1A6 in Monkey Small Intestine

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High Expression of UGT1A1/1A6 in Monkey Small Intestine: Comparison of Protein Expression Levels of Cytochromes P450, UDP-Glucuronosyltransferases, and Transporters in Small Intestine of Cynomolgus Monkey and Human Takanori Akazawa, Yasuo Uchida, Eisuke Miyauchi, Masanori Tachikawa, Sumio Ohtsuki, and Tetsuya Terasaki Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00772 • Publication Date (Web): 15 Nov 2017 Downloaded from http://pubs.acs.org on November 21, 2017

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Molecular Pharmaceutics

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High Expression of UGT1A1/1A6 in Monkey Small Intestine: Comparison of

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Protein Expression Levels of Cytochromes P450, UDP-Glucuronosyltransferases,

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and Transporters in Small Intestine of Cynomolgus Monkey and Human

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Takanori Akazawa†, Yasuo Uchida†, Eisuke Miyauchi†, Masanori Tachikawa†, Sumio

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Ohtsuki‡, and Tetsuya Terasaki*,†

7 8

†

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Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-ku, Sendai

Division of Membrane Transport and Drug Targeting, Graduate School of

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980-8578, Japan

11

‡

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University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan

Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto

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small

intestine,

drug

absorption,

cytochromes

P450,

14

KEYWORDS:

15

UDP-glucuronosyltransferase, transporter, cynomolgus monkey, species differences,

16

protein quantification, quantitative targeted absolute proteomics

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ABSTRACT

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Cynomolgus monkeys have been widely used for the prediction of drug absorption in

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humans. The purpose of this study was to clarify the regional protein expression levels

4

of cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UGTs), and transporters

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in small intestine of cynomolgus monkey using liquid chromatography–tandem mass

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spectrometry, and to compare them with the corresponding levels in human. UGT1A1 in

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jejunum and ileum were >4.57- and >3.11-fold, and UGT1A6 in jejunum and ileum

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were >16.1- and >8.57-fold, respectively, more highly expressed in monkey than in

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human. Also, jejunal expression of monkey CYP3A8 (homolog of human CYP3A4)

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was >3.34-fold higher than that of human CYP3A4. Among apical drug efflux

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transporters, BCRP showed the most abundant expression in monkey and human, and

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the expression levels of BCRP in monkey and human were >1.74- and >1.25-fold

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greater than those of P-gp, and >2.76- and >4.50-fold greater than those of MRP2,

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respectively. These findings should be helpful to understand species differences of the

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functions of CYPs, UGTs, and transporters between monkey and human. The

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UGT1A1/1A6 data would be especially important, because it is difficult to identify

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isoforms responsible for species differences of intestinal glucuronidation by means of

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functional studies, due to overlapping substrate specificity.

19


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INTRODUCTION

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Oral absorption of many drugs is influenced by transporters and metabolic

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enzymes expressed in small intestine. Drug efflux transporters, such as P-gp，BCRP, and

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MRP2, are expressed in the apical membrane of intestinal epithelial cells, and restrict

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the permeability of their substrates from the intestinal lumen into epithelial cells.1 P-gp

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and BCRP are especially important transporters for drug discovery and development,

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because there is clear clinical evidence that the oral absorption of some P-gp and BCRP

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substrates, such as fexofenadine, aliskiren, dabigatran (P-gp substrates),2-4 sulfasalazine,

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atorvastatin, and rosuvastatin (BCRP substrates),5, 6 is influenced by efflux transport

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mediated by P-gp and BCRP in human small intestine. Other drug transporters, such as

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OSTα/β, MRP1, 3, 4, and 5, are also expressed in the basolateral membrane of intestinal

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epithelial cells.1, 7 It was reported that methotrexate and cefadroxil are transported by

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MRP3 and/or MRP4 in mouse small intestine,8, 9 and digoxin is a substrate of OSTα/β.10

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However, the impact of intestinal OSTα/β, MRP1, 3, 4, and 5 on the pharmacokinetics

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of drugs remains unclear in human. In addition to drug transporters, metabolic enzymes,

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such as CYP3A4 and UGTs, are involved in limiting the absorption of many drugs in

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human small intestine.11, 12 Among intestinal CYPs, CYP3A4 is mainly expressed in

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human small intestine (70-80% of total intestinal CYP isoforms),13-15 and contributes to

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the first-pass metabolism of many substrates, such as midazolam and nifedipine.16, 17 In

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addition, many UGT isoforms, such as UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9,

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1A10, 2B4, 2B7, 2B15, and 2B17, are expressed in human small intestine.14, 15, 18, 19

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Since the substrate specificities of UGT isoforms often overlap, multiple UGT isoforms

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can be involved in the glucuronidation of a single drug, for example, ezetimibe

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(substrate of UGT1A1, 1A3, and 2B15), acetaminophen (substrate of UGT1A1, 1A6, 3 ACS Paragon Plus Environment


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1A9, 2B7, and 2B15), and morphine (substrate of UGT1A3, 1A10, 2B4, 2B7).12 As

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mentioned above, since many transporters and metabolic enzymes can affect intestinal

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drug absorption, it is crucial to estimate the extent of their impact on the bioavailability

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of drug candidates during drug discovery and development.

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Cynomolgus monkeys have been widely used as an experimental animal for

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prediction of the pharmacokinetics of candidate drugs in humans during drug

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development, because monkeys are evolutionarily close to humans, and the amino acid

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sequences of monkey cytochromes P450 show high degrees of homology (>91 %) with

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human cytochromes P450.20 Ward and Smith demonstrated that data obtained in

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monkey enabled the most accurate prediction of human clearance for 103 intravenously

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administered drugs, compared with rat and dog.21 However, cynomolgus monkey may

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not be an ideal model to predict oral drug absorption in humans for compounds that are

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substrates of metabolic enzymes and transporters. Takahashi et al. compared the values

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of Fa × Fg, which is the product of the fraction of intestinal absorption (Fa) and the

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fraction escaping intestinal extraction (Fg), of several drugs between monkey and

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human.22 They found that the values for drugs permeating across epithelial cells in the

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small intestine by passive diffusion (e.g., antipyrine, piroxicam, naproxen, and atenolol)

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in monkey were consistent with those in human, whereas the values of Fa × Fg for

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substrates of CYP3A4 (e.g. midazolam, nifedipine, propafenone, and verapamil), UGTs

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(e.g. acetaminophen and propafenone), and P-gp (e.g. verapamil) tended to be smaller in

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monkey than in human.22 Therefore, a quantitative understanding of species differences

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of intestinal transporter and metabolic enzyme functions between monkey and human is

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important to enable more reliable prediction of drug absorption in humans.

