IF a sequential, low-dose co-administration regimen consisting of sub-ototoxic 2-hydroxypropyl-β-cyclodextrin (HPβCD; 200–400 mg/kg/week subcutaneous, below the ~2,000 mg/kg ototoxic threshold) followed within 4 hours by UDP-003 (cyclodextrin dimer targeting 7KC; dose TBD from IND-enabling studies, subcutaneous) is administered to aged (20-month-old) male and female C57BL/6J mice over 12 weeks,

THEN a statistically significant reduction (≥40%) in lysosomal 7-ketocholesterol (7KC) burden — quantified by filipin/BODIPY-oxysterol staining, LC-MS/MS oxysterol profiling, and lysosomal membrane permeability (LMP) assay — will be observed simultaneously across four tissue compartments (aortic macrophage foam cells, vascular smooth muscle cells [VSMCs], retinal pigment epithelium [RPE], and cortical neurons), with a native cholesterol depletion ratio (7KC extracted : cholesterol extracted) significantly superior to HPβCD monotherapy, and with preserved outer hair cell viability (DPOAE audiometry),

BECAUSE the following causal chain operates:

1. HPβCD activates TFEB at sub-ototoxic concentrations. Even at doses below those required for direct bulk sterol extraction, HPβCD robustly activates Transcription Factor EB (TFEB), inducing the Coordinated Lysosomal Expression and Regulation (CLEAR) network, upregulating V-ATPase subunits, cathepsins, and NPC1/NPC2 transporters, and dramatically enhancing autophagic flux and lysosomal biogenesis (HPβCD activates TFEB-mediated autophagy)[https://doi.org/10.1074/jbc.m113.506246]. This creates a primed, high-capacity lysosomal compartment with increased sterol-exporting machinery — not more sterol extraction per se, but more cellular infrastructure ready to efflux mobilized lipid.

2. TFEB-driven NPC1/NPC2 upregulation opens the lysosomal exit channel for 7KC. The CLEAR network directly transcribes NPC1 and NPC2, the lysosomal proteins responsible for sterol egress. By upregulating these transporters, HPβCD's TFEB activation widens the bottleneck through which 7KC must pass to reach the cytosol and plasma membrane for subsequent cyclodextrin capture (HPβCD activates CLEAR network)[https://doi.org/10.1074/jbc.m113.506246]. [SPECULATIVE: The degree to which NPC1/NPC2 transports 7KC vs. native cholesterol preferentially under conditions of elevated 7KC lysosomal load is not yet quantified and requires validation.]

3. UDP-003's dimeric architecture selectively captures mobilized 7KC at the plasma membrane interface, sparing native cholesterol. Once 7KC is exported from the lysosome via the TFEB-upregulated NPC1/NPC2 machinery, it reaches the plasma membrane inner leaflet. UDP-003's computationally designed cyclodextrin dimer cavity is engineered to exploit the steric and electronic asymmetry of the 7-ketone group on the B-ring of the sterol backbone, conferring substantially higher binding affinity for 7KC over unoxidized cholesterol compared to monomeric HPβCD, whose indiscriminate sterol extraction force...

SENS category: LysoSENS

Key references: • doi.org/10.1074/jbc.m113.506246]. • doi.org/10.1126/scitranslmed.aad6100]. • doi.org/10.1111/bph.15209]. • doi.org/10.1038/s41598-017-02387-8]. • doi.org/10.1111/1440-1681.12285].