AgNPs reduce reproductive capability of female mouse for their toxic effects on mouse early embryo development.

Silver nanoparticles (AgNPs) are widely applied in the field of personal protection for their powerful toxic effects on cells, and recently, a new type of vaginal gel with AgNPs is used to protect the female reproductive tract from microbes and viruses. However, a high risk of AgNPs to the fetus and the underlying mechanism of AgNPs to interfere in embryo development still remain unclear. Thus, this study investigated the impact of two drugs of vaginal gel with AgNPs on reproductive capability of the female mouse by animal experiment. Then, kinetics of AgNPs affecting embryo development was investigated by in vitro embryos culturing, and cell membrane potential (CMP) of zygotes was analyzed by DiBAC4(3) staining. Results indicated that one of the drugs of vaginal gel certainly injured embryo development in spite of no apparent histological change found in ovaries and uteruses of drug-treated mice. In vitro embryo culturing discovered that the toxic effect of AgNPs on embryo development presented particle sizes and dose dependent, and AgNP treatment could rapidly trigger depolarization of the cell membrane of zygotes. Moreover, AgNPs changed the gene expression pattern of Oct-4 and Cdx2 in blastocysts. All these findings suggest that AgNPs can interfere with normal cellular status including cell membrane potential, which has not been noticed in previous studies on the impact of AgNPs on mammalian embryos. Thus, findings of this study alarm us the risk of applying vaginal gel with AgNPs in individual caring and protection of the female reproductive system.

Development and in vitro evaluation of a vaginal microbicide gel formulation for UAMC01398, a novel diaryltriazine NNRTI against HIV-1.

Diaryltriazines (DATAs) constitute a class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that are being investigated for use as anti-HIV microbicides. The aim of the present study was (1) to assess the biopharmaceutical properties of the DATA series, (2) to select the lead candidate as vaginal microbicide and (3) to develop and evaluate gel formulations of the lead candidate. First, the vaginal tissue permeation potential of the different DATAs was screened by performing permeability and solubility measurements. To obtain a suitable formulation of the lead microbicide candidate, several hydroxyethylcellulose-based gels were assessed for their cellular toxicity, stability and ability to enable UAMC01398 epithelial permeation. Also, attention was given to appropriate preservative selection. Because of its favourable in vitro activity, safety and biopharmaceutical profile, UAMC01398 was chosen as the lead microbicide candidate among the DATA series. Formulating UAMC01398 as a vaginal gel did not affect its anti-HIV activity. Safe and chemically stable gel formulations of UAMC01398 (0.02%) included a non-solubilizing gel and a gel containing sulfobutyl ether-ß-cyclodextrin (SBE-ßCD, 5%) as solubilizing excipient. Inclusion of SBE-ßCD in the gel formulation resulted in enhanced microbicide flux across HEC-1A epithelial cell layers, to an extent that could not be achieved by simply increasing the dose of UAMC01398. The applied rational (pre)formulation approach resulted in the development of aqueous-based gel formulations that are appropriate for further in vivo investigation of the anti-HIV microbicide potential of the novel NNRTI UAMC01398.

Safety and efficacy of tenofovir/IQP-0528 combination gels - a dual compartment microbicide for HIV-1 prevention.

Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor and IQP-0528 is a non-nucleoside reverse transcriptase inhibitor that also blocks virus entry. TFV and IQP-0528 alone have shown antiviral activity as microbicide gels. Because combination therapy will likely be more potent than mono-therapy, these drugs have been chosen to make a combination microbicide gel containing 2.5% TFV/1% IQP-0528. Safety and efficacy testing was done to evaluate five prototype combination gels. The gels retained TZM-bl cell and ectocervical and colorectal tissue viability. Further, the epithelium of the ectocervical and colorectal tissue remained intact after a 24h exposure. The ED(50) calculated from the formulations for IQP-0528 was ~32nM and for TFV was ~59nM and their inhibitory activity was not affected by semen. The ED(50) of TFV in the combination gels was ~100-fold lower than when calculated for the drug substance alone reflecting the activity of the more potent IQP-0528. When ectocervical and colorectal tissue were treated with the combination gels, HIV-1 p24 release was reduced by ≥1log(10) and ≥2log(10), respectively. Immunohistochemistry for the ectocervical tissues treated with combination gels showed no HIV-1 infected cells at study end. With the increased realization of receptive anal intercourse among heterosexual couples often in conjunction with vaginal intercourse, having a safe and effective microbicide for both mucosal sites is critical. The safety and efficacy profiles of the gels were similar for ectocervical and colorectal tissues suggesting these gels have the potential for dual compartment use.

No increase in cervicovaginal proinflammatory cytokines after Carraguard use in a placebo-controlled randomized clinical trial.

BACKGROUND: Assessment of cervicovaginal cytokine levels may be helpful to evaluate subclinical epithelial inflammation during safety evaluations of candidate microbicides. METHODS: Fifty-five HIV-seronegative Thai women were enrolled in a safety trial of the candidate microbicide Carraguard and were randomized to use Carraguard or placebo gel before vaginal sex. Cervicovaginal lavages were collected at baseline and after 1 month of gel use; levels of interleukin (IL)-1beta, IL-6, IL-8, and secretory leukocyte protease inhibitor (SLPI) were measured using microwell plate-based enzyme immunoassays. Median levels were compared between the baseline and 1-month follow-up visits using paired t tests; the median change between groups was compared using Wilcoxon rank sum tests. Women were examined for the presence of genital findings; the association between genital findings and cytokine levels was studied. RESULTS: No increase in levels of proinflammatory cytokines after use of Carraguard gel or placebo gel was observed during the study. The median change from the baseline to 1 month of follow-up was not significantly different between Carraguard and placebo groups (IL-1beta: -0.3 pg/mL vs. -3.93 pg/mL; P = 0.4, IL-6: -0.3 pg/mL vs. 0 pg/mL; P = 0.3, IL-8: -40.1 pg/mL vs. -53.2 pg/mL; P = 0.8, and SLPI: -26.5 pg/mL vs. 12.6 pg/mL; P = 0.07). Genital findings with intact epithelium were found in 16 (29%) women; these women tended to have somewhat higher IL-6 levels than those with normal epithelium (14.9 pg/mL vs. 8.8 pg/mL; P = 0.08). CONCLUSION: We found no increase in proinflammatory cytokines after Carraguard and placebo gel use, suggesting that neither gel causes inflammation. Further studies to assess the role of cytokines in microbicide safety studies are warranted.

Evaluation of local tolerance of the antiretroviral spermicide (WHI-07)-loaded gel-microemulsion in the porcine female reproductive tract.

The local tolerance of the antiretroviral spermicide, WHI-07 (5-bromo-6-methoxy-5,6-dihydro-3'-azidothymidine-5'-(p-bromophenyl)-methoxyalaninyl phosphate)-loaded gel-microemulsion was evaluated in a physiologically relevant and sensitive porcine model. Gilts (Duroc) in nonestrus stages of the reproductive cycle received either a single or a daily intravaginal application of 2.0% WHI-07 via a gel-microemulsion for 6 days. Cervicovaginal lavage (CVL) fluid was obtained for up to 72 h after a single exposure and the cellular profile and levels of inflammatory cytokines (IL-1beta, IL-8, IFN-gamma and TNF-alpha) were quantitated by flow cytometry and chemiluminescence-based multiplex immunoassay, respectively. The reproductive tract (vagina, cervix, uteri and Fallopian tubes) harvested on day 7 was scored histologically for evidence of mucosal irritation using a new scoring criterion for ten histological endpoints that reflect pathological changes in the epithelial/ subepithelial and vascular/perivascular compartments. When compared with irritant reactions caused by the detergent-type spermicide, benzalkonium chloride (BZK), the scatter profile of CVL immune cells and basal levels of proinflammatory cytokines (IL-1beta, IL-8, IFN-gamma and TNF-alpha) in CVL fluid were unaffected by intravaginal exposure to 2% WHI-07. Unlike BZK, endpoint histology of the proximal and distal regions of the reproductive tract from gilts treated with 2.0% WHI-07 via gel-microemulsion for 6 days did not result in mucosal irritation or alteration in the epithelium, subepithelium/lamina propria, vessels/perivascular tissues and underlying/surrounding muscles. Based on surrogate markers for inflammation, leukocyte profile and histologic data for local tolerance, repeated intravaginal administration of WHI-07 via gel-microemulsion as a prophylactic contraceptive is unlikely to cause vaginal irritation.