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Species differences of transporter and metabolic enzyme functions in small 4 ACS Paragon Plus Environment

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intestine between monkey and human have generally been discussed based on the

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differences of intestinal absorption of various substrates after oral administration.22-24

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It is not easy to clarify species differences precisely, because the functional specificities

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of many metabolic enzymes and transporters overlap to a greater or lesser extent.12, 25-28

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Expression of metabolic enzymes and transporters has been comprehensively examined

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at the mRNA level in monkey and human,29-33 but it is well established that mRNA

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expression levels of metabolic enzymes and transporters are not necessarily well

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correlated with protein expression levels or functional activities.34-36

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Protein expression levels of metabolic enzymes and transporters are considered

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to be highly correlated with functional activities.35-38 Paine et al. employed quantitative

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Western blotting analysis to provide the first protein expression profiles of CYP

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enzymes in human small intestine.13 A few years later, we developed the

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LC-MS/MS-based quantitative targeted absolute proteomics (QTAP) technique, which

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allows us to determine the protein expression levels of target molecules for which a

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target-specific peptide can be selected for quantitative analysis by using the amino acid

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sequence information in protein databases.39 In recent years, many researchers have

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reported protein quantification of metabolic enzymes and transporters in human small

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intestine by means of the QTAP technique.14,

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quantifications were performed by using several types of protein samples, such as

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whole-cell lysate and microsomal / plasma membrane fractions. It is important to select

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appropriate types of protein samples for a proper consideration of the functions of

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metabolic enzymes and transporters in the small intestine. The sites of functional

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activities of CYPs and UGTs are the microsomal membrane, and those of transporters

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are the plasma membrane. Since the protein expression levels of metabolic enzymes and

15, 19, 40-44


However, the protein


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transporters in whole-cell lysate include digested / inactive protein in cytosol, these

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expression data may be overestimated. We recently quantified the protein expression

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levels of transporters in plasma membrane fractions of the epithelial cells in mouse

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small intestine by means of the QTAP technique.45 We found that the protein expression

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level of mdr1a well reflected the transport activity in mouse small intestine.45 Thus,

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comprehensive quantification of protein expression levels at the sites of the functional

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activities should be an effective strategy to understand species differences in the

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functions of intestinal metabolic enzymes and transporters between monkey and human.

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The purpose of this study was to determine the regional protein expression

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levels of CYPs, UGTs, and transporters in small intestine of cynomolgus monkey, and

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to compare them with the corresponding levels in human. The results are expected to

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provide valuable insight into species differences of intestinal metabolic enzyme and

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transporter function between monkey and human. Many researchers have reported

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protein quantification of metabolic enzymes and transporters in human small intestine

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by means of QTAP technique,14, 15, 19, 40-44 but for the discussion of species differences, it

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is essential to compare protein expression levels between samples obtained by using

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identical methods to isolate epithelial cells from small intestine and to prepare

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membrane fractions.46 Therefore, in this study we also quantified CYPs, UGTs and

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transporters in human jejunum and ileum using the same procedures established for

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cynomolgus monkey.


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EXPERIMENTAL SECTION

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Reagents. Unlabeled (standard) and stable-isotope labeled peptides (internal

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standard) listed in Table S1 and S2 were designed according to our previous reports,39

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and synthesized by Thermo Electron Corporation (Sedanstrabe, Germany) with > 95%

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peptide purity. The concentrations of peptide solutions were determined by means of

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quantitative amino acid analysis, using an HPLC-UV system with post-column

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ninhydrin derivatization (Lachrom Elite, Hitachi, Tokyo, Japan). Other chemicals were

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commercial analytical-grade products.

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Monkey small intestine. Adult cynomolgus monkey (4 years 3 months after

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birth, male), which had originated from Indonesia (Table 1), was purchased from Shin

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Nippon Biomedical Laboratories, Ltd. (Kagoshima, Japan). This monkey was the same

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individual used in our previous report.47 We previously quantified the protein expression

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levels of transporters in small intestine of male mice by the same methods used in the

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present study.45 In order to allow comparison of the data in monkey with the reported

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data in mice, we used only male monkeys in this study, to rule out possible effects of

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gender difference. Duodenum, jejunum, and ileum were excised at Shin Nippon

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Biomedical Laboratories, Ltd. as follows. The monkey was anesthetized with

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pentobarbital (25.9 mg/kg weight) and perfused with saline containing sodium heparin

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to remove blood. After the perfusion, the duodenum, jejunum, and ileum were excised

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and frozen in liquid nitrogen. Duodenum was defined as the region from the pylorus end

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point to the pancreatic duct opening together with the same length from the pancreatic

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duct opening. Jejunum and ileum were respectively defined as the proximal two-fifths

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and distal three-fifths of the rest of the small intestine, excluding duodenum. The 7 ACS Paragon Plus Environment


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duodenum, jejunum, and ileum were each excised, and cut into 5 cm pieces. We used

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the most central 5 cm pieces of duodenum, jejunum, and ileum as analytical samples in

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the present study. The intestine was stored at −80 °C, shipped to Tohoku University on

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dry ice, and stored at −80 °C until isolation of epithelial cells. The protocols were

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performed in accordance with the animal welfare requirements of the Drug Safety

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Research Laboratories, Shin Nippon Biomedical Laboratories, Ltd., and were approved

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by the Institutional Animal Care and Use Committee.

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Human small intestine. Protein quantification in human intestine was

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performed with two different donor intestines, to take account of individual differences

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in human samples. Frozen jejuna and ilea of two human donors (jejunum and ileum of

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donor #1, and jejunum and ileum of donor #2; intra-subject; Table 1) were purchased

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from Analytical Biological Services (Wilmington, DE, USA). Although human jejunum

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and ileum are organs of several meters length, detailed information about the positions

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of the excised sites of the jejunum and ileum we used was not available from the

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supplier (Analytical Biological Services). The intestine was shipped to Tohoku

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University on dry ice, and stored at −80 °C until isolation of epithelial cells.

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The research protocols for this study were approved by the Ethics Committees

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of Tohoku University School of Medicine and the Graduate School of Pharmaceutical

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Sciences, Tohoku University.

