Lactobacillus crispatus Capsules: Single-Use Safety Study in the Macaca nemestrina Model DOROTHY L. PATTON, PHD,* YVONNE T. COSGROVE SWEENEY,* MAY A. D. ANTONIO, † LORNA K. RABE, † AND SHARON L. HILLIER, PHD † Background: Lactobacillus crispatus is a part of the normal vaginal microflora of humans. Goal: The goal of this study was to assess whether a capsule con- taining an H 2 O 2 -producing strain of L crispatus (CTV-05) would alter the vaginal microflora and/or epithelial tissues when applied intravag- inally in the pig-tailed macaque model. Study Design: Ten sexually mature female Macaca nemestrina were assessed at baseline for quantitative vaginal microbiology and vaginal pH and with colposcopy. One capsule containing 10 8 colony forming units of desiccated L crispatus CTV-05 was inserted into the vaginal fornix of each animal. Vaginal assessments were repeated on days 1 and 2 after capsule insertion. The L crispatus CTV-05 strain was identified with use of a DNA fingerprinting method. Results: Before product use, four of 10 animals had detectable levels of H 2 O 2 -producing lactobacilli. L crispatus CTV-05 was detected in 1 of 10 animals on day 1 and in 3 of 10 animals on day 2 following insertion of the capsule. There were no tissue changes observed by colposcopy. Vaginal pH decreased in two animals colonized by CTV- 05, from 7.0 at baseline to 4.5  0.5 on days 1 and 2 after product use. Conclusions: A single intravaginal application of capsules contain- ing 10 8 L crispatus CTV-05 resulted in vaginal colonization in three of 10 animals 2 days after use. The absence of colposcopic changes in the vagina/cervical tissues indicates that L crispatus capsules are well tolerated. LACTOBACILLUS SPECIES ARE THE predominant aerobes of the vaginal microflora in women of child-bearing age. While all lactobacilli produce lactic acid, some lactobacilli also produce hydrogen peroxide (H 2 O 2 ), a known antibacterial compound. 1 Because of this activity, H 2 O 2 -producing lactobacilli are believed to act as endogenous microbicides in the vagina. Women having H 2 O 2 -producing lactobacilli are less likely to have bacterial vagi- nosis 1,2 and more likely to be persistently colonized by Lactoba- cillus species over several months. 3 H 2 O 2 -producing lactobacilli may protect against genital infections, including Neisseria gonor- rhoeae and HIV infection. 4,5 Of the many species of Lactobacillus described, L crispatus and L jensenii are the most prevalent species among women having Lactobacillus-predominant vaginal micro- flora, and 94% to 95% of strains of these two species produce H 2 O 2 . 2,3,6 Recently, a capsule containing L crispatus has been developed for use in humans to promote and/or increase vaginal colonization by H 2 O 2 -producing lactobacilli. Use of this probiotic as a new intravaginal agent is now being evaluated. In a pilot study involv- ing nine women aged 18 to 40 years, vaginal colonization by L crispatus CTV-05 was evaluated at 1 to 3 days and 6 to 8 days following insertion of one capsule intravaginally twice daily for three days. 7 L crispatus CTV-05 was detected in five of nine women at the first follow-up and six of nine women at the second follow-up. The pig-tailed macaque (Macaca nemestrina) was chosen as the model for testing the use of the L crispatus capsule as a topical microbicide, because this macaque has been extensively utilized as a model for human vaginal physiology and sexually transmitted infections (STIs). 8 –11 While new vaginal products are usually evaluated for toxicity in the rabbit vaginal irritation model, this model has limited relevance for testing of Lactobacillus species. Rabbit vaginal flora, pH, and anatomy are markedly different from those of humans. 12,13 The vaginal ecosystem of the pig-tailed macaque has been shown to be quite similar to that of humans. 8 The objective of the study was to determine if intravaginal appli- cation of capsules containing 10 8 L crispatus significantly altered the epithelial tissues when applied as a topical microbicide. Here we report that the L crispatus CTV-05 strain can colonize the vagina of the pig-tailed macaque, as detected 48 hours after application. Methods and Materials Experimental Animals Ten breeding-age female Macaca nemestrina were obtained from colony animals at the Washington National Primate Research Center for assignment to this study. Prior approval for use of monkeys in this protocol was obtained from the Institutional Animal Care and Use Committee at the University of Washington. Animals were housed and cared for under conditions that meet NIH standards as stated in the Guide for the Care and Use of Laboratory Animals. This project was supported in part by grant P01-AI39061 and the University of Washington National Primate Research Center (RR-00166). Reprint requests: Dorothy L. Patton, PHD, University of Washington, Department of Obstetrics and Gynecology, Box 356460, Seattle, WA 98195-6460. E-mail: dpatton@u.washington.edu Received October 31, 2002, revised February 5, 2003, and accepted February 10, 2003. From the *Department of Obstetrics and Gynecology, University of Washington, Seattle, Washington; and † Department of Obstetrics and Gynecology and Reproductive Sciences, University of Pittsburgh and the Magee-Womens Research Institute, Pittsburgh, Pennsylvania 568