Blood-Brain Barrier (BBB) Pharmacoproteomics: Reconstruction of In Vivo Brain Distribution of 11 P-Glycoprotein Substrates Based on the BBB Transporter Protein Concentration, In Vitro Intrinsic Transport Activity, and Unbound Fraction in Plasma and Brain in Mice

The purpose of this study was to examine whether in vivo drug distribution to the brain can be reconstructed by integrating P-glycoprotein (P-gp)/mdr1a expression levels, P-gp in vitro activity, and drug unbound fractions in mouse plasma and brain. For 11 P-gp substrates, in vitro P-gp transport activities were determined by measuring transcellular transport across monolayers of mouse P-gp-transfected LLC-PK1 (L-mdr1a) and parental cells. P-gp expression amounts were determined by quantitative targeted absolute proteomics. Unbound drug fractions in plasma and brain were obtained from the literature and by measuring brain slice uptake, respectively. Brain-to-plasma concentration ratios (K-p brain) and its ratios between wild-type and mdr1a/1b(-/-) mice (K-p brain ratio) were obtained from the literature or determined by intravenous constant infusion. Unbound brain-to-plasma concentration ratios (K-p,K-uu,K-brain) were estimated from K-p brain and unbound fractions. Based on pharmacokinetic theory, K-p brain ratios were reconstructed from in vitro P-gp transport activities and P-gp expression amounts in L-mdr1a cells and mouse brain capillaries. All reconstructed K-p brain ratios were within a 1.6-fold range of observed values. K-p brain then was reconstructed from the reconstructed K-p brain ratios and unbound fractions. K-p,K-uu,K-brain was reconstructed as the reciprocal of the reconstructed K-p brain ratios. For quinidine, loperamide, risperidone, indinavir, dexamethasone, paclitaxel, verapamil, loratadine, and diazepam, the reconstructed K-p brain and K-p,K-uu,K-brain agreed with observed and estimated in vivo values within a 3-fold range, respectively. Thus, brain distributions of P-gp substrates can be reconstructed from P-gp expression levels, in vitro activity, and drug unbound fractions.