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Preparation of Microsomal and Plasma Membrane Fractions of Small Intestinal Epithelial Cells. Isolation of epithelial cells from small intestine was performed by agitation in 8 ACS Paragon Plus Environment

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phosphate-buffered saline (PBS) containing 2 mM EDTA and 0.5 mM dithiothreitol

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according to our previous report,45 and we subsequently prepared individual microsomal

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and plasma membrane fractions from small intestine of the monkey and the two human

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donors according to our previous reports.15

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Protein Quantification by Using LC-MS/MS. Membrane samples were

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digested with lysyl endopeptidase and trypsin to afford peptides, and protein expression

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levels were determined by quantification of specific peptides from the target molecules

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by using LC-MS/MS according to our previous report.45 The mass spectrometer was a

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triple quadrupole mass spectrometer (API5000 or QTRAP5500; AB SCIEX,

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Framingham, MA, USA) or a quadrupole time-of-flight mass spectrometer (Triple

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TOF5600; AB SCIEX). Although we used different mass spectrometers (API5000,

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QTRAP5500, and TripleTOF5600) for the quantification of transporter proteins, we

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considered that the measured protein expression levels were not instrument-dependent,

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based on our previous comparison of the transporter expression levels in human liver,

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kidney, and intestinal microsomes quantified by using API5000 (triple quadrupole mass

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spectrometer) and TripleTOF5600 (quadrupole time-of-flight mass spectrometer)

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instruments.44 The differences of 11 transporters (ABCA8, ABCC2, ABCC6, SLC5A1,

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SLC7A8, SLC16A1, SLC22A6, SLC22A8, SLC22A13, SLC22A18, and SLC27A2)

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were all within 1.35-fold between the two mass spectrometers. Several monkey

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molecules (Table S1) and human Na+/K+-ATPase were quantified by triple quadrupole

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mass spectrometry with an Agilent 1100 HPLC system (Agilent Technologies, Santa

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Clara, CA, USA) coupled to the API5000 or QTRAP5500 spectrometer equipped with a

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Turbo V ion source (AB SCIEX). HPLC was performed with C18 columns (XBridge 9 ACS Paragon Plus Environment


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BEH130 C18, 1.0 mm ID × 100 mm, 3.5 µm particles) (Waters, Milford, USA). Probe

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peptides of the quantified molecules and their SRM/MRM transitions are listed in Table

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S1. The measurement and analysis were carried out according to our previous report.48

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Other monkey and human molecules (Table S2) were quantified by employing

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the Triple TOF5600, which is a high-resolution MS analyzer, because the signal peak of

6

the target peptide was occluded by background noise in analysis using the triple

7

quadrupole mass spectrometer. Triple TOF5600 system analysis was performed with a

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NanoLC-Ultra 1D plus system (Eksigent Technologies, Dublin, CA, USA) coupled with

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a cHiPLC-nanoflex system (Eksigent Technologies) and a Triple-TOF5600 (AB

10

SCIEX) equipped with a NanoSpray III ion source (AB SCIEX). NanoLC was

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performed with a trap column (Nano cHiPLC Trap column 200 µm x 0.5 mm ChromXP

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C18-CL 3 µm 120Å) (Eksigent Technologies) and a Nano cHiPLC analytical column

13

(Nano cHiPLC column 75 µm × 15 cm ChromXP C18-CL 3 µm 120Å) (Eksigent

14

Technologies). The measurement and data analysis were conducted according to our

15

previous reports.45 The m/z values of Q1 and four SRM/MRM transitions of target

16

peptides are summarized in Tables S2.

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In this study, we could not determine the protein expression levels of some

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metabolic enzymes and transporters, such as CYP2C19, OATP2B1, and OSTβ, that

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have been reported to be expressed in the small intestine.13-15, 41, 43 The reason for this

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was that the synthesized probe peptides (OATP2B1 ； VLLQTLR ， CYP2C19 ；

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GHFPLAER ， OSTβ ； DVLSVFLPDVPETES) were not detected in the present

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LC-MS/MS system, and so we could not generate a calibration curve. However, the

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same peptides of OATP2B1 and CYP2C19 were at least detected in our previous

24

study.15 The difference between the present and previous studies was the LC set-up. In 10 ACS Paragon Plus Environment

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the previous study, after sample injection using the autosampler, the peptides were

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directly loaded onto the main analytical column (Nano cHiPLC column 75 µm × 15 cm

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ChromXP C18-CL 3 µm 120 Å) (Eksigent Technologies), which is longer and has a

4

higher capacity to retain hydrophilic peptides than the trap column (Nano cHiPLC Trap

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column 200 µm × 0.5 mm ChromXP C18-CL 3 µm 120 Å) (Eksigent Technologies,

6

Dublin, CA, USA). However, the present study employed the trap column for sample

7

loading in order to further clean up the samples before passing them to the main

8

analytical column and mass spectrometer. The two peptides are hydrophilic, and so

9

might have not been retained on the short trap column, which would account for the

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failure of detection in the mass spectrometer.

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For measurement and analysis, each peptide for a target protein was monitored

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with four kinds of SRM/MRM transitions. When positive peaks were observed in three

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or four sets of transitions, the protein expression levels (fmol/ fmol/µg microsomal or

14

plasma membrane protein) were determined as the average (mean) ± variability

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(S.E.M.) of three or four sets of transitions for one sample, according to our previous

16

report.45 When two or more among the four sets of transitions gave no signal peak in the

17

chromatogram of the target protein samples, the value of the limit of quantification (LQ)

18

was calculated according to ref. 48 for the API5000 or QTRAP5500 and according to

19

ref. 49 for the Triple TOF 5600.48, 49

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Statistical Analysis. The statistical significance of differences between two

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groups was determined by Student’s t-test and that among three groups was determined

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by one-way ANOVA with Tukey’s post hoc test. A value of p < 0.05 was considered

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statistically significant. 11 ACS Paragon Plus Environment


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1

RESULTS

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Quantification of CYP and UGT Proteins in Microsomal Membrane

3

Fraction of Monkey Small Intestine. The protein expression levels of 9 proteins in

4

microsomal membrane fractions, including 5 CYPs and 3 UGTs, were examined by

5

LC-MS/MS (Table S1). Among the 9 target proteins, 4 proteins, including 1 CYP and 2

6

UGTs, were detected in all intestinal segments (Table 2), and the other 5 proteins were

7

not detected in any intestinal segment. The LQs of target proteins that were not detected

8

in microsomal membrane fractions of monkey small intestine were calculated according

9

to our previous reports,48 as described in the Experimental Section (Table S3).

10

Comparing the protein expression levels across the three intestinal segments,

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CYP3A8 and NADPH-cytochrome P450 reductase (P450R) showed higher expression

12

in duodenum and jejunum than in ileum; their protein expression levels in duodenum

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were 3.01- and 1.62-fold higher than those in ileum, while those in jejunum were 3.30-

14

and 2.21-fold higher than those in ileum, respectively.

15 16

Quantification of Transporter Proteins in Plasma Membrane Fraction of

17

Monkey Small Intestine. The protein expression levels of 45 proteins in plasma

18

membrane fraction, including 11 ABC transporters and 31 SLC transporters, were

19

examined by LC-MS/MS (Tables S1 and S2). Among the 45 target proteins, 21 proteins,

20

including 7 ABC transporters and 12 SLC transporters, were detected in at least one of

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three intestinal segments (duodenum, jejunum, and ileum) (Tables 3 and 4), and the

22

other 24 proteins were not detected in any intestinal segment. The LQs of target proteins

23

that were not detected in plasma membrane fractions of monkey small intestine were

24

calculated according to our previous reports,48,

49

as described in the Experimental


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Section (Table S4).

