Utilising a dual human transporter MDCKII-MDR1-BCRP cell line to assess efflux at the Blood Brain Barrier (BBB)

To facilitate the design of drugs readily able to cross the blood brain barrier (BBB) a MDCK cell line was established that over expresses both P‑glycoprotein (Pgp) and Breast Cancer Resistance Protein (BCRP) the main human efflux transporters of the BBB. Proteomics analyses indicate BCRP is expressed at a higher level than Pgp in this cell line. This cell line shows good activity for both transporters (BCRP substrate dantrolene efflux ratio (ER) 16.3 {plus minus} 0.9, Pgp substrate quinidine ER 27.5 {plus minus} 1.2) and use of selective transport inhibitors enables an assessment of the relative contributions to overall ERs. The MDCKII-MDR1-BCRP ER negatively correlates with rat unbound brain/unbound plasma ratio, K_puu. Highly brain penetrant compounds with rat K_puu {greater than or equal to} 0.3 show ERs {less than or equal to} 2 in the MDCKII-MDR1-BCRP assay whilst compounds predominantly excluded from the brain, K_puu {less than or equal to} 0.05, demonstrate ERs {greater than or equal to} 20. A subset of compounds with MDCKII-MDR1-BCRP ER <2 and rat K_puu < 0.3 were shown to be substrates of rat Pgp using a rat transfected cell line, MDCKII-rMdr1a. These compounds also showed ERs >2 in the human NIH MDCKI-MDR1 (high Pgp expression) cell line which suggests that they are weak human Pgp substrates. Characterisation of 37 drugs targeting the central nervous system (CNS) in the MDCKII-MDR1-BCRP efflux assay show 36 have ERs < 2. In drug discovery, use of the MDCKII-MDR1-BCRP in parallel with the NIH MDCKI-MDR1 cell line is useful for identification of compounds with high brain penetration. Significance Statement A single cell line, that includes both the major human efflux transporters of the BBB, (MDCKII-MDR1-BCRP) has been established facilitating the rapid identification of efflux substrates and enabling the design of brain penetrant molecules. Efflux ratios using this cell line demonstrate a clear relationship with brain penetration as defined by rat brain K_puu.