Organotypic human vaginal-ectocervical tissue model for irritation studies of spermicides, microbicides, and feminine-care products.

A three-dimensional organotypic vaginal-ectocervical (VEC) tissue model has been developed to test the irritation of topically applied spermicides, microbicides, and vaginal-care products. The in vitro tissue model was reconstructed using normal VEC epithelial cells and is well stratified, containing differentiated basal, suprabasal, intermediate, and superficial cell layers similar to in vivo tissue. The intermediate and superficial cell layers contain glycogen, and the expression of cytokeratins 13 and 14 in the tissue also parallels that of native tissue. The MTT viability assay and histological assessment were used to test inter-lot and intra-lot reproducibility. The MTT average intra-lot coefficient of variation (CV) was less than 10% and the time required to reduce tissue viability by 50% (ET-50) following application of 1% Triton X-100 averaged 1.25+/-0.24h (n=23) upon completion of the 11-day culture period and 1.30 h+/- 0.19 for the same tissues stored overnight at 4 degrees C on agarose gels. The utility of the VEC model for irritation studies was examined by testing commercially available products using the MTT assay and histological assessment. The average ET-50 values ranged between 1.8 and 2.7h for feminine washes, 3.9-6.7 h for spermicides, 6.8-18 h for anti-itch creams, and >18 h for douches, lubricants, and anti-fungal creams. Studies of cytokines released from VEC cultures following product application showed that elevated concentrations of IL-1alpha and IL-1beta were associated with toxicity of test materials. In conclusion, the VEC tissue model is a highly reproducible, non-animal means to assess the irritation of contraceptives, microbicides, and vaginal-care products.

Comparative safety evaluation of the candidate vaginal microbicide C31G.

C31G is currently the focus of clinical trials designed to evaluate this agent as a microbicidal and spermicidal agent. In the following studies, the in vivo safety of C31G was assessed with a Swiss Webster mouse model of cervicovaginal toxicity and correlated with results from in vitro cytotoxicity experiments and published clinical observations. A single exposure of unformulated 1% C31G resulted in mild-to-moderate epithelial disruption and inflammation at 2 and 4 h postapplication. The columnar epithelium of the cervix was the primary site of damage, while no perturbation of the vaginal mucosa was observed. In contrast, application of unformulated 1.7% C31G resulted in greater levels of inflammation in the cervical epithelium at 2 h postapplication and severe epithelial disruption that persisted to 8 h postapplication. Application of a nonionic aqueous gel formulation containing 1% C31G resulted in no apparent cervicovaginal toxicity at any time point evaluated. However, formulation of 1.7% C31G did not substantially reduce the toxicity associated with unformulated C31G at that concentration. These observations correlate with findings gathered during a recent clinical trial, in which once-daily applications resulted in no adverse events in women receiving the formulation containing 1% C31G, compared to moderate-to-severe adverse events in 30% of women receiving the 1.7% C31G formulation. The Swiss Webster mouse model was able to effectively discriminate between concentrations and formulations of C31G that produced distinct clinical effects in human trials. The Swiss Webster animal model may be a highly valuable tool for preclinical evaluation of candidate vaginal microbicides.