2

Among apical drug efflux transporters (BCRP, P-gp, and MRP2), BCRP was

3

expressed at the highest level in all intestinal segments. Among basolateral drug

4

transporters (OSTα, OSTβ, MRP3, MRP4, MRP1, and MRP5), OSTα showed the

5

greatest protein expression level in all intestinal segments. Among hexose and

6

monocarboxylate transporters (SGLT1, GLUT5, GLUT2, GLUT1, and MCT1), SGLT1

7

showed the most abundant protein expression levels in duodenum and ileum, and

8

GLUT5 exhibited the greatest protein expression levels in jejunum.

9

Comparing the protein expression levels across the three intestinal segments,

10

BCRP, P-gp, OSTα, and ABCG8 were the most abundant proteins in ileum, and their

11

expression levels were 1.55, 1.44-, 4.85-, and 1.97-fold greater than those in duodenum,

12

and 1.63, 1.59-, 6.78-, and 1.09-fold greater than those in jejunum, respectively. MRP2,

13

MRP4, SGLT1, MCT1, PCFT, MRP6, OAT2, and Na+/K+-ATPase were the most

14

abundant proteins in duodenum, and their expression levels were 1.18-, 1.34-, 1.31-,

15

2.45-, 1.56-, 1.37-, 1.20-, and 1.37-fold greater than those in jejunum, and 1.94-, 1.57-,

16

2.16-, >2.87-, >12.7-, 2.30-, >1.09-, and 1.87-fold greater than those in ileum,

17

respectively.

18 19

Quantification of CYP and UGT Protein in Microsomal Membrane

20

Fraction of Human Small Intestine. The protein expression levels of 23 proteins in

21

microsomal membrane fractions, including 15 CYPs and 7 UGTs, were examined by

22

LC-MS/MS (Table S2). Among 23 target proteins, 12 proteins, including 7 CYPs and 4

23

UGTs, were detected in either jejunum or ileum (Table 2), and the other 11 proteins

24

were not detected in any intestinal segment of either of the donors. The LQs of targeted 13 ACS Paragon Plus Environment


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proteins that were not detected in microsomal membrane fractions of human small

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intestine were calculated according to our previous reports,49 as described in the

3

Experimental Section (Table S5).

4

CYP3A4 were more highly expressed in ileum than in jejunum in both donors;

5

the protein expression in ileum was 1.30-fold greater than that in jejunum in donor #1,

6

and 1.31-fold greater in donor #2.

7 8

Quantification of Transporter Proteins in Plasma Membrane Fraction of

9

Human Small Intestine. The protein expression levels of 43 proteins in plasma

10

membrane fraction, including 10 ABC transporters and 30 SLC transporters, were

11

examined by LC-MS/MS (Tables S1 and S2). Among 43 target proteins, 34 proteins,

12

including 10 ABC transporters and 21 SLC transporters, were detected in either jejunum

13

or ileum of two donors (Tables 3 and 4), and the other 9 proteins were not detected in

14

any intestinal segment of either of the donors. The LQs of target proteins that were not

15

detected in plasma membrane fractions of human small intestine were calculated

16

according to our previous reports,49 as described in the Experimental Section (Table S6).

17

Among apical drug efflux transporters (BCRP, P-gp, and MRP2), BCRP was

18

the most abundant protein in jejunum and ileum in both human donors. Among

19

basolateral drug transporters (OSTα, OSTβ, MRP3, MRP4, MRP1, and MRP5), MRP4

20

exhibited the highest protein expression in jejunum and ileum of donor #1, and OSTα

21

showed the greatest protein expression level in jejunum and ileum of donor #2. Among

22

hexose and monocarboxylate transporters (SGLT1, GLUT5, GLUT2, GLUT1, and

23

MCT1), SGLT1 was expressed at the highest levels in jejunum of donor #1 and in ileum

24

of both donors, and GLUT1 was expressed at the highest level in ileum of donor #1. 14 ACS Paragon Plus Environment

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1

P-gp, MCT1, and ASBT were more highly expressed in ileum than in jejunum

2

in both donors; their expression levels in ileum were 1.77-, 1.61-, and >3.52-fold greater

3

than those in jejunum of donor #1, and 2.13-, 1.55-, and >3.72-fold greater than those in

4

jejunum of donor #2, respectively. villin-1 was more highly expressed in jejunum than

5

in ileum in both donors; the protein expression in jejunum was 1.58-fold greater than

6

that in ileum in donor #1, and 1.82-fold greater in donor #2.

7 8

Comparison of Absolute Protein Expression Levels of CYPs, UGTs, and

9

Transporters in Small Intestine between Monkey and Human. It is important to

10

consider whether expression levels in different samples and species should be compared

11

after normalization by an enterocyte marker such as villin-1, in order to mitigate the

12

effects of various potential inconsistencies, including contamination with other tissues

13

during sample preparation. To investigate this issue, we quantified villin-1 in plasma

14

membrane fractions, and normalized the expression level of each transporter by that of

15

villin-1 (Tables S7 and S8). This normalization reduced the expression differences

16

between the two human donors (Tables 3, 4, S7, and S8). However, in order to compare

17

different samples and species in this way, it is necessary to confirm that there are no

18

individual differences or species differences in villin-1 expression. A previous report

19

found individual differences of villin-1 expression among human donors,50 and in

20

addition, it is not yet known whether there is a species difference of villin-1 expression

21

between monkey and human small intestine. Therefore, the validity of comparison of

22

villin-1-normalized expression levels is unclear. On the other hand, we previously

23

reported that the mdr1a transport activities in mouse small intestine can be reconstructed

24

from transport activity per protein amount of mdt1a and absolute protein expression 15 ACS Paragon Plus Environment


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1

levels of mdr1a, determined by the same methods as used in the present study.45 This at

2

least suggests that the absolute protein expression determined by using our methods

3

does reflect the intestinal functions of transporters, and thus that the absolute protein

4

expression levels may be appropriate for assessing species differences of intestinal

5

functions of transporters and metabolic enzymes. However, for this purpose, it is very

6

important to use pure samples uncontaminated with other tissues. One approach to

7

obtain intestinal epithelial cells with high purity might be purification with a

8

fluorescence-activated cell sorter (FACS) based on a suitable epithelial cell marker.

9

The absolute protein expression levels of CYPs, UGTs, and transporters in

10

jejunum and ileum in monkey were compared with those in human (Tables 2-4). Since

11

the present quantitative analyses were performed using only one male monkey and two

12

human donors, and the likelihood of significant inter-individual differences in both

13

monkey and human should be taken into account, our discussion of species differences

14

here is limited to the following cases: (1) when the molecule showed higher expression

15

in monkey than in both human donors #1 and #2 (e.g. BCRP in jejunum), and (2) when

16

the molecule showed lower expression in monkey than in both human donors #1 and #2

17

(e.g. SGLT1 in jejunum and ileum). If a molecule showed higher (lower) expression in

18

monkey than only one of the two human donors (e.g. BCRP in ileum), we considered

19

that a species difference was unlikely.

