Intermittent rapamycin feeding recapitulates some effects of continuous treatment while maintaining lifespan extension

**Background:** Rapamycin is a potent geroprotective drug that extends lifespan in mice but can cause adverse effects such as glucose intolerance, testicular degeneration, and cataracts when administered continuously. Intermittent dosing may reduce these side effects while preserving benefits. This study compared intermittent versus continuous rapamycin feeding in both male and female C3B6F1 hybrid mice, assessing lifespan, metabolic health, fitness, organ pathology, and circulating immune proteins.

**Methods:** From 6 months of age, mice were assigned to control (Eudragit S100), continuous rapamycin (42 mg/kg diet), or intermittent rapamycin (alternating weekly 42 mg/kg rapamycin and control food). Survival was monitored in 309 mice (155 males, 154 females). Longitudinal phenotyping included glucose tolerance tests (GTT), insulin tolerance tests (ITT), body composition, indirect calorimetry, rotarod, treadmill, open field, and electrocardiography (ECG). Organ pathology was assessed at 24 months in heart, liver, kidney, brown adipose tissue (BAT), pancreas, white adipose tissue (WAT), and spleen. Plasma levels of 92 immune-related proteins were measured using Olink proximity extension assay.

**Key Results:** - **Lifespan:** In males, intermittent and continuous rapamycin increased median lifespan by 18% and 28%, respectively, with no significant difference between regimens (log-rank P=0.13). In females, median lifespan increased by 14% (intermittent) and 25% (continuous); restricted mean time lost analysis showed a trend (P=0.082) and became significant when including additional cohorts (P=0.015). Maximum lifespan was extended similarly by both regimens in both sexes. - **Glucose tolerance:** Continuous rapamycin impaired glucose tolerance in both sexes (AUC significantly increased). Intermittent treatment partially rescued this: inter-off females had glucose tolerance similar to controls, while inter-on females and inter-off males showed improvement over continuous but remained worse than controls. - **Motor coordination:** In males, both intermittent and continuous rapamycin improved rotarod performance at 12 and 20 months (P<0.05). In females, only continuous treatment improved performance at 20 months. - **Heart function:** Both regimens prevented age-related decline in heart rate and QT interval prolongation at 20 months in both sexes. Heart weight was reduced only by continuous treatment in males and females. - **Organ pathology:** Continuous rapamycin reduced liver tumors in males (P<0.05), chronic progressive nephropathy in males (P<0.01), and inflammation in BAT, pancreas, kidney, and WAT in females. Intermittent treatment showed trends but generally less effect. Testicular degeneration was not rescued by intermittent treatment. Spleen weight was reduced by both regimens in both sexes. - **Circulating immune proteins:** Rapamycin downregulated several SASP-associated proteins (e.g., Ccl3, Ccl2, Tgfb1) in a sex- and regimen-specific manner, with more changes in females and under continuous treatment.

**Clinical Implications:** Intermittent rapamycin feeding can extend lifespan similarly to continuous treatment in males and nearly as much in females, while partially mitigating metabolic side effects like glucose intolerance. However, continuous treatment provides greater benefits for healthspan, including reduced age-related pathology and inflammation. These findings suggest that intermittent dosing may be a viable strategy for human translation, but the trade-off between lifespan extension and healthspan improvement must be carefully considered. The sex-specific effects highlight the need for personalized dosing regimens.