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.

Absence of Effect of Intravaginal Miconazole, Clindamycin, Nonoxynol-9, and Tampons on the Pharmacokinetics of an Anastrozole/Levonorgestrel Intravaginal Ring.

A study was performed to investigate the effect of an intravaginally administered antimycotic, an antibiotic, and a spermicide plus the co-usage of tampons on the pharmacokinetics (PK) of levonorgestrel (LNG) and anastrozole (ATZ) administered as an intravaginal ring (IVR) releasing 1050 µg ATZ per day and 40 µg LNG per day. In this parallel-group, randomized, open-label study, healthy premenopausal women received an IVR as the main treatment. Comedications were administered on 3 consecutive evenings during treatment with IVR on days 9-11 (group A, 400 mg miconazole; group B, 100 mg clindamycin; group C, 75 mg nonoxynol-9); tampon co-usage (group D) was performed on days 20-23. The primary PK parameter was the average plasma concentration (Cav,ss ) of ATZ and LNG at defined intervals, mainly prior to, during, and up to 7 days after the start of comedication. Fifty-two subjects were included, and at least 11 subjects per group completed the treatments. Overall, the medications and comedications were safe and well tolerated. Very similar ATZ and LNG plasma levels were observed across all groups. The calculated ratios of Cav,ss confirmed the absence of PK interactions because all relevant point estimates and 90% confidence intervals were within the range of 0.800-1.250, which is typically used in bioequivalence studies. These results demonstrate the absence of PK interactions between ATZ/LNG released from IVR and the tested antibiotic, antimycotic, spermicide, and tampons. Therefore, no restrictions for the use of the IVR are needed to continue the clinical program intended to treat endometriosis symptoms.

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

OBJECTIVE: To determine the feasibility of using quantitative changes in vaginal permeability to small molecules as a measure of candidate microbicide toxicity. STUDY DESIGN: Controlled, 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 ((99m)Tc-DTPA) followed by 12-h blood and urine collection. Pharmacokinetic (PK) parameters of (99m)Tc-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. RESULTS: Vaginal permeability of (99m)Tc-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(-4)µCi (27.90-152.00) following HEC dosing, 103.00 × 10(-4)µCi (98.20-684.00) following N-9 gel dosing and 20.30 × 10(-4)µCi (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. CONCLUSIONS: It 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. IMPLICATIONS: Establishing 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.

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)-1ß, 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-1ß, 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.

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.

Potential for revitalisation of the diaphragm for family planning in Uganda: a rapid assessment of the feasibility of introducing the SILCS Diaphragm.

This health systems assessment evaluated the feasibility of introducing a new contraceptive device, the SILCS single-size diaphragm, into the existing family planning method mix in Uganda. A total of 26 focus group discussions with 201 female and 77 male potential users and 98 key informant interviews with policymakers and providers were conducted between June and August 2010. Potential users, providers, and policymakers recognised that the SILCS Diaphragm could fill a gap in the method mix and expressed eagerness to make the SILCS Diaphragm available, particularly because it is nonhormonal and woman initiated. The diaphragm was viewed by all stakeholders as a method that would increase choice and could improve women's reproductive health in Uganda. Like many countries, Uganda's family planning programme is financially stretched, and clear support for the SILCS Diaphragm by end-users will need to be demonstrated before the product will be considered for public-sector introduction.

Spermicide used alone for contraception.

