzer and scanning electron microscopy (SEM) to evaluate the effect of tideglusib on human sperm samples. In experiment 4, we evaluated the effect of tideglusib on lactobacillus and vaginal cell growth in vitro. ResultsThe total irritation score of tideglusib vs. N-9 was 3.4 ± 2.07 vs. 7.8 ± 3.82, p<.05. The pregnancy rate of tideglusib, N-9 and control group was 11.1%, 0% and 88.9%, respectively. Tideglusib exhibited a dose-dependent spermostatic/spermicidal activity, and the minimum effective concentrations of tideglusib and N-9 were 8.724 ± 3.047 μM and 219.75 ± 41.78 μM, respectively. SEM and transmission electron microscopy revealed acrosomal membrane impairments caused by tideglusib. Tideglusib was much less toxic to vaginal cells and L. acidophilus than N-9 in vitro. ConclusionsEvaluation using rabbit models indicated that tideglusib is a prospective spermicidal contraceptive with low vaginal irritation effects. ImplicationsTideglusib or tideglusib analogues may be a contraceptive with perspective to replace N-9. It is possible for a spermicide to balance spermicidal activity and vaginal/cervical irritation effects very well.

Homopiperazine Derived Female Controlled Vaginal Trichomonacidal Contraceptive: An Addition to Structure-Activity Relationship.

In our previous work, several piperazine derived bis(dialkylaminethiocarbonyl) disulfides and disulfide esters of dithiocarbamic acid have been synthesized and evaluated for their spermicidal and microbicidal efficacy. These studies have provided some promising compounds for developing a dually active vaginal microbicidal contraceptive which is under pre-clinical stage. The main objective of this study was the design synthesis and biological evaluation of bis(dialkylaminethiocarbonyl) disulfides (4-15) and 2,2'-disulfanediylbis (3-(substituted-1-yl) propane-2,1-diyl) disubstituted-1-carbodithioates (19-28) as non-surfactant molecules capable of eliminating Trichomonas vaginalis as well as irreversibly immobilizing 100% human sperm promptly. Spermicidal, anti-trichomonas, cytotoxicity and biocompatibility study of the synthesized compounds was done as per the reported methodologies. Among bis(dialkylaminethiocarbonyl) disulfides (4-15, Table 1), compound 4 (MEC 0.02 mM) was found to be the most desirable for spermicidal activity as it was 40 times more active than Nonoxynol-9 (N-9), and also active against Trichomonas vaginalis (MIC 0.02 &1.10 mM). 2, 2'-disulfanediylbis (3-(substituted- 1-yl) propane-2, 1-diyl) disubstituted-1-carbodithioates (19-28, Table 2), and compounds (19, 22, 23, and 24 MEC 0.05 mM) were sixteen times more active than N-9 with promising Trichomonacidal activity. This study suggested that the disulfide linkage alone and dithiocarbamate along with disulfide group within the same chemical entity impart the desired multiple activities of compounds.

Testing vaginal irritation with the Hen’s Egg Test-Chorioallantoic Membrane assay

The HET-CAM (Hen’s Egg Test-Chorioallantoic Membrane) assay is an in vitro alternative to the in vivo Draize rabbit eye test. This qualitative method assesses the irritancy potential of chemicals. The chorioallantoic membrane responds to injury with an inflammatory process similar to that in the rabbit eye’s conjunctival tissue. Regarding topical toxicity assessment of medical devices, ISO 10993-10 states that any skin or eye irritant material shall be directly labelled as a potential vaginal irritant without animal testing, suggesting that the irri­tation potentials for the eye and the vaginal epithelia are similar. The aim of this work was to apply the HET-CAM assay to test the irritancy potential of vaginal formulations. Vaginal semisolid medicines and lubricants currently marketed were tested along with the Universal Placebo formulation that has been shown to be clinically safe. Nonoxynol-9 (N-9), a known vaginal irritant, was enrolled as positive control (concentrations ranging from 0.001 to 100% (v/v)). The assay was conducted according to the ICCVAM – Recommended Test Method (NIH Publication No. 10-7553 – 2010). Formulations were then classified according to irritation score (IS), using the analysis methods (A) and (B). The studied vaginal formulations showed low potential for irritation. N-9 was classified as a severe irritant at concentrations above 2%, which is in line with clinical data, envisaging a possible in vitro/in vivo correlation. IS (B) was considered a more detailed classification output. Although still requiring further validation, the HET-CAM assay seems an ideal prospect for in vitro vaginal irritancy testing.

