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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,
3
and Transporters in Small Intestine of Cynomolgus Monkey and Human
4 5
Takanori Akazawa†, Yasuo Uchida†, Eisuke Miyauchi†, Masanori Tachikawa†, Sumio
6
Ohtsuki‡, and Tetsuya Terasaki*,†
7 8
†
9
Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-ku, Sendai
Division of Membrane Transport and Drug Targeting, Graduate School of
10
980-8578, Japan
11
‡
12
University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto 862-0973, Japan
Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto
13
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
3
humans. The purpose of this study was to clarify the regional protein expression levels
4
of cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UGTs), and transporters
5
in small intestine of cynomolgus monkey using liquid chromatography–tandem mass
6
spectrometry, and to compare them with the corresponding levels in human. UGT1A1 in
7
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
11
transporters, BCRP showed the most abundant expression in monkey and human, and
12
the expression levels of BCRP in monkey and human were >1.74- and >1.25-fold
13
greater than those of P-gp, and >2.76- and >4.50-fold greater than those of MRP2,
14
respectively. These findings should be helpful to understand species differences of the
15
functions of CYPs, UGTs, and transporters between monkey and human. The
16
UGT1A1/1A6 data would be especially important, because it is difficult to identify
17
isoforms responsible for species differences of intestinal glucuronidation by means of
18
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
3
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
5
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
8
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
12
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
15
of drugs remains unclear in human. In addition to drug transporters, metabolic enzymes,
16
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
19
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
22
Since the substrate specificities of UGT isoforms often overlap, multiple UGT isoforms
23
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
6
prediction of the pharmacokinetics of candidate drugs in humans during drug
7
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
11
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
13
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
15
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
19
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
21
monkey than in human.22 Therefore, a quantitative understanding of species differences
22
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
2
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
12
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
14
allows us to determine the protein expression levels of target molecules for which a
15
target-specific peptide can be selected for quantitative analysis by using the amino acid
16
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
18
intestine by means of the QTAP technique.14,
19
quantifications were performed by using several types of protein samples, such as
20
whole-cell lysate and microsomal / plasma membrane fractions. It is important to select
21
appropriate types of protein samples for a proper consideration of the functions of
22
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
24
are the plasma membrane. Since the protein expression levels of metabolic enzymes and
15, 19, 40-44
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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,
6
comprehensive quantification of protein expression levels at the sites of the functional
7
activities should be an effective strategy to understand species differences in the
8
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
10
levels of CYPs, UGTs, and transporters in small intestine of cynomolgus monkey, and
11
to compare them with the corresponding levels in human. The results are expected to
12
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
15
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
17
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
19
transporters in human jejunum and ileum using the same procedures established for
20
cynomolgus monkey.
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EXPERIMENTAL SECTION
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Reagents. Unlabeled (standard) and stable-isotope labeled peptides (internal
3
standard) listed in Table S1 and S2 were designed according to our previous reports,39
4
and synthesized by Thermo Electron Corporation (Sedanstrabe, Germany) with > 95%
5
peptide purity. The concentrations of peptide solutions were determined by means of
6
quantitative amino acid analysis, using an HPLC-UV system with post-column
7
ninhydrin derivatization (Lachrom Elite, Hitachi, Tokyo, Japan). Other chemicals were
8
commercial analytical-grade products.
9 10
Monkey small intestine. Adult cynomolgus monkey (4 years 3 months after
11
birth, male), which had originated from Indonesia (Table 1), was purchased from Shin
12
Nippon Biomedical Laboratories, Ltd. (Kagoshima, Japan). This monkey was the same
13
individual used in our previous report.47 We previously quantified the protein expression
14
levels of transporters in small intestine of male mice by the same methods used in the
15
present study.45 In order to allow comparison of the data in monkey with the reported
16
data in mice, we used only male monkeys in this study, to rule out possible effects of
17
gender difference. Duodenum, jejunum, and ileum were excised at Shin Nippon
18
Biomedical Laboratories, Ltd. as follows. The monkey was anesthetized with
19
pentobarbital (25.9 mg/kg weight) and perfused with saline containing sodium heparin
20
to remove blood. After the perfusion, the duodenum, jejunum, and ileum were excised
21
and frozen in liquid nitrogen. Duodenum was defined as the region from the pylorus end
22
point to the pancreatic duct opening together with the same length from the pancreatic
23
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
2
the most central 5 cm pieces of duodenum, jejunum, and ileum as analytical samples in
3
the present study. The intestine was stored at −80 °C, shipped to Tohoku University on
4
dry ice, and stored at −80 °C until isolation of epithelial cells. The protocols were
5
performed in accordance with the animal welfare requirements of the Drug Safety
6
Research Laboratories, Shin Nippon Biomedical Laboratories, Ltd., and were approved
7
by the Institutional Animal Care and Use Committee.