BACKGROUND: Spermicides have been used as contraceptives for thousands of years. Despite this long use, only recently have studies examined the comparative efficacy and acceptability of these vaginal medications. Spermicides contain an active ingredient (most commonly nonoxynol-9) and a formulation used to disperse the product, such as foam or vaginal suppository. OBJECTIVES: This review examined all known randomized controlled trials of a spermicide used alone for contraception. SEARCH METHODS: In August 2013, we searched the following computerized databases for randomized controlled trials of spermicides for contraception: CENTRAL, MEDLINE, POPLINE, LILACS, EMBASE, ClinicalTrials.gov, and ICTRP. For the initial review, we examined the reference lists of trials found as well as those of review articles and textbook chapters. SELECTION CRITERIA: We included any trial of a commercial product used alone for contraception. Each included trial must have provided sufficient information to determine pregnancy rates. DATA COLLECTION AND ANALYSIS: Two authors independently extracted information from the trials identified. We did not conduct a meta-analysis, since most trials had large losses to follow up. We entered the data into tables and presented the results descriptively. MAIN RESULTS: We located reports from 14 trials for the initial review, but have not identified any new trials since then. In the largest trial to date, the gel (Advantage S) containing the lowest dose of nonoxynol-9 (52.5 mg) was significantly less effective in preventing pregnancy than were gels with higher doses of the same agent (100 mg and 150 mg). Probabilities of pregnancy by six months were 22% for the 52.5 mg gel, 16% for the 100 mg dose, and 14% for the 150 mg dose. In the same trial, the three different vehicles with 100 mg of nonoxynol-9 had similar efficacy. Interpretation of these figures is limited, since 39% of participants discontinued the method or were lost from the trial. Few important differences in efficacy emerged in other trials. AUTHORS' CONCLUSIONS: The probability of pregnancy varied widely in reported trials. A gel containing nonoxynol-9 52.5 mg was inferior to two other products tested in the largest trial. Aside from this finding, personal characteristics and behavior of users may be more important than characteristics of the spermicide products in determining the probability of pregnancy. Gel was liked more than the film or vaginal suppository in the largest trial. Spermicide trials have the dual challenges of difficult recruitment and high discontinuation rates; the latter threatens trial validity.

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.

Effectiveness of an optimized benzalkonium chloride gel as vaginal contraceptive: a randomized controlled trial among Chinese women.

BACKGROUND: This study was conducted to evaluate the efficacy, safety and acceptability of a newly developed benzalkonium chloride (BZK) contraceptive gel which was compared to nonoxynol-9 (N-9) gel. STUDY DESIGN: A Phase II, multicenter, randomized, controlled study at three Chinese centers was conducted to compare 120 women who used BZK gel with 120 women who used N-9 gel for 6 months. Contraceptive efficacy was assessed by pregnancy rate, and safety was evaluated by adverse events report, gynecologic examination, Papanicolaou smears, leukorrhea test, and blood and urine tests. The acceptability was assessed through follow-up visit forms and a questionnaire at the 6-month visit. RESULTS: Net cumulative rates in the BZK group at 6 months were as follows: follow-up 100%, terminations 5.1%, pregnancy 1.7%, medical reasons 0% and fear of failure 3.4%. At 6 months, the rates in the N-9 group were as follows: follow-up 99.2%, terminations 9.4%, pregnancy 0.9%, medical reasons 2.5%, fear of failure 3.4% and other personal reasons 2.6%. No significant difference in pregnancy rate and termination rate between the two groups was found (p>.05). Seven cases in the BZK group (5.8%) complained about leukorrhagia and vaginal irritation symptoms (itching and burning) at 6 months, while 16 cases in the N-9 group (13.3%) had similar complaints (p<.05). This significant difference continued to exist until the 6-month visit. The general satisfaction rate for BZK gel use (72.8%) is significantly higher than that for N-9 gel (42.5%). CONCLUSION: The optimized BZK gel is comparable to N-9 gel in terms of contraceptive efficacy and safety, and may be more acceptable to Chinese users.

Decreased cervical epithelial sensitivity to nonoxynol-9 (N-9) after four daily applications in a murine model of topical vaginal microbicide safety.

