Neutropenic thigh infection model.

The mouse neutropenic thigh infection model described by Craig and Andes (21) was used to assess the efficacy of ND-421. Briefly, the mice (n = 10 mice per group) were rendered neutropenic by intraperitoneal treatment with 100 μl cyclophosphamide (Alfa Aesar, Haverhill, MA) at 200 mg/kg of body weight, 4 days and 1 day prior to the infection. MRSA strains NRS70 and NRS119 were grown in brain heart infusion broth to an OD₅₄₀ of 0.5 and then diluted to a final concentration of approximately 10⁶ CFU/ml in fresh brain heart infusion broth. An 0.1-ml aliquot of the bacterial inoculum was injected intramuscularly into the right thigh. One hour after infection, the animals were administered a single oral dose of 40 mg/kg of ND-421, linezolid, or vehicle (10% dimethyl sulfoxide [DMSO], 25% Tween 80, 65% water) by oral gavage. The animals were sacrificed by CO₂ asphyxiation 48 h after infection. Terminal plasma was collected by cardiac puncture, followed by aseptic removal of both thigh muscles (uninfected and infected). The infected thigh was weighed, homogenized in phosphate-buffered saline using a Bullet blender (Next Advance, Inc., Averill Park, NY), serially diluted, and plated on LB agar plates (Fisher Scientific, Waltham, MA) to determine colony counts. The plates were incubated at 37°C for 24 h. The colony counts were expressed as log CFU/gram of tissue. Groups were compared using the Mann-Whitney U test.