| China is currently facing a dual challenge on retaining low birth rate and preventing Acquired immune deficiency syndrome (AIDS) and other sexually transmitted diseases (STDs). It is urgent to develop new topical contraceptive drugs which can replace nonoxynol-9 (N-9) in the field of population health.Acetic acid thermoreversible gel (Ace-Acid Gel) is a novel spermicide. It is a bio-adhesive gel with acetic acid as major ingredient and in situ gel as formulation. The gel can restore and maintain the balance of vaginal micro-environment and microbial through its acid buffering system, thus prevent unwanted pregnancy and enhance vaginal pathogen-defense capabilities.In the present study, we conducted the vaginal pharmacokinetics study, contraceptive efficacy study, vaginal epithelial cell toxicity study and the rabbit vaginal mucosa irritation study to preliminarily evaluate the pre-clinical efficacy and safety of the Ace-Acid Gel, and explore more sensitive methods and indicators for vaginal toxicity evaluation.In the pharmacokinetics study, female rabbits were divided into 11 groups randomly and administered with Ace-Acid Gel through vagina. At 0.25,0.50,1.0,1.5, 2.0,4.0,8.0,12.0,16.0,20.0 and 24.0 h after administertion, acetic acid contents in the rabbit vaginal lavage fluid and in the homogenated vaginal tissues were measured, vaginal pH was detected with a microelectrode inserted into the vagina, and pharmacokinetic parameters were obtained using nonlinear least squares fitting model. The results showed that Ace-Acid Gel can keep vagina acidity (pH 3.5-5.0) for about 20 hours, and pharmacokinetic parameters fit in with two-compartment model.The contraceptive efficacy studies include in vitro spermicidal test, in vivo spermicidal test and rabbit contraceptive test to evaluate the contraceptive efficacy of Ace-Acid Gel. In vitro spermicidal tests include the minimum effective spermicidal concentration (MEC) test, sperm motility recovery test, sperm morphology staining test and in vitro spermicidal effect of different dilution (1:1,1:5,1:10) of Ace-Acid Gel. The results showed that MEC of acetic acid was 1.21 mg/ml. Acetic acid can not only inhibit sperm motility but also inactivate the sperms at concentrations of 1.21 mg/ml or higher. Meanwhile, acetic acid showed inhibitive effects on sperm motility parameters and sperm movement pattern parameters. The results indicated that Ace-Acid Gel has similar spermicidal effects compared with N-9 Jelly.For in vivo spermicidal test, forty female estrus rabbits were divided into 4 groups randomly, which were saline group, gel base group, N-9 Jelly group and Ace-Acid Gel group (12.10 mg/ml). The rabbits were administered with test substances through vagina, and then mated with male rabbits (1:1) after 10 minutes. At 5,10,15 and 20 minutes after mating, semen samples were obtained from vagina to observe sperm motility using phase contrast microscope. Results showed that no mobile sperm was founded in the rabbits'vagina of Ace-Acid Gel group, while mobile sperms were founded in rabbits'vagina of the blank control group and gel base control group.For rabbit contraceptive test, sixty female rabbits were randomly divided into 6 groups which were saline group, gel base group, N-9 Jelly group and Ace-Acid Gel group (1.10 mg/ml,2.01 kg/ml,12.10 mg/ml). The rabbits were administered with test substances, and then mated with male rabbits (1:1) after 10 minutes. The number of pups was recorded and the infertile rate was calculated. Results showed that the infertile rates of middle-dose and high-dose Ace-Acid gel group were 100%, which were similar to that of N-9 Jelly goup.For vaginal epithelial cell toxicity study, three vaginal epithelial cell lines VK2, Ect and End were used and exposed to blank control, Ace-Acid Gel (1% and 10%), and N-9 Jelly (1% and 10%) for 4 h. Cell morphology was observed under inverted microscope, cell viability was evaluated with MTT method, and apoptosis were detected using flow cytometry (FCM). Results showed that morphologically 1% Ace-Acid Gel had no adverse effects on three cell lines,10% Ace-Acid Gel slightly increased suspension cells, while N-9 Jelly induced a lot of suspension cells and cell debris. Cell viability in 1% and 10% Ace-Acid Gel groups (except on End cell line) showed no significant difference (P>0.05) compared with the blank control, while cell viability in 1% and 10% N-9 Jelly groups showed significant decrease (P<0.001). Cell apoptosis in 1% and 10% Ace-Acid Gel groups also showed no significant change, while late stage apoptotic cells or dead cells increased dramatically in 1% and 10% N-9 Jelly groups.In vitro quantitative sterilization test of Lactobacillus was conducted to examine whether Ace-Acid Gel inhibits the growth of Lactobacillus. Lactobacillus were exposed to Ace-Acid Gel (25 mg/ml,12.5 mg/ml,6.25 mg/ml), N-9 Jelly (25 mg/ml, 12.5 mg/ml,6.25 mg/ml) and saline control. The number of colonies of each group was counted and recorded for statistical analysis. The results showed that Ace-Acid Gel did not inhibit the growth and propagation of Lactobacillus compared to saline control (P>0.05), while N-9 Jelly inhibited the growth and propagation of Lactocillus significantly (P<0.01).Vaginal mucosa irritation study was conducted according to WHO criteria. Thirty female rabbits were randomly divided into 5 groups, which were gel base group, N-9 Jelly group and Ace-Acid Gel group (6.05 mg/ml,12.10 kg/ml,18.14 mg/ml). The rabbits were administered 2.5 ml gel through vagina once per day for continuous 10 days, and sacrificed on 11th day. Vagina tissue was dissected for histopathological examination. Vaginal mucous lesions were scored in terms of edema, vascular congestion, cellular infiltration and epithelial ulceration. The total scores of low-dose, middle-dose and high-dose Acd-Acid Gel were 2.13,3.50 and 4.50, respectively. Compared with N-9 Jelly (which was scored 7.93), Ace-Acid Gel showed much lower irritation to vaginal mucosa. The structure of vaginal mucosa in Ace-Acid Gel groups and gel base group were basically normal, while in N-9 Jelly group the vaginal mucosa showed serious injury including ulceration, necrosis and epithelium loss.In summary, the present study showed that Ace-Acid Gel can retain in the vagina for about 20 hours, it had similar spermicidal and contraceptive efficacy compared with N-9 Jelly, but had much less toxicity to vaginal mucosa and did not inhibit the growth and propagation of lactobacillus. Results indicated that Ace-Acid Gel meets the requirements of vaginal application, and has good prospects for further research and development as a vaginal contraceptive. |
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