A phase I randomized postcoital testing and safety study of the Caya diaphragm used with 3% Nonoxynol-9 gel, ContraGel or no gel

ObjectivesThe Caya® Diaphragm is a newly approved single-size, nonlatex diaphragm. Contragel® is a personal lubricant containing lactic acid approved in Europe and other countries for use with vaginal barrier devices. This study assessed the effectiveness in preventing sperm from penetrating midcycle cervical mucus of Caya with Contragel, Caya with 3% nonoxynol-9 (N-9) and Caya alone. Study designPhase I multicenter, single-blind, randomized, crossover, nonsignificant risk study at two sites: Eastern Virginia Medical School, Norfolk, VA, USA, and Profamilia, Santo Domingo, Dominican Republic. Healthy, sexually active women 18–45years old, not at risk for pregnancy due to tubal occlusion, were eligible. Each participant was seen in nine visits, completing a baseline cycle (without product use) followed by three test cycles (sequence determined by randomization), each consisting of a cervical mucus check visit and a postcoital test visit. To proceed to test cycles, the baseline postcoital test had to show adequate cervical mucus and >5 progressively motile sperm per high power field (PMS/HPF). ResultsAll women had an average of <5 PMS/HPF during the test cycle of each study arm, the primary endpoint. Caya with ContraGel and Caya with N-9 reduced the average number of PMS/HPF from 22.5 to 0. Caya alone reduced the average number of PMS/HPF from 22.5 to 0.4. There were two possibly product-related mild adverse events. ConclusionThis study supports that Caya with ContraGel is safe and functions as well as Caya with N-9 in preventing PMS from reaching midcycle cervical mucus. ImplicationsA single-size diaphragm used with a personal lubricant gel containing lactic acid appears to be safe and to function as well as the same diaphragm used with N-9 in preventing PMS from reaching midcycle cervical mucus.

Antimicrobial effects of novel fluorous and non-fluorous surfactants

Novel fluorous and non-fluorous surfactants have been synthesized and examined for their potential as microbicides compared with nonoxynol-9 (N-9) by testing their effects on Candida albicans (C. albicans) and Escherichia coli (E. coli). These compounds include nonionic surfactants consisting of F5-triethylene glycol (F5-TEG), F7-triethylene glycol (F7-TEG), C5-triethylene glycol (C5-TEG), C7-triethylene glycol (C7-TEG), and anionic surfactants consisting of F5-propane sultone (F5-PS), and F7-propane sultone (F7-PS). In this study, we investigated the possible effects of fluorous and non-fluorous surfactants on the growth of C. albicans and E. coli cultured in vitro, which were treated individually with different concentrations of these novel surfactants and nonoxynol-9 (N-9). Then, each sample was incubated for 3, 6, 24 and 48 h at 35°C for C. albicans and 37°C for E. coli. After incubation, C. albicans and E. coli colonies were evaluated compared with the control. N-9 and F5-PS had only small effects (25% growth inhibition or lower) on C. albicans but F7-PS, F7-TEG, F5-TEG, and C5-TEG notably inhibited C. albicans growth, and had potential to control their population. C. albicans cells treated with 10% F7-TEG, F5-TEG, or C5-TEG showed no growth; especially, C5-TEG gave the maximum growth inhibition of C. albicans. For E. coli, N-9 had no growth inhibition but F7-PS, F5-TEG, C5-TEG, C7-TEG, and F7-TEG inhibited E. coli growth. Interestingly, F7-TEG showed the maximum inhibition for E. coli starting at a concentration of 1%. Therefore, these surfactants might have potential for prevention or treatment of genital and urinary tract infection from C. albicans and E. coli. Key words: Candida albicans, Escherichia coli, nonoxynol-9, fluorous surfactants, non-fluorous surfactants.

