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

Abstract

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-gptransfected 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(Ϫ/Ϫ) mice (K p brain ratio) were obtained from the literature or determined by intravenous constant infusion. Unbound brain-to-plasma concentration ratios (K p,uu,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,uu,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,uu,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.

Quantitative Targeted Proteomics for Membrane Transporter Proteins: Method and Application

“…In SIL methods such as the absolute quantification (AQUA) method (30), the IS is commonly spiked during or postdigestion and is the most feasible and rapid approach. Indeed, SIL peptides are among the most common IS used to evaluate the expression of drug transporters (5)(6)(7)18,19,(46)(47)(48)(49)(50)(51). A comparison of the SIL peptide with SILAM approach was recently carried out in the LC-MS/ MS method development in our laboratory (37).…”

Setting-up an <em>In Vitro</em> Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

“…In some cases, transport parameters were first verified in isolated microvessels (closest to the in vivo situation), and then studied in cell culture systems 76,61 . Molecular biology research has allowed the characterization of gene and protein expression in isolated microvessels and low passage endothelial cell cultures from the same species, and between different species, most often from small animals such as rodents (mice and rats) or from cow and pig in comparison to humans 77,78,75,15 . Transcriptomic comparison between in vivo and in vitro brain microvascular endothelial cells showed numerous gene transcripts that were differentially expressed and most often significantly downregulated in vitro.…”

Pharmacoproteomics-Based Reconstruction of In Vivo P-Glycoprotein Function at Blood-Brain Barrier and Brain Distribution of Substrate Verapamil in Pentylenetetrazole-Kindled Epilepsy, Spontaneous Epilepsy, and Phenytoin Treatment Models

“…3) to take account of the unbound fractions of P-gp/mdr1a substrates in plasma (f u,plasma ) and brain (f u,brain ) (Uchida et al, 2011a).…”

“…Using normal mice as a model, we have previously demonstrated that the K p brain ratio can be reconstructed using the equation presented below (eq. 2) from the in vitro P-gp/mdr1a efflux activity (in vitro P-gp efflux ratio) and protein expression levels of P-gp/mdr1a in mouse P-gp/mdr1a-transfected LLC-PK1 cell monolayer and isolated mouse brain capillaries, according to in vivo reconstruction theory (Uchida et al, 2011a). …”

“…To solve these problems, we proposed the pharmacoproteomic concept that the in vivo function of a target transporter at the BBB can be reconstructed by integrating the transport activity per target transporter protein measured in in vitro transfected cell lines (transport rate/mole) and the absolute protein expression level of the target transporter in brain capillaries isolated from brain (mole), and we experimentally demonstrated the validity of this approach by using P-gp/mdr1a as a model transporter in normal mice (Uchida et al, 2011a). This result raised the possibility that the in vivo functions of multiple transporters at the human BBB in disease states could be reconstructed from in vitro measured transport activity per target human transporter protein and its absolute expression levels in brain capillaries isolated from diseased brains after surgery or at autopsy, as well as under normal conditions.…”