20

CYP3A8 with those of human CYP3A4, because it was reported that CYP3A8 in

21

cynomolgus monkey is a homolog of CYP3A4 in human; the amino acid sequence

22

homology of the two cytochromes P450 is 93 %.20

We compared the expression levels of monkey

23

Molecules that showed higher expression levels in monkey than in both of the

24

human donors #1 and #2 were CYP3A8/CYP3A4, UGT1A1, UGT1A6, BCRP, MRP2, 16 ACS Paragon Plus Environment

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1

OATα, GLUT2, PCFT, TAUT, MRP6, OAT2, and Na+/K+-ATPase in jejunum (>3.34-,

2

>4.57-, >16.1-, >1.15-, >1.68-, >2.79-, >10.5-, >2.46-, >4.68-, >4.34-, >1.41-, and

3

>2.36- fold higher in monkey than human, respectively), and UGT1A1, UGT1A6,

4

OATα, and MRP6 in ileum (>3.11-, >8.57-, >9.18-, and >3.88-fold higher in monkey

5

than human, respectively), although the differences of UGT1A6 in jejunum and ileum

6

were not statistically significant. In contrast, the molecules that showed lower

7

expression levels in monkey than in both of the human donors #1 and #2 were SGLT1,

8

GLUT1, CNT2, and γ-GTP in jejunum (>3.01-, >6.09-, >2.21-, and >5.53-fold lower in

9

monkey than human, respectively), and MRP1, SGLT1, GLUT1, MCT1, PCFT, ASBT,

10

and γ-GTP in ileum (>1.56-, >3.85-, >1.54-, >1.34, >1.35-, >1.28-, and >5.04-fold

11

lower in monkey than human, respectively). Molecules other than those mentioned

12

above did not exhibit species differences between in monkey and human in either

13

jejunum or ileum.



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1

DISCUSSION

2

The present study is the ﬁrst to comprehensively quantify the protein

3

expression levels of CYPs, UGTs, and transporters in epithelial cells of monkey small

4

intestine, and to compare them with the corresponding levels in human. Among

5

metabolic enzymes, UGT1A1 and UGT1A6 in jejunum and ileum were more highly

6

expressed in monkey than in human, and the jejunal expression level of monkey

7

CYP3A8 was higher than that of human CYP3A4 (Table 2). Among apical drug efflux

8

transporters (P-gp, BCRP, and MRP2), BCRP showed the most abundant expression in

9

monkey and human small intestine (Table 3).

10

UGTs play a role in glucuronide conjugation of drugs having carboxylic acid,

11

hydroxyl, thiol, and amino groups. For example, buprenorphine and ezetimibe are

12

conjugated by UGT1A1,51 and acetaminophen is conjugated by UGT1A1 and

13

UGT1A6.52 The intrinsic clearances of buprenorphine and ezetimibe in small intestinal

14

microsomes were reported to be 8.75- and 17.2-fold higher in monkey than in human,

15

respectively.51 Also, the Fa × Fg values of acetaminophen were reported to be 0.19 in

16

monkey and 1 in human.22 These data suggested that the glucuronidation of

17

buprenorphine, ezetimibe, and acetaminophen in small intestine is more extensive in

18

monkey than in human. UGT isoforms frequently share substrate specificities; for

19

example, buprenorphine is also a substrate of UGT1A3 and 2B7, ezetimibe is also a

20

substrate of UGT1A3 and 2B15, and acetaminophen is also a substrate of UGT1A1,

21

1A9, 2B7, 2B15.12,

22

contributing to species differences of glucuronidation in small intestine between

23

monkey and human by means of studies of intestinal glucuronidation activity using

24

UGT substrates, due to this overlap in the substrate specificity of UGT isoforms. Our

51, 52

So far, it has been difficult to identify the UGT isoforms


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1

results indicate that UGT1A1 and UGT1A6 are more highly expressed in jejunum and

2

ileum in monkey than in human (Table 2), and this may at least in part explain the

3

species differences in intestinal glucuronidation of buprenorphine, ezetimibe, and

4

acetaminophen between monkey and human.

5

CYP3A4 was reported to be mainly expressed in human small intestine among

6

CYP subfamilies.13-15 The intestinal absorptions of CYP3A substrates are known to

7

show species differences between monkey and human.24, 53 Nishimuta et al. reported

8

that the intrinsic clearances of 14 substrates of CYP3A in intestinal microsomes were 2-

9

to 4-fold higher in monkey than in human,53 and Akabane et al. reported that the Fa ×

10

Fg values of several substrates (dexamethasone, nifedipine, midazolam, quinidine, and

11

tacrolimus) for CYP3A were smaller in monkey than in human.24 We found that

12

monkey CYP3A8 was at least 3.34-fold more highly expressed in jejunum than human

13

CYP3A4 (Table 2), and the differences of intestinal absorption of CYP3A substrates

14

between monkey and human tended to be in agreement with the differences of protein

15

expression levels. However, besides the protein expression levels, the differences of

16

metabolizing activities per protein amount between human CYP3A4 and monkey

17

CYP3A8 can also affect the differences of intestinal absorption of CYP3A substrates

18

between monkey and human. Carr et al. examined the differences of metabolizing

19

activities per protein amount between human CYP3A4 and rhesus monkey CYP3A64

20

by means of metabolic studies with recombinant enzymes.54 Since the amino acid

21

sequence of CYP3A64 in rhesus monkey is the same as that of CYP3A8 in cynomolgus

22

monkey, the metabolizing activity per protein amount of CYP3A64 was equal to that of

23

CYP3A8.54 This report demonstrated that the metabolizing activities of midazolam and

24

nifedipine per protein amount of rhesus monkey CYP3A64 (cynomolgus monkey 19 ACS Paragon Plus Environment


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1

CYP3A8) were 1.7- and 2.0-fold higher than those of human CYP3A4.54 The Fa × Fg

2

values of midazolam and nifedipine were reported to be 15.0- and 4.68-fold smaller in

3

monkey than in human.24 Although we found that the jejunal expression level of

4

monkey CYP3A8 was more than 3.34-fold higher than that of human CYP3A4 (Table

5

2), the species differences of intestinal absorption of these CYP3A substrates between

6

monkey and human could be caused by differences of not only the protein expression

7

levels, but also metabolizing activities per protein amount between monkey CYP3A8

8

and human CYP3A4.

