Safety of multiple administrations of spermicidal LL-37 antimicrobial peptide into the mouse female reproductive tract.

We have previously demonstrated spermicidal activity of LL-37 antimicrobial peptide on mouse/human sperm and its contraceptive effects in female mice. With its microbicidal action against Neisseria gonorrhoeae, LL-37 warrants development into a multipurpose prevention technology (MPT) agent for administering into the female reproductive tract (FRT). However, it is important to verify that multiple administrations of LL-37 do not lead to damage of FRT tissues and/or irreversible loss of fecundity. Herein, we transcervically injected LL-37 (36 µM-10× spermicidal dose) into female mice in estrus in three consecutive estrous cycles. A set of mice were sacrificed for histological assessment of the vagina/cervix/uterus 24 h after the last injection, while the second set were artificially inseminated with sperm from fertile males 1 week afterwards, and then monitored for pregnancy. Mice injected with PBS in parallel were regarded as negative controls, whereas those injected with vaginal contraceptive foam (VCF, available over the counter), containing 12.5% nonoxynol-9, served as positive controls for vaginal epithelium disruption. We demonstrated that the vagina/cervix/uterus remained normal in both LL-37-injected and PBS-injected mice, which also showed 100% resumption of fecundity. In contrast, VCF-injected mice showed histological abnormalities in the vagina/cervix/uterus and only 50% of them resumed fecundity. Similarly, LL-37 multiply administered intravaginally caused no damage to FRT tissues. While our results indicate the safety of multiple treatments of LL-37 in the mouse model, similar studies have to be conducted in non-human primates and then humans. Regardless, our study provides an experimental model for studying in vivo safety of other vaginal MPT/spermicide candidates.

Surrogate post-coital testing for contraceptive efficacy against human sperm activity in the ovine vaginal model†.

High unintended pregnancy rates are partially due to lack of effective nonhormonal contraceptives; development of safe, effective topical vaginal methods will address this need. Preclinical product safety and efficacy assessment requires in vivo testing in appropriate models. The sheep is a good model for the evaluation of vaginally delivered products due to its close similarities to humans. The study objective was to develop an ovine model for efficacy testing of female nonhormonal contraceptives that target human sperm. Fresh human semen was pooled from male volunteers. Nonpregnant female Merino sheep were treated with control or vaginal contraceptive product (IgG antibody with action against sperm or nonoxynol-9 [N9]). Pooled semen was added to the sheep vagina and mixed with product and vaginal secretions. Microscopic assessment of samples was performed immediately and progressive motility (PM) of sperm was compared between treatments. Cytokines CXCL8 and IL1B were assessed in vaginal fluid after instillation of human semen. No adverse reactions or elevations in proinflammatory cytokines occurred in response to human semen. N9 produced signs of acute cellular toxicity while there were no cellular changes after IgG treatment. N9 and IgG had dose-related effects with the highest dose achieving complete sperm immobilization (no sperm with PM). Surrogate post-coital testing of vaginally administered contraceptives that target human semen was developed in an ovine model established for vaginal product preclinical testing. This expanded model can aid the development of much needed nonhormonal topical vaginal contraceptives, providing opportunities for rapid iterative drug development prior to costly, time-intensive human testing.

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.

Pretreatment of human cervicovaginal mucus with pluronic F127 enhances nanoparticle penetration without compromising mucus barrier properties to herpes simplex virus.

Mucosal drug delivery nanotechnologies are limited by the mucus barrier that protects nearly all epithelial surfaces not covered with skin. Most polymeric nanoparticles, including polystyrene nanoparticles (PS), strongly adhere to mucus, thereby limiting penetration and facilitating rapid clearance from the body. Here, we demonstrate that PS rapidly penetrate human cervicovaginal mucus (CVM), if the CVM has been pretreated with sufficient concentrations of Pluronic F127. Importantly, the diffusion rate of large polyethylene glycol (PEG)-coated, nonmucoadhesive nanoparticles (PS-PEG) did not change in F127-pretreated CVM, implying that F127 did not significantly alter the native pore structure of CVM. Additionally, herpes simplex virus type 1 (HSV-1) remains adherent in F127-pretreated CVM, indicating that the presence of F127 did not reduce adhesive interactions between CVM and the virions. In contrast to treatment with a surfactant that has been approved for vaginal use as a spermicide (nonoxynol-9 or N9), there was no increase in inflammatory cytokine release in the vaginal tract of mice after daily application of 1% F127 for 1 week. Pluronic F127 pretreatment holds potential as a method to safely improve the distribution, retention, and efficacy of nanoparticle formulations without compromising CVM barrier properties to pathogens.