BACKGROUND: The disappointing clinical failures of five topical vaginal microbicides have provided new insights into factors that impact microbicide safety and efficacy. Specifically, the greater risk for human immunodeficiency virus type 1 (HIV-1) acquisition associated with multiple uses of a nonoxynol-9 (N-9)-containing product has highlighted the importance of application frequency as a variable during pre-clinical microbicide development, particularly in animal model studies. METHODS: To evaluate an association between application frequency and N-9 toxicity, experiments were performed using a mouse model of cervicovaginal microbicide safety. In this model system, changes in cervical and vaginal epithelial integrity, cytokine release, and immune cell infiltration were assessed after single and multiple exposures to N-9. RESULTS: After the initial application of N-9 (aqueous, 1%), considerable damage to the cervical epithelium (but not the vaginal epithelium) was observed as early as 10 min post-exposure and up to 8 h post-exposure. Subsequent daily exposures (up to 4 days) were characterized by diminished cervical toxicity relative to single exposures of like duration. Levels of pro-inflammatory cytokines released into the cervicovaginal lumen and the degree of CD14-positive immune cell infiltration proximal to the cervical epithelium were also dependent on the number of N-9 exposures. CONCLUSIONS: Rather than causing cumulative cervical epithelial damage, repeated applications of N-9 were characterized by decreased sensitivity to N-9-associated toxicity and lower levels of immune cell recruitment. These results provide new insights into the failure of N-9-based microbicides and illustrate the importance of considering multiple exposure protocols in pre-clinical microbicide development strategies.

Colposcopy: still useful in microbicide safety trials?

BACKGROUND: Colposcopy is used to evaluate vaginal microbicides, but its link to risk of HIV is unknown. This reanalysis of 9 safety studies determined the impact of omitting colposcopy on the number of findings detected and assessed whether colposcopy was useful in identifying nonoxynol-9 (N-9) as an unsafe product in one study. METHODS: Product-related findings seen with naked eye and colposcopy or by colposcopy alone were evaluated. Using data from one study, the ratio of findings in N-9 users to those in hydroxyethylcellulose (HEC) users was compared for findings seen by naked eye and colposcopy versus findings detected only by colposcopy. RESULTS: Of the 403 finding observations in the 9 studies, 173 (43%) would have been missed without colposcopy. Data from the N-9/HEC study showed that without colposcopy, there would have been 7 times as many observations in the N-9 group as in the HEC group (63 vs. 9). With colposcopy, the N-9/HEC ratio was 13:9 or 1.4. Considering epithelial integrity, finding type, and size showed similar patterns, except that among the smallest findings (<5 mm), the N-9/HEC ratio was 1.2 by naked eye and nearly the same at 1.4 by colposcopy. CONCLUSION: Colposcopy was not helpful in identifying an unsafe product: the conclusions reached using naked eye examination alone were more alarming regarding the safety of N-9 than reached by including colposcopy. Recommendations include: (1) naked eye examinations should be continued in microbicide studies; (2) colposcopy may be considered for early studies, such as first-in-human studies, but has no place in large studies; and (3) colposcopy should be replaced as soon as possible with a more objective validated biomarker of HIV risk.

Acceptability of optical coherence tomography and abstinence requirements among women participating in microbicide safety trials.

BACKGROUND: Developing effective and safe microbicides requires study procedures (e.g., technology used, abstinence requirements, and product use) that are acceptable to participants. METHODS: Thirty women completed 4 study visits including pelvic examination, colposcopy, optical coherence tomography (OCT), and semistructured, qualitative interviews. Additional requirements included abstinence (for approximately 16 days) and twice daily vaginal product use (for 5.5 days). Interviews were audio-recorded, transcribed, and analyzed using framework analysis. Themes addressing OCT experiences, acceptability of abstinence, and vaginal product use were examined. RESULTS: OCT was viewed favorably as an imaging technology. Some women reported feeling the fiber-optic probe "poking" them and more than one-third spontaneously reported feeling pressure or pinching upon rotation of the speculum in connection with the OCT evaluation. Compliance with vaginal gel use was high, but for many women assigned to use a product containing nonoxynol-9 (vs. placebo), the postproduct use examination was more uncomfortable, relative to the initial examination or 1 week following product discontinuation. Nearly all women experienced product leakage; acceptability of leakage varied. Two women were not abstinent and several more found abstinence challenging. Some women involved their partner in decision making regarding trial enrollment. Strategies to remain abstinent included participating when the partner was away, avoiding early intimacy, and engaging in alternative sexual activities. CONCLUSIONS: Qualitative interviews in early-phase studies provide insights and capture information that would be missed by behavioral inference alone. Understanding participant's experiences is important in order to provide anticipatory guidance and plan future microbicide studies that facilitate adherence with trial requirements.

Mucosal integrity and inflammatory markers in the female lower genital tract as potential screening tools for vaginal microbicides.