Influence of Nonoxynol-9 on the Corrosion Inhibition of Carbon Steel in 1.0 M Hydrochloric Acid Solution

Abstract The inhibitory performance of nonoxynol-9 (N9) as a corrosion inhibitor for carbon steel was evaluated in 1.0 M HCl solution at different temperatures. Potentiodynamic polarization, electrochemical impedance spectroscopy (EIS) and quantum chemical calculation methods were used in this study. The results indicated that the corrosion rate decreased with increasing concentration of N9 up 150 ppm and decreased with the increase in temperature of the medium. The comparison between the results obtained by polarization and EIS methods showed a good agreement. The corrosion inhibition effect of N9 could be related to the adsorption of N9 molecules on the metal surface. Polarization curves indicated that N9 behaves as a mixed type inhibitor. EIS exhibited one capacitive loop which indicates that the corrosion reaction is controlled by charge transfer process. The inhibition mechanism of N9 involves physical interaction between the inhibitor and metal surface. The adsorption of N9 on carbon steel affords physisorption process and obeyed the Langmuir adsorption isotherm.

Potential Use of Antimicrobial Peptides as Vaginal Spermicides/Microbicides.

The concurrent increases in global population and sexually transmitted infection (STI) demand a search for agents with dual spermicidal and microbicidal properties for topical vaginal application. Previous attempts to develop the surfactant spermicide, nonoxynol-9 (N-9), into a vaginal microbicide were unsuccessful largely due to its inefficiency to kill microbes. Furthermore, N-9 causes damage to the vaginal epithelium, thus accelerating microbes to enter the women’s body. For this reason, antimicrobial peptides (AMPs), naturally secreted by all forms of life as part of innate immunity, deserve evaluation for their potential spermicidal effects. To date, twelve spermicidal AMPs have been described including LL-37, magainin 2 and nisin A. Human cathelicidin LL-37 is the most promising spermicidal AMP to be further developed for vaginal use for the following reasons. First, it is a human AMP naturally produced in the vagina after intercourse. Second, LL-37 exerts microbicidal effects to numerous microbes including those that cause STI. Third, its cytotoxicity is selective to sperm and not to the female reproductive tract. Furthermore, the spermicidal effects of LL-37 have been demonstrated in vivo in mice. Therefore, the availability of LL-37 as a vaginal spermicide/microbicide will empower women for self-protection against unwanted pregnancies and STI.

Feasibility of radiolabeled small molecule permeability as a quantitative measure of microbicide candidate toxicity

ObjectiveTo determine the feasibility of using quantitative changes in vaginal permeability to small molecules as a measure of candidate microbicide toxicity. Study designControlled, open-labeled, prospective study. Seven healthy women received a single vaginal dose of hydroxyethylcellulose gel (HEC), nonoxynol-9 (N-9) or K-Y Jelly. Each gel was radiolabeled with a small molecule (99mTc-DTPA) followed by 12-h blood and urine collection. Pharmacokinetic (PK) parameters of 99mTc-DTPA were calculated to compare the impact of each gel on vaginal permeability. Each woman served as her own control. The Friedman test with post hoc Wilcoxon test was used to detect differences among the gels. ResultsVaginal permeability of 99mTc-DTPA was highest for the N-9 radiolabel. N-9 plasma area under the concentration curve was 2.7-fold higher (p=.04), and peak concentration was threefold higher (p=.04) compared to HEC. There were no significant PK parameter differences between HEC and K-Y Jelly or between N-9 and K-Y Jelly. Cumulative dose-adjusted median (interquartile range) 12-h timed urine gamma activity was 66.70×10−4μCi (27.90–152.00) following HEC dosing, 103.00×10−4μCi (98.20–684.00) following N-9 gel dosing and 20.30×10−4μCi (11.10–55.90) following K-Y gel dosing. The differences between urine HEC and K-Y Jelly (p=.047) and between N-9 and K-Y Jelly (p=.016) were statistically significant. ConclusionsIt is feasible to measure differences in vaginal permeability among vaginal gels using a radiolabeled small molecule, though there are permeability differences that require a nuanced understanding of gel composition to interpret the results. ImplicationsEstablishing the safety of both vehicle and active pharmaceutical ingredient is an essential task in microbicide development, to be determined as soon as possible. This study suggests that a combination of microbicide toxicity assessments, that is, cervicovaginal permeability, inspection and histopathology, may need to be studied simultaneously.