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.

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.

Monitoring vaginal epithelial thickness changes noninvasively in sheep using optical coherence tomography.

OBJECTIVE: High-resolution optical coherence tomography can be used noninvasively to evaluate vaginal morphologic features, including epithelial thickness, to assess this protective barrier in transmission of sexually transmitted infections and to monitor tissue response to topical medications and hormonal fluctuations. We examined the use of optical coherence tomography to measure epithelial thickness noninvasively before and after topical treatment with a drug that causes epithelial thinning. STUDY DESIGN: Twelve female sheep were treated with intravaginal placebo (n = 4) or nonoxynol-9 (n = 8). Vaginal optical coherence tomography images were obtained before and 24 hours after treatment. Four sheep in the nonoxynol-9 group were also examined on days 3 and 7. Vaginal biopsies were obtained on the last examination day. Epithelial thickness was measured in optical coherence tomography images and in hematoxylin and eosin-stained histologic sections from biopsies. Statistical analysis was performed using analyses of variance (significance P < .05). RESULTS: Baseline optical coherence tomography epithelial thickness measurements were similar (85 ± 19 µm placebo, 78 ± 20 µm nonoxynol-9; P = .52). Epithelial thinning was significant after nonoxynol-9 (32 ± 22 µm) compared with placebo (80 ± 15 µm) 24 hours after treatment (P < .0001). In the 4 nonoxynol-9-treated sheep followed for 7 days, epithelial thickness returned to baseline by day 3, and increased significantly on day 7. Epithelial thickness measurements from histology were not significantly different than optical coherence tomography (P = .98 nonoxynol-9, P = .93 hydroxyethyl cellulose). CONCLUSION: Drug-induced changes in the epithelium were clearly detectable using optical coherence tomography imaging. Optical coherence tomography and histology epithelial thickness measurements were similar, validating optical coherence tomography as a noninvasive method for epithelial thickness measurement, providing an important tool for quantitative and longitudinal monitoring of vaginal epithelial changes.

Therapeutic efficacy of platelet transfusion treated with amotosalen/UVA pathogen inactivation technology (INTERCEPTTM Blood System) in acute myeloid leukemia patients undergoing chemotherapy with curative intent: a single center experience.

BACKGROUND: The INTERCEPTTM Blood System (Intercept Blood System, Cerus Europe BV, Amersfoort, the Netherlands) has been used to reduce or inactivate pathogen load in platelet concentrates in France for three years. MATERIALS AND METHODS: After comparing the transfusion efficiency between pathogen-reduced platelets (PR_PLT) and untreated platelet products (U_PLT), our single-center observational study assessed the effectiveness of PR_PLT for the prevention of bleeding and for therapeutic treatment of WHO grade 2 bleeding in 176 patients undergoing chemotherapy with curative intent for acute myeloid leukemia (AML). The main endpoints were the 24-hour (h) corrected count increment (24h_CCI) after each transfusion, and time to next transfusion. RESULTS: Whereas the transfused doses tended to be higher in the PR_PLT group compared to U_PLT, there was a significant difference in intertransfusion interval (ITI) and 24h_CCI. In prophylactic transfusions, PR_PLT transfusions of >0.65×1011/10 kg, regardless of the age of the product (day 2 to day 5), resulted in a 24h_CCI similar to that of the untreated platelet product; this meant the patient could be transfused at least every 48h. In contrast, most PR_PLT transfusions of <0.55×1011/10 kg did not achieve a transfusion interval of 48h. In the context of WHO grade 2 bleeding, PR_PLT transfusions >0.65×1011/10 kg and storage of less than 4 days seems more effective in stopping bleeding. DISCUSSION: These results, which must be confirmed by prospective studies, indicate the need for vigilance regarding the quantity and quality of PR_PLT products used to treat patients at risk of bleeding crisis. Future prospective studies are needed to confirm these findings.