Mucosal toxicity studies of a gel formulation of native pokeweed antiviral protein.

Pokeweed antiviral protein (PAP), a 29-kDa plant-derived protein isolated from Phytolacca americana, is a promising nonspermicidal broad-spectrum antiviral microbicide. This study evaluated the mucosal toxicity potential of native PAP in the in vivo rabbit vaginal irritation model as well as the in vitro reconstituted human vaginal epithelial tissue model. Twenty-two New Zealand white rabbits in 4 subgroups were exposed intravaginally to a gel with and without 0.01, 0.1, or 1.0% native PAP for 10 consecutive days. The dose of PAP used represented nearly 200- to 20,000 times its in vitro anti-HIV IC50 value. Animals were euthanized on day 11 and vaginal tissues were evaluated for histologic and immunohistochemical evidence of mucosal toxicity, cellular inflammation, and hyperplasia. Blood was analyzed for changes in hematology and clinical chemistry profiles. Reconstituted human vaginal epithelial tissue grown on membrane filters was exposed to 0.01, 0.1, or 1.0% native PAP in medium or topically via a gel for 24 hours and tissue damage was evaluated by histological assessment. In the in vivo rabbit vaginal irritation model, half of all PAP-treated rabbits (8/16) exhibited an acceptable range of vaginal mucosal irritation (total score <8 out of a possible 16), whereas nearly a third of PAP-treated rabbits (5/16) developed moderate to marked vaginal mucosal irritation (total score >11). However, no treatment-related adverse effects were seen in hematological or clinical chemistry measurements. Furthermore, in vitro exposure of a 3-dimensional human vaginal tissue grown on polycarbonate membrane filters to identical concentrations of PAP either added to culture medium or applied topically via gel formulation did not result in direct toxicity as determined by histologic evaluation. These findings indicate careful monitoring of vaginal irritation will be required in the clinical development of PAP as a nonspermicidal microbicide.

Mouse model of cervicovaginal toxicity and inflammation for preclinical evaluation of topical vaginal microbicides.

Clinical trials evaluating the efficacy of nonoxynol-9 (N-9) as a topical microbicide concluded that N-9 offers no in vivo protection against human immunodeficiency virus type 1 (HIV-1) infection, despite demonstrated in vitro inactivation of HIV-1 by N-9. These trials emphasize the need for better model systems to determine candidate microbicide effectiveness and safety in a preclinical setting. To that end, time-dependent in vitro cytotoxicity, as well as in vivo toxicity and inflammation, associated with N-9 exposure were characterized with the goal of validating a mouse model of microbicide toxicity. In vitro studies using submerged cell cultures indicated that human cervical epithelial cells were inherently more sensitive to N-9-mediated damage than human vaginal epithelial cells. These results correlated with in vivo findings obtained by using Swiss Webster mice in which intravaginal inoculation of 1% N-9 or Conceptrol gel (containing 4% N-9) resulted in selective and acute disruption of the cervical columnar epithelial cells 2 h postapplication accompanied by intense inflammatory infiltrates within the lamina propria. Although damage to the cervical epithelium was apparent out to 8 h postapplication, these tissues resembled control tissue by 24 h postapplication. In contrast, minimal damage and infiltration were associated with both short- and long-term exposure of the vaginal mucosa to either N-9 or Conceptrol. These analyses were extended to examine the relative toxicity of polyethylene hexamethylene biguanide (PEHMB), a polybiguanide compound under evaluation as a candidate topical microbicide. In similar studies, in vivo exposure to 1% PEHMB caused minimal damage and inflammation of the genital mucosa, a finding consistent with the demonstration that PEHMB was >350-fold less cytotoxic than N-9 in vitro. Collectively, these studies highlight the murine model of toxicity as a valuable tool for the preclinical assessment of toxicity and inflammation associated with exposure to candidate topical microbicides.