9

BCRP and P-gp are involved in restricting the oral absorption of various drugs

10

in small intestine, and the intestinal absorption of substrates of BCRP and P-gp is

11

increased by coadministration of inhibitors of these transporters.1-5 Our findings indicate

12

that the protein expression level of BCRP is higher than that of P-gp in monkey and

13

human small intestine (Table 3). In contrast, our previous study showed that bcrp

14

expression was smaller than mdr1a (P-gp) expression in mouse small intestine.45 These

15

results suggest that BCRP plays a greater functional role relative to P-gp in monkey and

16

human small intestine as compared with mouse small intestine. P-gp and BCRP share

17

many substrates.27, 28 Thus, for compounds that are common substrates of P-gp and

18

BCRP, the influence of drug-drug interaction (DDI) at BCRP upon oral absorption may

19

be greater in monkey and human than in mouse.

20

BCRP protein expression in jejunum was significantly higher in monkey than

21

in human (Table 3). Therefore, BCRP may have a greater influence on small intestinal

22

absorption in monkey than in human. Indeed, bioavailability of sulfasalazine, which is a

23

selective substrate for BCRP, was reported to be smaller in monkey (2.75 %) than in

24

human (8 %),55,

56

suggesting that the BCRP efflux activity for orally administered 20 ACS Paragon Plus Environment

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1

sulfasalazine is higher in monkey than in human. The difference of BCRP protein

2

expression levels in jejunum between monkey and human might therefore account for

3

the species difference of oral absorption of this BCRP substrate.

4

As regards P-gp, we found no species difference of P-gp expression levels in

5

jejunum and ileum between monkey and human (Table 3). The influence of P-gp on

6

intestinal absorption is often investigated by measuring changes of the plasma

7

concentration of probe substrates upon coadministration with P-gp inhibitors. The

8

plasma AUC of fexofenadine (a P-gp substrate) after oral administration was increased

9

2.6-fold by coadministration with ketoconazole (a P-gp inhibitor) in both monkey and

10

human2, 57 This suggests that P-gp has similar effects on fexofenadine absorption in

11

monkey and human, supporting our findings regarding the protein expression

12

differences of P-gp between monkey and human.

13

The OSTα and OSTβ heterodimer is known to mediate transport activity,10 but

14

in the present work, we were not able to design specific quantifiable peptides for OSTβ

15

in human, so we quantified only OSTα in human. In monkey small intestine, OSTα and

16

OSTβ were more highly expressed than MRP3 and MRP4 (Table 3). The previous

17

QTAP study in human jejunum found that the protein expression levels of OSTα and

18

OSTβ was greater than those of MRP1, 4, 5.15 In the present study, there were

19

differences of relative OSTα and MRP family protein expression levels between the two

20

human donors. In jejunum and ileum of human donor #2, the protein expression levels

21

of OSTα were higher than those of MRP1, MRP3, MRP4, and MRP5, as in monkey

22

(Table 3). In contrast, the protein expression level of OSTα was almost equal to that of

23

MRP4 in the ileum of human donor #1 (Table 3). In the jejunum of human donor #1, the

24

expression level of OSTα was under the limit of quantification, but comparison between 21 ACS Paragon Plus Environment


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1

OSTα and MRP4 expression levels is problematic because the limit of quantification of

2

OSTα (0.557 fmol/µg plasma membrane protein) is higher than the protein expression

3

level of MRP4 (0.323 fmol/µg plasma membrane protein), which was expressed most

4

abundantly among MRP family members in jejunum of human donor #1 (Tables 3 and

5

S6). It has been reported that OSTα/β recognizes digoxin as a substrate, and

6

OSTα/β-mediated transport activity of estrone 3-sulfate was inhibited by various

7

organic anionic drugs.10 Hence, OSTα/β might play a role in the oral absorption of

8

anionic drugs. Here, we found that OSTα was more highly expressed in monkey than in

9

both human donors, indicating that the transport rate of OSTα/β substrate in oral

10

absorption might be greater in monkey than in human.

11

Conflicting data about the intestinal expressions of OATP1A2 and OATP1B3

12

have been reported. Glaseser et al. showed that OATP1A2 is expressed in apical

13

membrane of human small intestinal epithelial cells by immunostaining,58 whereas

14

Gröer et al. found no expression of OATP1A2 in the crude membrane fraction of human

15

small intestine by means of QTAP analysis.41 In the present study, protein expression of

16

OATP1A2 was detected in jejunum and ileum of human donor #2 in the plasma

17

membrane fraction, in which OATP1A2 would be concentrated, compared with the

18

crude membrane fraction (Table 3). However, the expression level was quite small

19

(0.336 and 0.189 fmol/µg plasma membrane protein in jejunum and ileum, respectively;

20

Table 3). These data suggest that OATP1A2 protein is expressed in the small intestine,

21

but its level is very low, so that it may be below the detection limit, depending on the

22

experimental method employed. OATP1B3 could be also expressed in the small

23

intestine, because its gene expression was detected58 and its protein expression was also

24

detected in the present study (Table 3). However, Miyauchi et al. reported that, even in 22 ACS Paragon Plus Environment

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1

the plasma membrane fraction, the OATP1A2 and OATP1B3 expression levels were

2

below the limit of quantification in QTAP analysis.15 The probe peptides they used were

3

different from our peptides, among many tryptic peptides formed from these transporter

4

proteins. Therefore, differences in digestion efficacy with lysyl endopeptidase and

5

trypsin might account for the different results between their study and our study.

6

Glucose and short-chain fatty acids (e.g., butyric acid, lactic acid, and pyruvic

7

acid) are important physiological energy sources. Hexose transporters SGLT1 (a glucose

8

transporter) and GLUT5 (a fructose transporter) were highly expressed in monkey and

9

human small intestine (Table 4). An earlier QTAP study also found that the protein

10

expression levels of SGLT1 and GLUT5 were greater than that of MCT1 in human

11

jejunum.15 In contrast, we previously found that the expression level of mct1, which

12

transports short-chain fatty acids, was greater than that of sglt1 in mouse small

13

intestine.45 Dietary fibers are digested by intestinal bacteria to generate short-chain fatty

14

acids, and the population density of intestinal bacteria in small intestine of mouse is

15

greater than that in human,59 suggesting that short-chain fatty acid production might be

16

greater in mouse small intestine than in human small intestine. Therefore, the expression

17

differences of hexose and monocarboxylate transporters in monkey, human, and mouse

18

could be favorable for effective absorption of energy sources in the respective species.

