Hypothesis

Combining intermittent dasatinib+quercetin (D+Q) dosing with early time‑restricted eating (eTRE) creates a circadian‑gated autophagic surge that selectively targets senescent cells for clearance, thereby lowering SASP burden and improving tissue function more than either intervention alone.

Mechanistic Rationale

Senolytics induce apoptosis in senescent cells by disrupting SCAP–NET pathways, but many senescent cells resist death due to elevated anti‑apoptotic Bcl‑2 family members. eTRE, by restricting food intake to a 6‑hour window aligned with the active phase, amplifies hepatic SIRT1 activity and drives a robust autophagic flux marked by LC3‑II accumulation and p62 degradation. Autophagy can degrade mitochondrial antigens and lysosomal regulators that sustain senescent cell survival, effectively lowering the threshold for D+Q‑induced apoptosis. Moreover, the circadian clock component REV‑ERBα, upregulated during the fasting window, directly represses mTORC1 signaling, synergizing with D+Q‑mediated transient mTOR inhibition observed after senolytic exposure. This dual hit—autophagy‑mediated sensitisation plus mTOR suppression—should produce synthetic lethality specifically in cells exhibiting high p16^INK4a^ and SA‑β‑Gal activity, while sparing proliferating progenitors that rely on basal mTOR for growth.

Testable Predictions

1. Participants receiving D+Q plus eTRE will show a greater reduction in circulating p16^INK4a^‑positive extracellular vesicles compared to D+Q alone or eTRE alone after four weeks.
2. The combination will produce a larger decline in SASP cytokines (IL‑6, TNFα, MCP‑1) than either monotherapy, correlating with increased LC3‑II/I ratio and decreased p62 in peripheral blood mononuclear cells.
3. Autophagy flux markers will peak during the fasting window and return to baseline within 2 h of refeeding, indicating a temporally restricted effect that aligns with senolytic administration timing.
4. Functional outcomes—gait speed and grip strength—will improve significantly only in the combination group, reflecting tissue‑level senescent cell clearance.

Experimental Design

A randomized, crossover trial in 60 adults aged 65‑80 with mild frailty (Fried criteria ≥2) will assign participants to four 4‑week sequences separated by 2‑week washouts: (a) placebo, (b) D+Q (dasatinib 100 mg + quercetin 1000 mg) on two consecutive days weekly, (c) eTRE (eating window 08:00‑14:00) daily, (d) D+Q on the same two days plus eTRE throughout the week. Primary endpoints: change in p16^INK4a^‑positive EVs (flow cytometry) and SASP cytokine panel (Luminex). Secondary endpoints: PBMC LC3‑II/I and p62 (Western blot), fasting glucose/HbA1c, and physical performance tests. Blood draws will occur at baseline, 2 h post‑refeeding, and 24 h post‑fast to capture autophagic dynamics. Statistical analysis will use mixed‑effects models with sequence and period as fixed effects, participant as random effect.

Falsifiability

If the combination fails to produce a statistically significant greater reduction in p16^INK4a^‑positive EVs or SASP cytokines compared to each monotherapy, or if autophagy markers do not show the predicted fasting‑phase elevation, the hypothesis is refuted. Conversely, confirmation would support a mechanistic link between circadian‑aligned autophagy enhancement and senolytic efficacy, justifying larger trials targeting healthspan extension.