Chronotherapy as a Potential Strategy to Reduce Ifosfamide-Induced Encephalopathy: A Preclinical Study in a Murine Model

Purpose

Ifosfamide (IFO), an alkylating anticancer drug, is clinically effective but limited by severe toxicities, notably encephalopathy and urotoxicity. Chronotherapy -optimizing drug delivery according to circadian timing-may improve its therapeutic index and reduce toxicity. This study investigated circadian variation in IFO-induced toxicities in mice.

Methods

A total of 160 male Swiss albino mice were synchronized to a 12:12 h light-dark cycle. In an earlier study, IFO chronotolerance assessed at LD50 (a lethal dose to 50 % of mice) showed circadian survival rhythms but high lethality, limiting organ-specific analyses. In this study, we used a sublethal dose LD30 of IFO (the dose lethal to 30% of mice) to allow detailed evaluation of tissue chronotoxicity while reducing lethality. 140 mice were treated at four circadian times (1, 7, 13, and 19 H ours A fter L ight O nset, HALO), while twenty controls received distilled water. Endpoints included hematology, hepatic enzymes, histopathology (brain, liver, kidney, bladder) and neurobehavioral performance (wire-hanging test).

Results

Significant circadian variation was observed across multiple endpoints. Survival (Fig.1) and organ injury (Fig.3) strongly dependent on dosing time. Administration at 7 HALO led to the worst outcomes, with ∼47% survival and severe multi-organ injury , whereas 13 HALO preserved survival (∼82%) and minimized organ injury . This six-hour shift represented a critical therapeutic window. Importantly, in mice (nocturnal species), 13 HALO corresponds to one hour after the onset of darkness (active phase), whereas in humans (diurnal species), the comparable stage corresponds to one hour after light onset (active phase), thus optimal treatment-timing is inverted between mice and humans . Assuming that for human the light onset occurs around 5a.m., 13 HALO in mice corresponds physiologically to 1 HALO, therefore, the optimal IFO administration time would be around 6 a.m.

Toxicity is expected to increase from 1 HALO until 13 HALO.

In rodent, hematological toxicity exhibited strong rhythmicity (Leukopenia: WBC: F(3,45)=52.4, p<0.001 and lymphopenia: lymphocytes: F(3,45)=61.8, p<0.001), with maximal suppression at 7 and 19 HALO and relative preservation at 13 HALO. Hepatic toxicity was time-dependent AST (F(3,45)=33.7, p<0.001); ALT (F(3,45)=28.5, p<0.001). Histopathological lesions varied by organ (bladder: Chi-square (3)=14.8, p=0.002; liver: Chi-square (3)=18.2, p<0.001; brain: Chi-square (3)=9.7, p=0.045; kidney: Chi-square (3)=11.6, p=0.021). Neurobehavior impairment was also modulated by circadian-.timing.

Conclusion

IFO-induced encephalopathy and multi-organ toxicities follow robust circadian rhythms in mice. Administration at 13 HALO (beginning of the active phase) minimized organ injury and improved survival. These findings support circadian-based chronotherapy as a promising strategy to improve IFO safety and therapeutic index for further clinical investigation.

Declarations

• Funding: No specific funding was received for this work.

• Competing interests: The authors declare no competing interests.

• Author contributions: MMC designed the study and wrote the first draft of the manuscript. DD conducted experiments and collected data. NAB supervised the project and revised the manuscript. All authors approved the final version.

• Data availability: The datasets generated and analyzed during this study are available from the corresponding author on reasonable request.

• Ethics: All procedures involving animals were conducted in accordance with institutional guidelines for the care and use of laboratory animals.

  Ethical approval was obtained from the Faculty of Medicine, Monastir.