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Practical Application of Rodent Transporter Knockout Models to assess Brain Penetration in Drug Discovery.

BACKGROUND & OBJECTIVE
Compound X is a drug candidate for the treatment of neurodegenerative diseases. Its brain distribution was evaluated as part of the lead identification and optimization of early drug discovery.


METHODS
The brain distribution of compound X was studied in genetic transporter knockout rodent models, in vivo models with a chemical inhibitor and in vitro transporter cell systems.


RESULTS
Compound X was found to be a substrate for human Breast Cancer-Resistance Protein (BCRP) in vitro (efflux ratio 8.1) and rodent Bcrp in vivo (Kp,uuKO/Kp,uuWT = 0.15/0.057 = 2.7, p < 0.05) but not a substrate for human P-glycoprotein (P-gp) in vitro (efflux ratio 1.0) nor rodent P-gp in vivo (Kp,uuKO/Kp,uuWT = 0.056/0.051 = 1.1, p > 0.05). When both transporters were knocked out in vivo, Kp,uu increased to 0.51 ± 0.02. Similar patterns observed across compounds with related chemistry corroborated structure-activity relationship.


CONCLUSION
While in vitro assays showed compound X to be a substrate for human BCRP and not P-gp, in vivo studies indicated a synergistic effect between rodent efflux transporters. However, this only accounted for ~50% of restricted BBB-transport, suggesting involvement from other efflux transporters. Given Kp,uu is a key criterion for assessing technical quality of CNS candidates before progression into clinical development, it is important to identify relevant screening assays for a better understanding of low Kp,uu and brain distribution in pre-clinical models for translation to humans.