Tetsuya Terasaki

Quantitative targeted absolute proteomics of human blood–brain barrier transporters and receptors

J. Neurochem. (2011) 117, 333–345.

Contribution of carrier-mediated transport systems to the blood-brain barrier as a supporting and protecting interface for the brain; Importance for CNS drug discovery and development

The blood–brain barrier (BBB) forms an interface between the circulating blood and the brain and possesses various carrier-mediated transport systems for small molecules to support and protect CNS function. For example, the blood-to-brain influx transport systems supply nutrients, such as glucose and amino acids. Consequently, xenobiotic drugs recognized by influx transporters are expected to have high permeability across the BBB. On the other hand, efflux transporters, including ATP-binding cassette transporters such as P-glycoprotein located at the luminal membrane of endothelial cells, function as clearance systems for metabolites and neurotoxic compounds produced in the brain. Drugs recognized by these transporters are expected to show low BBB permeability and low distribution to the brain. Despite recent progress, the transport mechanisms at the BBB have not been fully clarified yet, especially in humans. However, an understanding of the human BBB transport system is critical, because species differences mean that it can be difficult to extrapolate data obtained in experimental animals during drug development to humans. Recent progress in methodologies is allowing us to address this issue. Positron emission tomography can be used to evaluate the activity of human BBB transport systems in vivo. Proteomic studies may also provide important insights into human BBB function. Construction of a human BBB transporter atlas would be a most important advance from the viewpoint of CNS drug discovery and drug delivery to the brain.

P-glycoprotein as the drug efflux pump in primary cultured bovine brain capillary endothelial cells

The expression of a functional P-glycoprotein (P-gp) which pumps drugs out of brain capillary endothelial cells (BCEC) into blood was studied by evaluating the steady-state uptake and efflux of vincristine (VCR) by primary cultured bovine BCEC. The steady-state uptake of VCR was increased in the presence of metabolic inhibitors, and an anti-P-gp monoclonal antibody, MRK16, as well as verapamil and steroid hormones which are known to reverse multidrug resistance in tumor cells. Furthermore, efflux of VCR from BCEC was inhibited by verapamil. By immunohistochemistry, P-gp was localized at the luminal side of the capillary endothelial cells in both gray matter of bovine brain and primary cultured BCEC. These data suggest that P-gp functions as a drug efflux pump at the luminal side of BCEC and regulates the transfer of certain lipophilic drugs from the blood into the brain.

Simultaneous Absolute Protein Quantification of Transporters, Cytochromes P450, and UDP-Glucuronosyltransferases as a Novel Approach for the Characterization of Individual Human Liver: Comparison with mRNA Levels and Activities

The purpose of the present study was to determine the absolute protein expression levels of multiple drug-metabolizing enzymes and transporters in 17 human liver biopsies, and to compare them with the mRNA expression levels and functional activities to evaluate the suitability of the three measures as parameters of hepatic metabolism. Absolute protein expression levels of 13 cytochrome P450 (P450) enzymes, NADPH-P450 reductase (P450R) and 6 UDP-glucuronosyltransferase (UGT) enzymes in microsomal fraction, and 22 transporters in plasma membrane fraction were determined using liquid chromatography/tandem mass spectrometry. CYP2C9, CYP2E1, CYP3A4, CYP2A6, UGT1A6, UGT2B7, UGT2B15, and P450R were abundantly expressed (more than 50 pmol/mg protein) in human liver microsomes. The protein expression levels of CYP3A4, CYP2B6, and CYP2C8 were each highly correlated with the corresponding enzyme activity and mRNA expression levels, whereas for other P450s, the protein expression levels were better correlated with the enzyme activities than the mRNA expression levels were. Among transporters, the protein expression level of organic anion-transporting polypeptide 1B1 was relatively highly correlated with the mRNA expression level. However, other transporters showed almost no correlation. These findings indicate that protein expression levels determined by the present simultaneous quantification method are a useful parameter to assess differences of hepatic function between individuals.

A pericyte-derived angiopoietin-1 multimeric complex induces occludin gene expression in brain capillary endothelial cells through Tie-2 activation in vitro.

Although tight‐junctions (TJs) at the blood–brain barrier (BBB) are important to prevent non‐specific entry of compounds into the CNS, molecular mechanisms regulating TJ maintenance remain still unclear. The purpose of this study was therefore to identify molecules, which regulate occludin expression, derived from astrocytes and pericytes that ensheathe brain microvessels by using conditionally immortalized adult rat brain capillary endothelial (TR‐BBB13), type II astrocyte (TR‐AST4) and brain pericyte (TR‐PCT1) cell lines. Transfilter co‐culture with TR‐AST4 cells, and exposure to conditioned medium of TR‐AST4 cells (AST‐CM) or TR‐PCT1 cells (PCT‐CM) increased occludin mRNA in TR‐BBB13 cells. PCT‐CM‐induced occludin up‐regulation was significantly inhibited by an angiopoietin‐1‐neutralizing antibody, whereas the up‐regulation by AST‐CM was not. Immunoprecipitation and western blot analyses confirmed that multimeric angiopoietin‐1 is secreted from TR‐PCT1 cells, and induces occludin mRNA, acting through tyrosine phosphorylation of Tie‐2 in TR‐BBB13 cells. A fractionated AST‐CM study revealed that factors in the molecular weight range of 30–100 kDa led to occludin induction. Conversely, occludin mRNA was reduced by transforming growth factor β1, the mRNA of which was up‐regulated in TR‐AST4 cells following hypoxic treatment. In conclusion, in vitro BBB model studies revealed that the pericyte‐derived multimeric angiopoietin‐1/Tie‐2 pathway induces occludin expression.

