A surrogate barrier model for high-throughput blood-brain barrier permeability prediction: integrating LLC-PK1-MOCK/MDR1 Cells and lysosomal trapping correction

To mitigate risks in central nervous system (CNS) drug development, we established a high-throughput in vitro blood-brain barrier (BBB) model using LLC-PK1-MOCK and LLC-PK1-MDR1 cells in a Transwell system, aiming to replicate in vivo brain distribution and elucidate permeability mechanisms. Model integrity was assessed via transepithelial electrical resistance (TEER) and efflux functionality using control drugs (atenolol, digoxin). Bidirectional transport studies of 41 compounds quantified permeability (P_app), efflux ratios (ER), and recoveries, while in vivo brain distribution parameters (K_{<i>p</i>,uu,brain}) were derived from literature and rat studies. The model demonstrated critical BBB features: tight junction integrity (TEER &gt; 70 Ω·cm²), <i>P</i>-gp efflux activity (digoxin ER = 5.10 ~ 17.12), and discrimination of passive diffusion (63.41% of drugs) from transporter-mediated mechanisms (19.5% <i>P</i>-gp substrates). A training set of 20 randomly selected drugs revealed a robust correlation between MDR1-derived P_app(A-B) and K_{<i>p</i>,uu,brain} (R = 0.8886), with the remaining 21 compounds validating predictive accuracy (≤2-fold error). Four alkaloids exhibiting low recovery (&lt;80%) due to lysosomal trapping were corrected using Bafilomycin A1, aligning their permeability with in vivo outcomes. These results position the LLC-PK1-MOCK/MDR1 model as a reliable surrogate tool for early CNS drug screening, enabling rapid prioritization of candidates based on BBB penetration potential. Its integration into preclinical workflows promises to accelerate the development of therapeutics for neurological disorders. The development of physiologically relevant in vitro BBB models is pivotal to overcoming the high attrition rates in CNS drug discovery. Our study establishes a robust, high-throughput surrogate barrier model using LLC-PK1-MOCK/MDR1 cells in a Transwell system. This model recapitulates critical BBB features, including increased paracellular tightness and <i>P</i>-gp transporter functionality. It also addresses longstanding limitations of intracellular drug accumulation by lysosomal trapping. By validating the model with 41 structurally diverse compounds and correlating in vitro permeability (P_app) to in vivo brain distribution (K_{<i>p</i>,uu,brain}), we demonstrate its predictive accuracy and utility in distinguishing passive diffusion, transporter-mediated efflux, and lysosomal sequestration mechanisms. This cost and time -efficient platform streamlines early-stage CNS drug screening, enabling rapid identification of brain-penetrant candidates and reducing reliance on resource-intensive in vivo studies.