Mouse Neutropenic Thigh Infection Model

Mice (n = 8 per group) were rendered neutropenic by ip dosing with cyclophosphamide (200 mg/kg) 4 days and 1 day prior to infection. The bacterial inoculum of NRS70 was prepared by suspending colonies into brain–heart infusion and adjusting to approximately 1 × 10⁶ CFU/mL. A 100 μL aliquot of the bacterial inocula was injected intramuscularly into the right thigh. Mice were then administered three subcutaneous doses of 2 or 27, vehicle (negative control), or linezolid (positive control) every 8 h (1, 9, and 17 h after infection). Compounds 2 and 27 were administered at 20 mg/kg and linezolid at 10 mg/kg. After 24 h, the mice were euthanized by CO₂ asphyxiation, and then terminal blood was collected by cardiac puncture in heparinized syringes and both thighs were surgically removed and transferred to sterile tubes. The thighs were weighed and an equal volume of sterile PBS was added. The infected thighs were homogenized using a bullet blender (Next Advance Inc., Averill Park, NY), and dilutions of the homogenate were prepared in triplicate and plated onto LB agar and incubated overnight. The colonies were then counted and averaged for each group and reported as log₁₀(CFU/g tissue). Uninfected thighs were homogenized with a bullet blender and centrifuged at 15 000g for 5 min, and a 50 μL aliquot of the supernatant was mixed with 100 μL of acetonitrile containing internal standard and centrifuged at 21 000g for 15 min and the supernatant analyzed by UPLC with MRM detection. Plasma was analyzed following the procedure for PK samples.