19

It is important to consider regional differences of protein expression of

20

metabolic enzymes and transporters, because these are expected to influence drug

21

absorption sites in the small intestine. Several protein quantification studies of CYPs,

22

UGTs, and transporters in human small intestine have previously been done using

23

LC-MS/MS.14, 15, 19, 40-44

24

protein expression differences using jejunum and ileum obtained from the same human

However, the present study is the first to determine regional



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1

donors. The P-gp protein expression level in monkey was higher in ileum than in

2

duodenum and jejunum (Table 3). Nishimura et al. reported that the efflux activities of

3

P-gp substrates etoposide and digoxin in monkey small intestine were greater in ileum

4

than in duodenum and jejunum.60 Thus, there appears to be a good correlation between

5

regional differences in P-gp protein expression and function. We found here that the

6

protein expression levels of P-gp were higher in ileum than in jejunum in both human

7

donors (Table 3). Therefore, it appears that higher expression of P-gp in the lower

8

segment of small intestine is a common feature in monkey and human. In addition, we

9

found regional differences of expression of monkey CYP3A8 and human CYP3A4,

10

respectively. Monkey CYP3A8 was more highly expressed in duodenum and jejunum

11

than in ileum (Table 2). Since microsomes from the upper segment of monkey small

12

intestine show higher metabolic activity towards CYP3A substrates,61, 62 it appears that

13

regional differences of CYP3A activity tend to be in agreement with those of CYP3A8

14

protein expression. In human small intestine, several studies have found that protein

15

expression and activity of CYP3A4 are higher in jejunum than in ileum.63, 64 However,

16

Paine et al. found interindividual variability of the intestinal segment showing peak

17

CYP3A activity in human donors.63 In the present study, CYP3A4 expression was

18

slightly higher in ileum than in jejunum in both human donors (Table 2). Thus, our

19

results imply that the regional expression pattern of human CYP3A4 is different from

20

that of monkey CYP3A8, although the possibility of interindividual regional differences

21

in human CYP3A4 expression cannot be ruled out.

22

The present quantitative analyses were performed using small numbers of

23

samples (from one male monkey and two human donors). Further, there were

24

differences of age, sex, and disease status between the two human donors, and these 24 ACS Paragon Plus Environment

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1

differences might account in large part for the inter-individual differences observed in

2

the present study. Hence, we compared the inter-individual differences in our study with

3

previous protein quantification or functional analysis data of CYPs, UGTs, and

4

transporters in human small intestine, to see whether the differences are within a

5

reasonable range.

6

Miyauchi et al. quantified the protein expression levels of CYPs and UGTs in

7

microsomal fractions and transporters in plasma membrane fractions in the jejuna of

8

24−28 morbidly obese human subjects by using LC-MS/MS.15 The inter-individual

9

differences in the protein expression levels of CYPs, UGTs, and transporters in jejuna of

10

human donors #1 and #2 in the present study are compared with those in Miyauchi’s

11

subjects in Table S9. Twenty molecules were detected in jejuna of both donors in our

12

study, and in at least one of Miyauchi’s subjects (CYP3A4, CYP2C9, CYP51A1,

13

UGT2B17, P-gp, MRP1, MRP2, MRP4, BCRP, ABCG8, GLUT5, 4F2hc, SGLT1,

14

TAUT, PEPT1, MCT1, CNT2, PCFT, villin-1, and Na+/K+-ATPase). Among them, 14

15

molecules (CYP3A4, CYP51A1, UGT2B17, P-gp, MRP1, MRP2, MRP4, BCRP,

16

SGLT1, PEPT1, MCT1, CNT2, PCFT, and Na+/K+-ATPase) showed expression

17

differences between human donors #1 and #2 that were smaller than the maximum

18

inter-individual differences reported by Miyauchi et al., while the other 6 (CYP2C9,

19

ABCG8, GLUT5, 4F2hc, TAUT, and villin-1) showed larger differences (Table S9).

20

These data might imply that the inter-individual differences of the 14 molecules

21

described above are within a reasonable range in our quantification study, whereas the

22

expression levels of other 6 molecules might have been influenced by disease status (all

23

the intestinal samples used by Miyauchi et al. were from morbidly obese subjects).

24

Paine et al. reported that the protein expression of CYP2C9 and 3A4 showed 9.3- and 25 ACS Paragon Plus Environment


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1

17.0-fold differences in 31 human donors by means of quantitative western blotting,13

2

and Obach et al. reported that the activities of CYP2C9 and 3A4 showed 17.7- and

3

6.8-fold differences among 10 human donors by means of metabolic studies using

4

intestinal microsomes.65 In our study, the expression differences of CYP2C9 and 3A4 in

5

the two donors were 7.38- and 1.59-fold in jejunum and 4.47- and 1.60-fold in ileum,

6

respectively (Table 2), which are relatively small. Additionally, although protein

7

expressions of UGT1A1, 1A6, and 2B7 were not detected in donor #1 in our study

8

(Table 2), Strassburg et al. also reported that not all donors showed gene expressions of

9

UGT1A1, 1A6, and 2B7.18 Berggern et al. reported that the protein expression levels of

10

P-gp and MRP2 normalized by that of villin-1 showed approximately 10-fold and 2-fold

11

differences in jejuna from three human subjects,34 and these differences are larger than

12

the differences of normalized expression levels of P-gp (1.07-fold) and MRP2

13

(1.82-fold) in our study (Table S7). These data at least suggest that the inter-individual

14

differences in our study lie within a reasonable range. However, in order to confirm the

15

species differences between monkey and human, it will be necessary to conduct studies

16

using intestinal samples from larger numbers of monkeys and humans with differences

17

of gender, age, and disease status.

18

In the present study, we determined the expression levels of CYPs and UGTs

19

in microsomal membrane fractions, and those of transporters in plasma membrane

20

fractions. We prepared microsomal membrane fraction from whole-cell lysate, and

21

subsequently prepared plasma membrane fraction from microsomal membrane fraction.

22

However, some degree of contamination (e.g. cytosol in microsomal fraction) is

23

inevitable. Wegler et al. suggested that this contamination would cause underestimation

24

of the quantified protein expression levels, and suggested that protein quantifications 26 ACS Paragon Plus Environment

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1

should be performed with whole-cell lysate to avoid artifacts due to the membrane

2

preparation procedure.46 On the other hand, the protein expression levels of metabolic

3

enzymes and transporters in whole-cell lysate include digested / inactive protein in

4

cytosol, so the expression data may be overestimated. As described in Supporting

5

Information, it is theoretically possible to correct the quantitative values in pure

6

membrane fraction by taking account of contamination with other fractions, using the

7

quantitative values of marker proteins such as illin-1 (non-membrane marker) and

8

CYP3A4 (microsomal membrane marker). Unfortunately, we did not collect whole-cell

9

lysate samples in the present study, but this approach might be useful in the future for

10

comparing protein expression levels between different samples or species.

11

In conclusion, we have determined the protein expression levels of CYPs,

12

UGTs, and transporters in small intestine in monkey, and compared them with the

13

corresponding levels in human. We found that the jejunal and ileal expression levels of

14

UGT1A1 and 1A6 were greater in monkey than in human, and the jejunal expression

15

level of monkey CYP3A8 was greater than that of human CYP3A4. These species

16

difference of protein expression levels between monkey and human should be taken into

17

account in considering possible species differences in absorption of substrates of these

18

metabolic enzymes after oral administration in monkey and human. Among apical

19

efflux transporters, BCRP was more highly expressed than P-gp in monkey and human.

