Transporter expressions as part of required scaling factor to support in vitro in vivo extrapolation for blood-brain barrier drug permeability

Access of drugs to the central nervous system is limited by the blood–brain barrier, and this in turn affects drug efficacy/toxicity. To date, most drug discovery optimization paradigms have relied heavily on in vitro transporter assays and preclinical species pharmacokinetic evaluation to provide a qualitative assessment of human brain penetration. Because of the lack of human brain pharmacokinetic data, mechanistic models for preclinical species, combined with in vitro and in silico data, are useful for translation to human. These models require transporter expression data to be measured in both in vitro and in vivo systems. The purpose of this work was to quantify transporter expression and generate scaling factors (SFs) to enable in vitro in vivo extrapolation (IVIVE) of transporter-mediated processes and to support the development of PBPK model of the brain in rats. SF represents the ratio of abundance of the relevant transporter in the tissue relative to transporter expressing cells. Using quantitative proteomics with QconCAT technology, the expression of human and rat P-gp (ABCB1/Abcb1) and BCRP/Bcrp (ABCG2/Abcg2) was measured in rat brain microvessels, mock and transfected cell lines including, Madin-Darby Canine Kidney I (MDCK I), Madin-Darby Canine Kidney II (MDCK II) and Porcine Kidney epithelial cells (LLC-PK1). P-gp expression ranged from 32 to 71 pmol/mg in rat brain microvessels, exceeding literature values of 14.1–25.2 pmol/mg protein. Conversely, Bcrp expression ranged between 0.02–0.27 pmol/mg protein lower than the literature range (2–6.2 pmol/mg protein). P-gp expression in MDCK I and LLC-PK1 cells transfected with rat Mdr1a was similar (within 1.5-fold) as was human P-gp expression in MDR1 transfected LLC-PK1 and MDCK II cells. The generated SFs were 34.4 and 50.4 for brain P-gp (depending on the cell line used) and 0.53 for brain Bcrp. Endogenous P-gp transporter was detected in MDCK II cell lines when protein expression was measured using a surrogate peptide that was shared across species. The current work provides a framework for proteomics-informed translation of in vitro P-gp and BCRP-related kinetics of drugs and supports the development of PBPK models to predict drug disposition in the brain.