Ammonium salts of carbamodithioic acid as potent vaginal trichomonacides and fungicides

Chemical attenuation of the reactive oxygen species (ROS)-sensitive anaerobes Trichomonas vaginalis, which is the most prevalent non-viral sexually transmitted infection, and two often coexisting vaginal infections, namely Candida albicans and Staphylococcus aureus, which are opportunistic reproductive tract infections, was attempted with novel ammonium salts of carbamodithioic acid through inhibition of free thiols. In vitro and in vivo efficacies of the designed compounds were evaluated as topical vaginal microbicides. Five compounds showed exceptional activity against drug-resistant and -susceptible strains with negligible toxicity to host (HeLa) cells in vitro in comparison with the standard vaginal microbicide nonoxynol-9 (N-9), without disturbing the normal vaginal flora (i.e. Lactobacillus). The compounds significantly inhibited the cytopathic effects of Trichomonas on HeLa cells in vitro with efficacies comparable with metronidazole (MTZ); however, their efficacy to rescue host cells from co-infection (protozoal and fungal) was greater than that of MTZ. The compounds inhibited β-haemolysis of red blood cells caused by Trichomonas and were found to be active in vivo in the mouse subcutaneous abscess assay. Some compounds rapidly immobilized human sperm. A mechanism involving inhibition of free thiols and consequently the cysteine proteases of T. vaginalis by the new compounds has been proposed. Thus, a unique scaffold of antimicrobial agents has been discovered that warrants further investigation for development as contraceptive vaginal microbicides.

Innovative Disulfide Esters of Dithiocarbamic Acid as Women-Controlled Contraceptive Microbicides: A Bioisosterism Approach.

In an ongoing effort to discover an effective, topical, dual-function, non-surfactant contraceptive vaginal microbicide, a novel series of 2,2'-disulfanediylbis(3-(substituted-1-yl)propane-2,1-diyl) disubstituted-1-carbodithioates were designed by using a bioisosterism approach. Thirty-three compounds were synthesized, and interestingly, most demonstrated multiple activities: they were found to be spermicidal at a minimal effective concentration of 1-0.001 %, trichomonacidal against drug-susceptible and resistant Trichomonas strains at minimal inhibitory concentration (MIC) ranges of 10.81-377.64 and 10.81-754.14 μM, respectively, and fungicidal at MIC 7.93-86.50 μM. These compounds were also found to be non-cytotoxic to human cervical (HeLa) epithelial cells and vaginal microflora (Lactobacilli) in vitro. The most promising compound, 2,2'-disulfanediylbis(3-(pyrrolidin-1-yl)propane-2,1-diyl)dipyrrolidine-1-carbodithioate (5), exhibited spermicidal activity 15-fold higher than that of the marketed spermicide Nonoxynol-9 (N-9) and also demonstrated microbicidal potency. To identify common structural features required for spermicidal activity, a 3D-QSAR analysis was carried out, as well as in vivo efficacy studies and fluorescent labeling studies to determine the biological targets of compound 5.