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

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.

In vivo and in vitro blood-brain barrier transport of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors

Among the HMG-CoA reductase inhibitors, lovastatin and simvastatin have central nervous system (CNS) side effects, such as sleep disturbance, whereas pravastatin does not. This difference in CNS side effects may be due to a difference in blood–brain barrier (BBB) permeability among these inhibitors. To test this hypothesis, we compared the BBB transport ability of HMG-CoA reductase inhibitors by using an in vivo brain perfusion technique in rats and an in vitro culture system of bovine brain capillary endothelial cells. The in vivo BBB permeability coefficients of the lipophilic inhibitors, [14C]lovastatin and [14C]simvastatin, were high. In contrast, that of the hydrophilic inhibitor, [14C]pravastatin, was low and not significantly different from that of [14C]sucrose, an extracellular space marker. Similarly, the in vitro BBB permeability coefficients of [14C]lovastatin and [1C]simvastatin were high, while that of [14C]-pravastatin was low. The in vivo and in vitro transcellular permeabilities obtained for HMG-CoA reductase inhibitors were comparable. This study shows that the BBB permeability correlates with the CNS side effects of the HMG-CoA reductase inhibitors.

New approaches to in vitro models of blood-brain barrier drug transport.

The pharmaceutical industry has been searching for an in vitro blood-brain barrier (BBB) model that preserves in vivo transporter functions in CNS drug discovery and development. The application of conditionally immortalized cell lines derived from transgenic animals harboring temperature-sensitive SV40 large T-antigen gene, is a rational and promising approach to such a workable in vitro BBB model. The established brain capillary endothelial cell lines retain the in vivo transport rate of several compounds and various forms of gene expression. Furthermore, this new approach has enabled the development of stable and reproducible co-culture models with a pericyte cell line and/or an astrocyte cell line.

Role of blood-brain barrier organic anion transporter 3 (OAT3) in the efflux of indoxyl sulfate, a uremic toxin: Its involvement in neurotransmitter metabolite clearance from the brain

Renal impairment is associated with CNS dysfunctions and the accumulation of uremic toxins, such as indoxyl sulfate, in blood. To evaluate the relevance of indoxyl sulfate to CNS dysfunctions, we investigated the brain‐to‐blood transport of indoxyl sulfate at the blood–brain barrier (BBB) using the Brain Efflux Index method. [3H]Indoxyl sulfate undergoes efflux transport with an efflux transport rate of 1.08 × 10−2/min, and the process is saturable with a Km of 298 µm. This process is inhibited by para‐aminohippuric acid, probenecid, benzylpenicillin, cimetidine and uremic toxinins, such as hippuric acidand 3‐carboxy‐4‐methyl‐5‐propyl‐2‐furanpropanoic acid. RT–PCR revealed that an OAT3 mRNA is expressed in conditionally immortalized rat brain capillary endothelial cell lines and rat brain capillary fraction. Xenopus oocytes expressing OAT3 were found to exhibit [3H]indoxyl sulfate uptake, which was significantly inhibited by neurotransmitter metabolites, such as homovanillic acid and 3‐methoxy‐4‐hydroxymandelic acid, and by acyclovir, cefazolin, baclofen, 6‐mercaptopurine, benzoic acid, and ketoprofen. These results suggest that OAT3 mediates the brain‐to‐blood transport of indoxyl sulfate, and is also involved in the efflux transport of neurotransmitter metabolites and drugs. Therefore, inhibition of the brain‐to‐blood transport involving OAT3 would occur in uremia and lead to the accumulation of neurotransmitter metabolites and drugs in the brain.

Restricted transport of cyclosporin A across the blood-brain barrier by a multidrug transporter, P-glycoprotein

The blood-brain barrier permeability of cyclosporin A (CsA), an immunosuppressive cyclic peptide, is restricted despite its highly lipophilic nature. In this study, the uptake of CsA by primary cultured bovine brain capillary endothelial cells (BCEC) was investigated in order to clarify whether P-glycoprotein (P-gp), an ATP-dependent drug efflux pump expressed in the luminal surface of BCEC, causes the decreased transport of CsA into the brain. P-gp, having a molecular mass of 130-140 kDa, was detected with anti-P-gp monoclonal antibody, C219, using western blot analysis of the membrane fraction isolated from the bovine brain capillary. The uptake of CsA by primary cultured bovine BCEC was time-dependent, and the steady-state uptake of CsA was increased in the presence of several multidrug resistance reversing agents including verapamil and steroid hormones and the substrate of P-gp in BCEC, vincristine. The steady-stage uptake was increased significantly to approximately 4-fold when cellular ATP was depleted by treating with 2,4-dinitrophenol, suggesting that the efflux process is ATP dependent. Furthermore, in the presence of an anti-P-gp monoclonal antibody, MRK16, at a 10 micrograms/mL concentration, the uptake of CsA was increased approximately 3-fold. These results suggest that the low permeability of CsA into the brain is caused by the active efflux from BCEC by P-gp present in the luminal surface of cells.