Chronotolerance of ifosfamide in mice: Evidence for a circadian rhythm

Ifosfamide (IFO), developed in the 1970 s as a cyclophosphamide analog, is a cytotoxic alkylating agent of the oxazaphosphorine family. Despite its efficacy in sarcomas and germ-cell tumors, its clinical use remains limited by toxicities, particularly encephalopathy, for which no preventive or curative strategy exists. While cyclophosphamide has been extensively investigated in chronotherapy, IFO chronotolerance remains underexplored. This study aimed to determine circadian variation in IFO toxicity in mice. A total of 180 male Swiss Albino mice (8-10 weeks, ≈25 g) were synchronized for 3 weeks to a 12:12 light - dark cycle and housed in two separate rooms with inverted schedules to allow exploration of six circadian dosing times (1, 5, 9, 13, 17, and 21 HALO; Hours After Light Onset). LD₅₀ was determined by regression analysis of mortality rates at increasing doses (400-750 mg/kg). Survival, survival time, body weight, and rectal temperature were assessed following a single intraperitoneal LD₅₀ dose (520 mg/kg). The estimated LD₅₀ was 520 mg/kg (95% CI: 495-545; χ²=14.8, df=1, p < 0.001; R²=0.92). Circadian variation in survival was highly significant (p < 0.0001). The highest tolerance occurred at 13 HALO (72% survival, mean survival 12.2 d, minimal weight loss and hypothermia), whereas the lowest tolerance was observed at 9 HALO (22% survival, mean survival 7.6 d, maximal weight loss and hypothermia). A secondary peak of tolerance was detected at 1 HALO (64% survival). Rectal temperature rhythms confirmed proper circadian entrainment at baseline, but IFO administration induced marked desynchronization at toxic dosing times. IFO toxicity follows a bimodal circadian pattern, with a major tolerance peak at 13 HALO and a secondary peak at 1 HALO. Translational extrapolation suggests that 13 HALO in mice corresponds to early morning (≈06:00h) in humans, indicating that morning administration may represent the safest therapeutic window for IFO.