Unexpected Inflammatory Effects of Intravaginal Gels (Universal Placebo Gel and Nonoxynol-9) on the Upper Female Reproductive Tract: A Randomized Crossover Study.

Intravaginal anti-HIV microbicides could provide women with a self-controlled means for HIV prevention, but results from clinical trials have been largely disappointing. We postulated that unrecognized effects of intravaginal gels on the upper female reproductive tract might contribute to the lower-than-expected efficacy of HIV microbicides. Our objective was to study the effects of intravaginal gels on the immune microenvironment of the cervix and uterus. In this randomized crossover study, 27 healthy female volunteers used a nightly application of intravaginal nonoxynol-9 (N9) gel as a “failed” microbicide or the universal placebo gel (UPG) as a “safe” gel (intervention cycles), or nothing (control cycle) from the end of menses to the mid-luteal phase. At a specific time-point following ovulation, all participants underwent sample collection for measurements of T-cell phenotypes, gene expression, and cytokine/chemokine protein concentrations from 3 anatomic sites above the vagina: the cervical transformation zone, the endocervix and the endometrium. We used hierarchical statistical models to estimate mean (95% CI) intervention effects, for N9 and UPG relative to control. Exposure to N9 gel and UPG generated a common “harm signal” that included transcriptional up-regulation of inflammatory genes chemokine (C-C motif) ligand 20 (macrophage inflammatory factor-3alpha) and interleukin 8 in the cervix, decreased protein concentrations of secretory leukocyte protease inhibitor, and transcriptional up-regulation of inflammatory mediators glycodelin-A and osteopontin in the endometrium. These results need to be replicated with a larger sample, but underscore the need to consider the effects of microbicide agents and gel excipients on the upper female reproductive tract in studies of vaginal microbicides.

A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation

Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV+2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1β, CXCL8, epithelial membrane protein (EMP)-1 (P<0.05), and decreased levels of TLR2 (P<0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P<0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1β, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product.

New Candidate Biomarkers in the Female Genital Tract to Evaluate Microbicide Toxicity

Vaginal microbicides hold great promise for the prevention of viral diseases like HIV, but the failure of several microbicide candidates in clinical trials has raised important questions regarding the parameters to be evaluated to determine in vivo efficacy in humans. Clinical trials of the candidate microbicides nonoxynol-9 (N9) and cellulose sulfate revealed an increase in HIV infection, vaginal inflammation, and recruitment of HIV susceptible lymphocytes, highlighting the need to identify biomarkers that can accurately predict microbicide toxicity early in preclinical development and in human trials. We used quantitative proteomics and RT-PCR approaches in mice and rabbits to identify protein changes in vaginal fluid and tissue in response to treatment with N9 or benzalkonium chloride (BZK). We compared changes generated with N9 and BZK treatment to the changes generated in response to tenofovir gel, a candidate microbicide that holds promise as a safe and effective microbicide. Both compounds down regulated mucin 5 subtype B, and peptidoglycan recognition protein 1 in vaginal tissue; however, mucosal brush samples also showed upregulation of plasma proteins fibrinogen, plasminogen, apolipoprotein A-1, and apolipoprotein C-1, which may be a response to the erosive nature of N9 and BZK. Additional proteins down-regulated in vaginal tissue by N9 or BZK treatment include CD166 antigen, olfactomedin-4, and anterior gradient protein 2 homolog. We also observed increases in the expression of C-C chemokines CCL3, CCL5, and CCL7 in response to treatment. There was concordance in expression level changes for several of these proteins using both the mouse and rabbit models. Using a human vaginal epithelial cell line, the expression of mucin 5 subtype B and olfactomedin-4 were down-regulated in response to N9, suggesting these markers could apply to humans. These data identifies new proteins that after further validation could become part of a panel of biomarkers to effectively evaluate microbicide toxicity.

