In vivo efficacy testing in the gonorrhea mouse model

Groups of female BALB/cAnNCr mice (Charles River Laboratories) (6–7 weeks old) were treated with 17β-estradiol and antibiotics (streptomycin and trimethoprim) to increase susceptibility to long-term N. gonorrhoeae infection as described¹⁵. Mice were inoculated vaginally with N. gonorrhoeae strain H041 (10⁴ cfu/mouse) two days after estradiol pellet implantation and vaginal swabs were cultured for 2 days post-bacterial inoculation to confirm infection. On the afternoon of the second culture day (day 0), mice were given MBX-4132, GEN or the vehicle (n = 20–21 mice/group). Doses of MBX-4132 were prepared fresh in vehicle at the time of treatment and administered as a single oral dose (dose volume 10 ml/kg). The positive control GEN (48 mg/kg) was prepared and administered intraperitoneally as 5 daily doses as previously described¹⁵. Vaginal swabs were collected on 8 consecutive days following treatment and quantitatively cultured for N. gonorrhoeae to assess efficacy. The data are expressed as CFU/ml of vaginal swab suspension. Clearance was shown by Kaplan-Meier curves with log-rank (Mantel-Cox) statistical analysis. The average cfu/ml over time was compared by 2-way ANOVA with Bonferroni post-hoc analysis. Statistics were performed in GraphPad Prism Software. Mice were housed in Allentown cage units with 2–5 animals per cage at 20–26 °C with relative humidity 30–70% and a minimum of 10 air changes/h and 12 h light/12 h dark cycle. At the study endpoint (10 days post-inoculation), mice were euthanized using compressed CO₂ gas in a CO₂ gas chamber in the Laboratory Animal Medicine Facility. All animal experiments were conducted at the Uniformed Services University of the Health Sciences, a facility fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, under a protocol that was approved by the university’s Institutional Animal Care and Use Committee.