Rat sperm immobilisation effects of a protein from Ricinus communis (Linn.): an in vitro comparative study with nonoxynol-9.

Previous study conducted in our department showed that 50% ethanolic extract of the root of Ricinus communis possess reversible antifertility effect and a 62-kDa protein (Rp) from this extract is responsible for the antifertility effects. In this study, we compared the spermicidal effect of this Rp with nonoxynol-9 (N-9) in vitro. The sperm immobilisation studies showed that 100 µg ml(-1) of Rp was able to immobilise the sperms completely within 30 s. Sperm revival test revealed that the spermicidal effect was irreversible. There was also a significant reduction in sperm viability and hypo-osmotic swelling in Rp and N-9 treated groups in comparison with the control. In Rp and N-9 treated groups, the number of acrosome-reacted cells was found to be high and also caused agglutination of the spermatozoa, indicating the loss of intactness of the plasma membrane, which was further supported by the significant reduction in the activity of membrane bound 5'-nucleotidase, acrosomal acrosin. In short, the protein Rp possesses spermicidal activity in vitro and its effects are similar to that of nonoxynol 9.

Development and characterization of a three-dimensional organotypic human vaginal epithelial cell model.

We have developed an in vitro human vaginal epithelial cell (EC) model using the innovative rotating wall vessel (RWV) bioreactor technology that recapitulates in vivo structural and functional properties, including a stratified squamous epithelium with microvilli, tight junctions, microfolds, and mucus. This three-dimensional (3-D) vaginal model provides a platform for high-throughput toxicity testing of candidate microbicides targeted to combat sexually transmitted infections, effectively complementing and extending existing testing systems such as surgical explants or animal models. Vaginal ECs were grown on porous, collagen-coated microcarrier beads in a rotating, low fluid-shear environment; use of RWV bioreactor technology generated 3-D vaginal EC aggregates. Immunofluorescence and scanning and transmission electron microscopy confirmed differentiation and polarization of the 3-D EC aggregates among multiple cell layers and identified ultrastructural features important for nutrient absorption, cell-cell interactions, and pathogen defense. After treatment with a variety of toll-like receptor (TLR) agonists, cytokine production was quantified by cytometric bead array, confirming that TLRs 2, 3, 5, and 6 were expressed and functional. The 3-D vaginal aggregates were more resistant to nonoxynol-9 (N-9), a contraceptive and previous microbicide candidate, when compared to two-dimensional monolayers of the same cell line. A dose-dependent production of tumor necrosis factor-related apoptosis-inducing ligand and interleukin-1 receptor antagonist, biomarkers of cervicovaginal inflammation, correlated to microbicide toxicity in the 3-D model following N-9 treatment. These results indicate that this 3-D vaginal model could be used as a complementary tool for screening microbicide compounds for safety and efficacy, thus improving success in clinical trials.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: The study reveals a unique mechanism of action for DSE spermicides. DSE-37 holds promise as a safe vaginal contraceptive. CDRI Communication No. 7545.

Chlamydia trachomatis laboratory strains versus recent clinical isolates: implications for routine microbicide testing.

A topical microbicide that women can use to prevent sexually transmitted diseases (STDs) is essential, and many microbicide candidates are being tested for activity against human immunodeficiency virus and other STDs, including Chlamydia trachomatis. Screening assays for assessing the activity of microbicides against C. trachomatis are typically done with laboratory-adapted strains, but it is possible that recent clinical isolates may have different susceptibilities to microbicides, as has been seen with Neisseria gonorrhoeae and Lactobacillus spp. (B. J. Moncla and S. L. Hillier, Sex. Transm. Dis. 32:491-494, 2005). We utilized three types of microbicides to help define this aspect of our assay to test microbicides against C. trachomatis in vitro. To simulate conditions of transmission, we used an assay that we previously developed in which we exposed chlamydial elementary bodies to microbicides prior to contact with epithelial cells. We first determined the toxicity of microbicides to the cells used to culture Chlamydia trachomatis in the assay and, if necessary, modified the assay to eliminate toxicity at the concentrations tested. We compared the sensitivities of recent clinical isolates of Chlamydia trachomatis versus laboratory strains of the same serovar and found major differences in sensitivity to nonoxynol-9 (non-9), but only minor differences were seen with the other microbicides. We thus conclude that when assessing activity of potential topical microbicides versus the obligate intracellular bacteria C. trachomatis, the use of recent clinical isolates may not be necessary to draw a conclusion about a microbicide's effectiveness. However, it is important to keep in mind that differences (like those seen with non-9) are possible and that clinical isolates could be included in later stages of testing.

