A dual-GLB1-activated prodrug regimen combining sub-threshold Gal-Dox with Nav-Gal exploits synthetic senolytic letha...

IF a dual-agent GLB1-activated prodrug regimen — sub-therapeutic Gal-Dox at 0.5 mg/kg IV weekly (one-quarter of the proposed 2 mg/kg monotherapy dose) combined with Nav-Gal at 50 mg/kg oral, weekly — is administered to 20-month-old male C57BL/6J mice with established hepatic senescent cell burden for 4 consecutive weeks,

THEN hepatic senescent cell clearance (≥50% reduction in p16Ink4a transcript, SA-β-gal⁺ hepatocytes, IL-6, and PAI-1) will be achieved with hepatotoxicity significantly below that of 2 mg/kg unconjugated doxorubicin controls (ALT/AST remaining <2.5× ULN), exceeding the efficacy of either agent alone at matched doses,

BECAUSE of the following step-by-step causal chain:

1. Senescent hepatocytes in aged mice accumulate markedly elevated lysosomal GLB1 activity relative to proliferating hepatocytes, creating the enzymatic differential required for prodrug activation (GLB1 is the activating enzyme for both conjugates) (Galactose-modified duocarmycin prodrugs rely on GLB1-mediated cleavage for senolytic activity)[https://doi.org/10.1111/acel.13133].

2. Nav-Gal, upon internalization into senescent cells, is hydrolyzed by hyperactive GLB1 to release active navitoclax, which inhibits BCL-2 and BCL-xL, lowering the mitochondrial apoptotic threshold specifically within the senescent population while sparing platelets and non-senescent hepatocytes that cannot rapidly cleave the galactose cap (Nav-Gal is a GLB1-dependent senolytic prodrug with broad activity; pharmacologically inactive in non-senescent cells due to the intact galactose moiety)[https://doi.org/10.1111/acel.13142].

3. Simultaneously, sub-therapeutic Gal-Dox (0.5 mg/kg) is cleaved by the same elevated GLB1 within senescent hepatocytes, releasing doxorubicin locally to introduce DNA double-strand breaks — a dose insufficient to cause significant doxorubicin-mediated cardiotoxicity or hepatotoxicity systemically (ALT/AST exceeding 2.5× ULN is the hepatotoxicity benchmark for unconjugated doxorubicin at standard doses)[https://pmc.ncbi.nlm.nih.gov/articles/PMC8334288/].

4. [SPECULATIVE] The simultaneous presence of BCL-2/BCL-xL inhibition (from Nav-Gal–released navitoclax) and DNA damage (from Gal-Dox–released doxorubicin) constitutes a synthetic senolytic lethality: senescent cells — which depend on upregulated BCL-2 family proteins for the survival of their persistent DNA damage response — are rendered acutely apoptosis-competent by navitoclax precisely at the moment Gal-Dox introduces new genotoxic stress, creating a cooperativity exceeding either agent alone. This mirrors BH3-mimetic + genotoxin synthetic lethality established in oncology but has not been applied within a dual-GLB1-gated prodrug framework.

5. Because both cytotoxic payloads require GLB1-mediated galactose cleavage for activation, non-senescent hepatocytes — which lack sufficient GLB1 to rapidly hydrolyze both prodrugs — are spared from both the BCL-2 inhibition and the DNA damage, preserv...

SENS category: GlycoSENS

Key references: • doi.org/10.1111/acel.13133]. • doi.org/10.1111/acel.13142]. • doi.org/10.1038/s41467-020-18039-x]. • doi.org/10.1111/acel.13133] • doi.org/10.1111/acel.13142]