Spermicidal and antifertility effects of an imbibing and soluble nonoxynol-9 diaphragm (ISND) in rabbits

Objective To determine the minimum effective concentration (MEC) of an imbibing and soluble nonoxynol-9 (N-9) diaphragm (ISND) required for immobilisation of all spermatozoa in vitro and in vivo. The speed of semen absorbance, time of ISND to dissolution, and the antifertility effects were also investigated in rabbits.Methods In vitro spermicidal tests with ISND were conducted using fresh semen from humans and rabbits. Spermicidal and antifertility effects were observed in vivo after the ISND was placed directly into the vagina of rabbits.Results The MEC of N-9 required in the ISND to totally immobilise sperm within 20 seconds was 0.15 mg/ml for human sperm, and 0.5 mg/ml for rabbit sperm. The human semen was absorbed into the ISND in 45 minutes; the diaphragm dissolved in the vagina 3.5 hours later. In vivo, in rabbits, the MEC of N-9 required to immobilise sperm within five minutes of mating was 1 mg/kg in the ISND, and 10 mg/kg for the nonoxynol-9 film. The median effective dose of N-9 in the ISND was 1.07 mg/kg, whereas for the film it was 3.30 mg/kg.Conclusion The spermicidal and antifertility activities of a low dose N-9 in the ISND were high, with properties of imbibition and solubility confirmed.Chinese 摘要 目的 确定吸液可溶性壬丙醇醚-9(N-9)隔膜(ISND)在体内和体外使所有精子制动所需要的最低有效浓度(MEC)。同时研究了在家兔体内的精液吸收速度，ISND分解速度和避孕效果。方法 使用了人类和家兔的新鲜精液进行ISND体外杀精试验。将ISND直接置入家兔阴道后观察ISND在体内的杀精和避孕效果。结果 ISND使人类精液的精子在20秒内完全制动的N-9最低有效浓度为0.15 mg/ml，家兔精液中精子20秒内完全制动的N-9最低有效浓度为0.5 mg/ml。人类精液在45分钟内被吸收进ISND；隔膜于3.5小时后在阴道中分解。在家兔体内，交配5分钟内使精子制动的ISND中N-9最低有效浓度为1 mg/kg，壬丙醇醚-9膜中N-9最低有效浓度为10 mg/kg。ISND中N-9的中位有效剂量为1.07 mg/kg，N-9膜中N-9的中位有效剂量为3.30 mg/kg。结论 ISND中低剂量N-9具有高杀精和避孕效能，同时具有已被证实的吸液可溶性。

Quantitative determination of microbicidal spermicide 'nonoxynol-9' in rabbit plasma and vaginal fluid using LC-ESI-MS/MS: application to pharmacokinetic study.

Nonoxynol-9 (N-9), a microbicidal spermicide, has been in use as an over-the-counter contraceptive since the 1960s. A detailed account of its pharmacokinetic profile using highly sensitive detection method has not been reported yet. We developed and validated a rapid, selective and sensitive high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for N-9 detection in plasma and simulated vaginal fluid. The analytes were quantified using reverse phase Thermo Accucore C18 (150 mm × 4.6mm, 5 μm) column with isocratic elution using acetonitrile: 0.1% formic acid in triple distilled water (90:10, v/v) as mobile phase. The ionization was optimized using ESI (+) and selectivity was achieved by tandem mass spectrometric analysis using MRM transition, m/z 617.4→133.2 for N-9 and m/z 180.1→138.1 for phenacetin. The method was linear over the range 0.195-100 ng/mL. The method was accurate and precise with intra-batch and inter-batch accuracy (% bias) of less than ± 15% and precision (% CV) of <15% for N-9. The mean peak plasma concentration (Cmax) 4.87 ± 0.37 ng/mL was achieved 1.0h after vaginal application with terminal half-life 1.45 ± 0.07 h in rabbits. The validated method was successfully applied for pharmacokinetic study of N-9 in rabbits after vaginal administration.