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

BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.

Highly Soluble Drugs Directly Granulated by Water Dispersions of Insoluble Eudragit® Polymers as a Part of Hypromellose K100M Matrix Systems.

The aim of the present study was to investigate the suitability of insoluble Eudragit® water dispersions (NE, NM, RL, and RS) for direct high-shear granulation of very soluble levetiracetam in order to decrease its burst effect from HPMC K100M matrices. The process characteristics, ss-NMR analysis, in vitro dissolution behavior, drug release mechanism and kinetics, texture profile analysis of the gel layer, and PCA analysis were explored. An application of water dispersions directly on levetiracetam was feasible only in a multistep process. All prepared formulations exhibited a 12-hour sustained release profile characterized by a reduced burst effect in a concentration-dependent manner. No effect on swelling extent of HPMC K100M was observed in the presence of Eudragit®. Contrary, higher rigidity of formed gel layer was observed using combination of HPMC and Eudragit®. Not only the type and concentration of Eudragit®, but also the presence of the surfactant in water dispersions played a key role in the dissolution characteristics. The dissolution profile close to zero-order kinetic was achieved from the sample containing levetiracetam directly granulated by the water dispersion of Eudragit® NE (5% of solid polymer per tablet) with a relatively high amount of surfactant nonoxynol 100 (1.5%). The initial burst release of drug was reduced to 8.04% in 30 min (a 64.2% decrease) while the total amount of the released drug was retained (97.02%).

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.

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

BACKGROUND: 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. OBJECTIVE: 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. METHOD: Spermicidal, anti-trichomonas, cytotoxicity and biocompatibility study of the synthesized compounds was done as per the reported methodologies. RESULT: 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. CONCLUSION: 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.

Effects of long-term use of nonoxynol-9 on vaginal flora.

OBJECTIVE: Products containing nonoxynol-9 have been used as spermicidal contraceptives for many years, but limited data have been published describing the long-term effects of nonoxynol-9 use on the vaginal microbial ecosystem. This longitudinal study was conducted to examine the effects of nonoxynol-9 on the vaginal ecology. METHODS: Vaginal swabs were obtained from 235 women enrolled in a randomized clinical trial before initiation of use of 1 of 5 different formulations of nonoxynol-9 for contraception, and up to 3 more samples were gathered over 7 months of use. The swab samples were evaluated in a single laboratory. The prevalence of several constituents of the normal vaginal flora was evaluated. The associations between nonoxynol-9 dosage, formulation, average product use per week, and number of sex acts per week were calculated. RESULTS: The changes in prevalence of vaginal microbes after nonoxynol-9 use were minimal for each of the different nonoxynol-9 formulations. However, when both nonoxynol-9 concentration and number of product uses are taken into account, nonoxynol-9 did have dose-dependant effects on the increased prevalence of anaerobic gram-negative rods (odds ratio [OR] 2.4, 95% confidence interval [CI] 1.1-5.3), H2O2-negative lactobacilli (OR 2.0, 95% CI 1.0-4.1), and bacterial vaginosis (OR 2.3, 95% CI 1.1-4.7). CONCLUSION: This study demonstrated that most nonoxynol-9 users experienced minimal disruptions in their vaginal ecology. There were no differences between the different formulations evaluated with respect to changes in vaginal microflora. However, independent of the nonoxynol-9 formulation, there was a dose-dependent effect with increased exposure to nonoxynol-9 on the risk of bacterial vaginosis and its associated flora. LEVEL OF EVIDENCE: II-2.

Lactobacillus species isolated from the vagina: identification, hydrogen peroxide production and nonoxynol-9 resistance.

