Transcriptomic and Quantitative Proteomic Analysis of Transporters and Drug Metabolizing Enzymes in Freshly Isolated Human Brain Microvessels

Abstract

We have investigated the transcriptomic and/or proteomic patterns of 71 solute carrier (SLC) and organic solute (OST) transporters, 34 ATP-binding cassette (ABC) transporters, and 51 metabolizing enzymes in human brain microvessels. We used quantitative RT-PCR and LC-MS/MS to examine isolated brain microvessels and cortex biopsies from 12 patients with epilepsia or glioma. SLC2A1/GLUT1, SLC1A3/EAAT1, and SLC1A2/EAAT2 were the main SLC proteins whereas ABCG2/BCRP, ABCB1/MDR1, ABCA2 and ABCA8 were the main ABC quantified in isolated brain microvessels; ABCG2/BCRP was 1.6-fold more expressed than ABCB1/MDR1, and ABCC4/MRP4 was 10 times less abundant than ABCB1/MDR1. CYP1B1 and CYP2U1 were the only quantifiable CYPs. Finally, GSTP1, COMT, GSTM3, GSTO1 and GSTM2 proteins were the main phase II enzymes quantified; UGTs and NATs were not detected. Our extensive investigation of gene and protein patterns of transporters and metabolizing enzymes provides new molecular information for understanding drug entry and metabolism in the human blood-brain barrier.

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).…”

“…The protein quantification using LC-MS targeted proteomics approach can further be amended to a higher-throughput mode to compare the expressions of many proteins in different organs (19,48,49,77,78). For example, Kamiie et al (19) reported the simultaneous analysis of 36 membrane proteins in mouse BBB, and the follow-up studies extended upon the surrogate peptide library to target an extensive number of membrane proteins in human and monkey brain microvessels (48,50,78). These studies provide valuable information with respect to understand species differences in transporter expression at the BBB (74).…”

“…In vitro systems including isolated primary hepatocytes and heterogeneous gene overexpressed cell lines have been widely used to investigate transporter-involved drug disposition; however, translation of in vitro kinetics parameters obtained from different systems or different laboratories to in vivo is not always straightforward. For example, through a collaborative effort between 23 pharmaceutical, academic, and contract research laboratories, a minimum of 20-and 24-fold deviation was found to exist between the lowest and highest Pglycoprotein (P-gp/ABCB1) IC 50 values for sertraline and isradipine, respectively, to a maximum of 407-and 796-fold deviation for telmisartan and verapamil (2). The presence of digoxin uptake transporters and their differences in expression in in vitro systems used in different laboratories is considered as the major contributor to the variability of IC 50 values (2,3).…”

“…For example, through a collaborative effort between 23 pharmaceutical, academic, and contract research laboratories, a minimum of 20-and 24-fold deviation was found to exist between the lowest and highest Pglycoprotein (P-gp/ABCB1) IC 50 values for sertraline and isradipine, respectively, to a maximum of 407-and 796-fold deviation for telmisartan and verapamil (2). The presence of digoxin uptake transporters and their differences in expression in in vitro systems used in different laboratories is considered as the major contributor to the variability of IC 50 values (2,3). Moreover, IVIVE of biliary elimination that involves drug transporter-mediated processes is not well established from preclinical species to human.…”

A Pharm-Ecological Perspective of Terrestrial and Aquatic Plant-Herbivore Interactions

“…Certainly, shifts in generalism and omnivory will make linking laboratory pharm-ecological studies to field foraging patterns difficult as the link between diet and response is clearer in specialist herbivores. However, broadscale approaches such as state-of-the-art genomic and proteomic techniques can compare the diversity of detoxification mechanisms (Browning et al, 2010;Eyckmans et al, 2012;Glenn et al, 2010;Itokawa et al, 2010;Shawahna et al, 2011;Thiyagarajan and Qian, 2008;Whalen et al, 2008) and microbial communities (Langille et al, 2012;Masahira et al, 2009;Matteotti et al, 2011;Weinstock, 2012) relative to dietary diversity. These emerging approaches offer enormous potential to compare and contrast pharmecological mechanisms and foraging phenotypes across wide taxonomic groups 3.…”