Pharmacokinetics, Pharmacodynamics and Drug Metabolism
Quantitative Membrane Protein Expression at the Blood–Brain Barrier of Adult and Younger Cynomolgus Monkeys

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Abstract

Cynomolgus monkey has been used as a model for the prediction of drug disposition in human brain. The purpose of this study was to clarify protein expression levels of membrane proteins affecting drug distribution to brain, such as transporters, receptors, and junctional proteins, in cynomolgus monkey brain microvessels by using liquid chromatography tandem mass spectrometory. In adult monkeys, three ATP-binding cassette transporters (multidrug resistance 1 (MDR1), breast cancer resistance protein (BCRP), and multidrug resistance protein 4 (MRP4)), six solute carrier transporters (glucose transporter 1 (GLUT1), GLUT3/14, monocarboxylate transporter 1 (MCT1), MCT8, organic anion transporting polypeptide 1A2, and equilibrative nucleoside tranporter 1), two junctional proteins (claudin-5 and vascular endothelial cadherin), and two receptors (insulin receptor and low-density lipoprotein receptor-related protein 1) were detected. Comparison of the expression levels with those in mouse, which we reported previously, revealed a pronounced species difference. BCRP expression in monkey was greater by 3.52-fold than that in mouse, whereas MDR1 and MRP4 expression levels in monkey were lower by 0.304- and 0.180-fold, respectively, than that in mouse. This study also investigated the developmental changes in expression of membrane proteins in neonate and child monkeys. Expression of MDR1 was similar in neonate and adult monkeys, whereas in rat, P-glycoprotein expression was reported to be significantly lower in brain microvessels of neonate as compared with adult rat. These results will be helpful to understand and predict brain concentrations of drugs in different species and at different ages of primates. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3939–3950, 2011

Section snippets

INTRODUCTION

Cynomolgus monkey has been considered as a model animal for humans because monkeys are the second nearest animal to humans in the evolutionary tree, and consequently, it has been used for the prediction of drug disposition in human brain. Substrates for P-glycoprotein (P-gp), such as verapamil and GR205171, were reported to be distributed more extensively in monkey brain than in rat brain,1 and it was suggested that there are species differences in drug permeability across the blood–brain

Reagents

All peptides listed in Supplementary Table 1 were purchased from Thermo Electron Corporation (Sedantrabe, Germany). Peptide purity (>95%) was provided by the manufacturer, using reversed-phase high-performance liquid chromatography with UV detection (RP-HPLC–UV, with a detection wavelength of 215 nm) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses. Other chemicals were commercial products of analytical grade.

Animals

Brains of cynomolgus monkeys,

Isolation of Brain Microvessels from Monkey Brain

The brain microvessels were isolated by means of a combination of dextran density separation and size filtration from the brain of neonate, child, and adult monkeys. As shown in Figure 1, microscopic analysis revealed that brain microvessels were predominantly present in the preparations obtained from the brain at each age. The diameter of the microvessels was 4–8 μm, and there was no obvious age-related difference in the characteristics of the microvessels.

Quantitative Protein Expression of Membrane Proteins in Brain Microvessels of Adult Monkeys

Protein expression amounts of

DISCUSSION

This is the first study to determine quantitatively the expression levels of membrane proteins in cynomolgus monkey brain microvessels, which constitute the BBB. Comparison with our previously reported results for mouse brain microvessels10 indicates that there is a pronounced species difference between monkey and mouse. BCRP expression in the monkey was higher than that in mouse, whereas MDR1 and MRP4 expression levels were lower than those in mouse (Table 5). OATP1A2/OATP-A expression in

Acknowledgements

This study was supported in part by a Grant-in-Aid for JSPS Fellows, Grant-Scientific Research (S), and a Global Center of Excellence (COE) Program grant from the Japan Society for the Promotion of Science. This study was also supported in part by the Industrial Technology Research Grant Program from New Energy and the Industrial Technology Development Organization of Japan. We are most grateful to Drs. Yasuyuki Ishii, Masato Chiba, Tomoyuki Ohe, and Kentaro Wakayama, Banyu Pharmaceutical Co.,

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