BACKGROUND: Lactic acid production is considered to be the major protection mechanism of lactobacilli against vaginal infections due to genital pathogens. Some species of Lactobacillus are also hydrogen peroxide (H(2)O(2)) producers. Nonoxynol-9 (N-9) is a nonionic detergent and is the active component of many spermicidal preparations. It immobilizes sperm by disrupting the cell membrane and is believed to act similarly on a number of bacteria and viruses. It is known that N-9 inhibits Lactobacilli in vitro at concentrations of 0.1% to 1%. PURPOSE: The present study was conducted to identify the species of Lactobacillus isolated from vaginal fluids of reproductive-age women and to characterize the H(2)O(2)-producing and N-9-resistant strains in an Argentine population. RESULTS: We identified Lactobacillus acidophilus, L. fermentum, L. gasseri, L. jensenii, L. casei subsp. casei, L. brevis and L. delbrueckii subsp. delbrueckii as the most frequent species. In this Argentine, South American population, 62% of women had H(2)O(2)-producing vaginal lactobacilli. We found a high number of sensitive strains. Sixty-two H(2)O(2)-producer strains were detected, 50 (80.6%) strains were sensitive to N-9 and 12 (19.4%) strains were resistant to the inhibitory effect of N-9. DISCUSSION: The vaginal microecologic findings are comparable to those found in other populations and suggest that (1) vaginal microecologic conditions are likely to be similar among Argentine women as in other countries, and (2) N-9 may have deleterious effects as in other populations.

[Contraceptive efficacy of bioadhesive benzalkonium chloride gel in comparison with nonoxynol-9 gel].

OBJECTIVE: To compare the contraceptive efficacy of a new spermicide, bioadhesive benzalkonium chloride (BZK) gel, with the traditional nonoxynol (NP-9) gel. METHODS: A total of 240 child-bearing age women volunteers were randomly divided into two groups: 120 using the BZK gel for contraception, and the other 120 using the NP-9 gel. Using life table method and log-rank test, we compared the pregnancy rates and discontinuation rates after follow-up for 6 months in two groups. RESULTS: No abnormal findings of cervical smears were detected before and after this clinical trial in all 240 women. The follow-up rates at 6 months were 100.0% and 99.2% in the BZK group and the NP-9 group, respectively. The 6-month gross cumulative pregnancy rates of typical use were 1.72 and 0.91 per 100 women (P > 0.05), respectively. If we excluded the 2 pregnant women (1 in each group), who did not correctly or consistently use the spermicides for every intercourse, the cumulative pregnancy rates at 6 months in perfect use would be 0.87 and 0 per 100 women (P > 0.05). And the gross cumulative discontinuation rates due to allergy or other adverse reactions at 6 months in typical use were 0 and 2.68 per 100 women (P > 0.05), respectively. CONCLUSION: The contraceptive efficacy of bioadhesive BZK gel is the same as that of the NP-9 gel, and it is more acceptable in clinical use.

Prolonged exposure to the candidate microbicide C31G differentially reduces cellular sensitivity to agent re-exposure.

Comparative assays of in vitro cytotoxicity using nonoxynol-9 (N-9) and the candidate microbicides C31G and sodium dodecyl sulfate (SDS) demonstrated that these agents, which are, respectively, characterized as nonionic, amphoteric, and anionic surfactants, differed in their concentration-dependent effects on cell viability, especially after prolonged exposure. We hypothesized that differences in cellular sensitivity may have been due, in part, to cellular changes induced by long-term exposure to each agent. To examine this possibility, HeLa cells were exposed to N-9, C31G, or SDS for extended periods of time and subsequently reassessed for sensitivity to each of these agents. Following 10 continuous days of C31G exposure, HeLa cells were less sensitive to a subsequent C31G exposure compared to cells that had not undergone long-term C31G treatment. Interestingly, long-term C31G exposure also changed subsequent sensitivity to N-9 but not SDS. In contrast, prolonged exposure to either N-9 or SDS did not reduce sensitivity to re-exposure. The effect of long-term C31G exposure was both concentration-dependent and transient, as treated cells reverted to pre-exposure sensitivity in a time-dependent manner following the cessation of C31G exposure. Lipid analyses of cells exposed to C31G for extended durations revealed altered phospholipid profiles relative to C31G-naïve cells. Experiments examining the individual components of C31G demonstrated the involvement of the amine oxide moiety in reductions in cellular sensitivity. These studies, which provide new information concerning the cytotoxicity of surfactant microbicides, suggest that cervicovaginal epithelial cells may have greater in vivo tolerance for products containing C31G through unique interactions between C31G and components of the cellular membranes.