BACKGROUND: In the female genital tract, vaginal colposcopy, endometrial mucosal integrity and inflammatory mediators are potential in vivo biomarkers of microbicide and contraceptive safety. STUDY DESIGN: A randomized, blinded crossover trial of 18 subjects comparing effects of nonoxynol-9 vaginal gel (Gynol II; putative inflammatory gel), hydroxyethyl cellulose gel (HEC; putative inert gel) and no gel exposure on endometrial and vaginal epithelial integrity and endometrial and vaginal inflammatory markers [interleukin (IL) 1ß, IL-6, IL-8, MCP-1, MIP-1α, MIP-1ß, RANTES, tumor necrosis factor α, IL-1RA, IL-10, SLPI). RESULTS: Gynol II was associated with more vaginal lesions. No endometrial disruptions were observed across conditions. In the vagina, RANTES (p=.055) and IL-6 (p=.04) were higher after HEC exposure than at baseline. In the endometrium, IL-1ß (p=.003) and IL-8 (p=.025) were lower after Gynol II cycles than after no gel. CONCLUSIONS: Gynol II and HEC may modulate inflammatory markers in the vagina and endometrium. How these changes relate to infection susceptibility warrants further study.

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.

PROBLEM: 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. METHODS AND RESULTS: 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. CONCLUSION: 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.

The effect of a combination of 0.1% octenidine dihydrochloride and 2% 2-phenoxyethanol (octenisept) on wound healing in pigs in vivo and its in vitro percutaneous permeation through intact and barrier disrupted porcine skin.

A combination of 0.1% octenidine dihydrochloride and 2% 2-phenoxyethanol (octenisept) is a commonly used disinfectant in human medicine. As porcine skin represents an adequate model for human skin, the effect of octenidine dihydrochloride and phenoxyethanol on wound healing is studied in pigs. Furthermore, the in vitro percutaneous permeation of the test substances is studied. The impact of the test formulations on wound healing is examined (A) under non occlusive conditions and (B) in comparison to another disinfectant based on povidone-iodine under occlusive conditions, while wounds are treated daily with the test substances. The percutaneous permeation of octenidine dihydrochloride and phenoxyethanol is studied in Franz-type diffusion cells with intact skin as well as barrier disrupted after tape stripping. Compared with povidone-iodine or vehicle treatment as well as untreated control wounds the treatment of wounds with the test formulation has no influence on the healing rate in pigs and does not induce retardation of wound healing. The in vitro diffusion experiment reveals that octenidine dihydrochloride is only detectable in the acceptor chamber of three-barrier disrupted skin samples. Phenoxyethanol permeates through intact porcine skin in amounts of 11.3% and through barrier disrupted skin in amounts of 43.9%

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.

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

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

Rectal and vaginal immunization of mice with human papillomavirus L1 virus-like particles.

Human papillomavirus (HPV) vaccines based on L1 virus-like particle (VLP) can prevent genital HPV infection and associated lesions after three intramuscular injections. Needle-free administration might facilitate vaccine implementation, especially in developing countries. Here we have investigated rectal and vaginal administration of HPV16 L1 VLPs in mice and their ability to induce anti-VLP and HPV16-neutralizing antibodies in serum and in genital, rectal and oral secretions. Rectal and vaginal immunizations were not effective in the absence of adjuvant. Cholera toxin was able to enhance systemic and mucosal anti-VLPs responses after rectal immunization, but not after vaginal immunization. Rectal immunization with Resiquimod and to a lesser extent Imiquimod, but not monophosphoryl lipid A, induced anti-HPV16 VLP antibodies in serum and secretions. Vaginal immunization was immunogenic only if administered in mice treated with nonoxynol-9, a disrupter of the cervico-vaginal epithelium. Our findings show that rectal and vaginal administration of VLPs can induce significant HPV16-neutralizing antibody levels in secretions, despite the fact that low titers are induced in serum. Imidazoquinolines, largely used to treat genital and anal warts, and nonoxonol-9, used as genital microbicide/spermicide were identified as adjuvants that could be safely used by the rectal or vaginal route, respectively.