20

These findings should be helpful for understanding the functions of metabolic enzymes

21

and transporters in monkey small intestine and the differences from those in human. We

22

believe our quantification of UGT1A1/1A6 is particularly important. Since the substrate

23

specificities of UGT isoforms often overlap, it is difficult to identify isoforms

24

responsible for species differences of intestinal glucuronidation by means of functional 27 ACS Paragon Plus Environment


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1

studies using UGT substrates. Our findings on the expression differences of UGT1A1

2

and 1A6 between monkey and human could help to resolve this issue, and suggest that

3

the contributions of UGT1A1 and 1A6 to intestinal metabolism would be greater in

4

monkey than in human.


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1

ASSOCIATED CONTENT

2

Supporting Information

3

The Supporting Information is available free of charge on the ACS Publications

4

website.

5

Theoretical approach for determination of true quantitative values of proteins in pure

6

membrane fraction.

7

Table S1. Peptide probes and SRM/MRM transitions for quantification of monkey and

8

human molecules by using triple quadrupole mass spectrometer

9

Table S2. Peptide probes and SRM/MRM transitions for quantification of monkey and

10

human molecules by using triple TOF 5600

11

Table S3. Molecules under the limit of quantification in microsomal membrane fractions

12

of isolated epithelial cells in cynomolgus monkey small intestine

13

Table S4. Molecules under the limit of quantification in plasma membrane fractions of

14

isolated epithelial cells in cynomolgus monkey small intestine

15

Table S5. Molecules under the limit of quantification in microsomal membrane fractions

16

of isolated epithelial cells in human small intestine

17

Table S6. Molecules under the limit of quantification in plasma membrane fractions of

18

isolated epithelial cells in human small intestine

19

Table S7. Protein expression levels of drug transporters normalized by villin-1 in

20

plasma membrane fractions in monkey and human small intestine

21

Table S8. Protein expression levels of endogenous transporters normalized by villin-1 in

22

plasma membrane fractions in monkey and human small intestine

23

Table S9. Range of inter-individual expression differences in human jejunum

24



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1

AUTHOR INFORMATION

2

Corresponding Author

3

Tetsuya Terasaki

4

* Phone: +81-22-795-6831. Fax: +81-22-795-6886.

5

E-mail:[email protected].

6 7

Note

8

Regarding conflicts of interest, Tetsuya Terasaki and Sumio Ohtsuki are full professors

9

at Tohoku University and Kumamoto University, respectively, and are also directors of

10

Proteomedix Frontiers Co., Ltd. This study was not supported by Proteomedix Frontiers

11

Co., Ltd., and their positions at Proteomedix Frontiers Co., Ltd. did not influence the

12

design of the study, the collection of the data, the analysis or interpretation of the data,

13

the decision to submit the manuscript for publication, or the writing of the manuscript,

14

and did not present any financial conflicts. The other authors declare no competing

15

interests.

16 17

ACKNOWLEDGEMENTS

18

We thank Ms. Akiko Niitomi for her secretarial assistance. This study was supported in

19

part by three Grants-in-Aid from the Japan Society for the Promotion of Science (JSPS)

20

for Young Scientists (A) [KAKENHI: 16H06218], Scientific Research (S) [KAKENHI:

21

18109002] and Scientific Research (A) [KAKENHI: 24249011], and also supported in

22

part by the Nakatomi Foundation.

23 24

ABBREVIATIONS USED 30 ACS Paragon Plus Environment

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1

4F2hc, 4F2 cell-surface antigen heavy chain; ABC, ATP-binding cassette; ASBT, apical

2

sodium-dependent bile acid transporter; BCRP, breast cancer resistance protein; CNT,

3

concentrative nucleoside transporter; CYP, cytochrome P450; γ-gtp, γ-glutamyl

4

transpeptidase; ENT, equilibrative nucleoside transporter; GLUT, glucose transporter;

5

HPLC,

6

chromatography/tandem mass spectrometry; LQ, limit of quantification; MATE,

7

multidrug and toxin extrusion protein; MCT, monocarboxylate transporter; MDR,

8

multidrug resistance protein; MRP, multidrug resistance-associated protein; OAT,

9

organic anion transporter; OATP, organic anion-transporting polypeptide; OCT, organic

10

cation transporter; OCTN, organic cation/carnitine transporter; OST, organic solute

11

transporter; P450R, NADPH-P450 reductase; PCFT, proton-coupled folate transporter;

12

PEPT, peptide transporter; SGLT, sodium glucose cotransporter; SLC, solute carrier;

13

SRM/MRM, selected/multiple reaction monitoring; TAUT, taurine transporter; UGT,

14

UDP-glucuronosyltransferase; ULQ, under limit of quantification.

high-performance

liquid

chromatography;


LC/MS/MS,

liquid


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Table 1. Sample information of monkey and human small intestine Species

Monkey

Human donor #1

Strain (monkey)

Age

Sex

Drug history

Notes

Cynomolgus

4 years 3 months

Male

None

Origin; Indonesia

Caucasian

74 years

Female

Novocaine,

Cause of death; COPD

Codeine

BMI; 21.5

Race (human)

Levofloxacin, Human donor #2

Vancomycin, Caucasian

22 years

Male

Haloperidol,

Prednisone, Cyclophosphamide

2 3

BMI, body-mass index; COPD, chronic obstructive pulmonary disease

4

The monkey was the same individual used in our previous report.47

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Cause of death; Wegener’s disease BMI; 27.0


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Table 2. Protein expression levels of CYPs and UGTs in microsomal membrane fractions in monkey and human small intestine Expression level (fmol/µg microsomal membrane protein) Molecule

Region

Monkey

Human #1

Human #2

CYP CYP3A8 (monkey) / CYP3A4 (human)

Duodenum Jejunum Ileum Duodenum

CYP2C9

CYP2C18

CYP2D6

CYP2J2

CYP4A11

30.0 ± 0.9## 32.9 ± 0.7

## ¶¶ ‡‡

9.97 ± 0.54** †† ¶ ‡ Not measured

Not measured

Not measured

# §§ ‡‡

6.20 ± 0.36

9.84 ± 0.43## §§ ¶¶

8.03 ± 0.40† § ‡‡

12.9 ± 0.5†† § ¶¶

Not measured

Not measured

Jejunum

Not measured

0.507 ± 0.093

3.74 ± 0.25¶¶

Ileum

Not measured

0.722 ± 0.084‡‡

3.23 ± 0.08¶¶

Duodenum

Not measured

Not measured

Not measured

Jejunum

Not measured

ULQ (

1A6 in Monkey Small Intestine

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