8 9
Human small intestine. Protein quantification in human intestine was
10
performed with two different donor intestines, to take account of individual differences
11
in human samples. Frozen jejuna and ilea of two human donors (jejunum and ileum of
12
donor #1, and jejunum and ileum of donor #2; intra-subject; Table 1) were purchased
13
from Analytical Biological Services (Wilmington, DE, USA). Although human jejunum
14
and ileum are organs of several meters length, detailed information about the positions
15
of the excised sites of the jejunum and ileum we used was not available from the
16
supplier (Analytical Biological Services). The intestine was shipped to Tohoku
17
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
19
of Tohoku University School of Medicine and the Graduate School of Pharmaceutical
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Sciences, Tohoku University.
21 22 23 24
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
2
according to our previous report,45 and we subsequently prepared individual microsomal
3
and plasma membrane fractions from small intestine of the monkey and the two human
4
donors according to our previous reports.15
5 6
Protein Quantification by Using LC-MS/MS. Membrane samples were
7
digested with lysyl endopeptidase and trypsin to afford peptides, and protein expression
8
levels were determined by quantification of specific peptides from the target molecules
9
by using LC-MS/MS according to our previous report.45 The mass spectrometer was a
10
triple quadrupole mass spectrometer (API5000 or QTRAP5500; AB SCIEX,
11
Framingham, MA, USA) or a quadrupole time-of-flight mass spectrometer (Triple
12
TOF5600; AB SCIEX). Although we used different mass spectrometers (API5000,
13
QTRAP5500, and TripleTOF5600) for the quantification of transporter proteins, we
14
considered that the measured protein expression levels were not instrument-dependent,
15
based on our previous comparison of the transporter expression levels in human liver,
16
kidney, and intestinal microsomes quantified by using API5000 (triple quadrupole mass
17
spectrometer) and TripleTOF5600 (quadrupole time-of-flight mass spectrometer)
18
instruments.44 The differences of 11 transporters (ABCA8, ABCC2, ABCC6, SLC5A1,
19
SLC7A8, SLC16A1, SLC22A6, SLC22A8, SLC22A13, SLC22A18, and SLC27A2)
20
were all within 1.35-fold between the two mass spectrometers. Several monkey
21
molecules (Table S1) and human Na+/K+-ATPase were quantified by triple quadrupole
22
mass spectrometry with an Agilent 1100 HPLC system (Agilent Technologies, Santa
23
Clara, CA, USA) coupled to the API5000 or QTRAP5500 spectrometer equipped with a
24
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
2
peptides of the quantified molecules and their SRM/MRM transitions are listed in Table
3
S1. The measurement and analysis were carried out according to our previous report.48
4
Other monkey and human molecules (Table S2) were quantified by employing
5
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
8
NanoLC-Ultra 1D plus system (Eksigent Technologies, Dublin, CA, USA) coupled with
9
a cHiPLC-nanoflex system (Eksigent Technologies) and a Triple-TOF5600 (AB
10
SCIEX) equipped with a NanoSpray III ion source (AB SCIEX). NanoLC was
11
performed with a trap column (Nano cHiPLC Trap column 200 µm x 0.5 mm ChromXP
12
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.
17
In this study, we could not determine the protein expression levels of some
18
metabolic enzymes and transporters, such as CYP2C19, OATP2B1, and OSTβ, that
19
have been reported to be expressed in the small intestine.13-15, 41, 43 The reason for this
20
was that the synthesized probe peptides (OATP2B1 ； VLLQTLR ， CYP2C19 ；
21
GHFPLAER ， OSTβ ； DVLSVFLPDVPETES) were not detected in the present
22
LC-MS/MS system, and so we could not generate a calibration curve. However, the
23
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
2
directly loaded onto the main analytical column (Nano cHiPLC column 75 µm × 15 cm
3
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
5
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
10
failure of detection in the mass spectrometer.
11
For measurement and analysis, each peptide for a target protein was monitored
12
with four kinds of SRM/MRM transitions. When positive peaks were observed in three
13
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
15
(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
20 21
Statistical Analysis. The statistical significance of differences between two
22
groups was determined by Student’s t-test and that among three groups was determined
23
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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RESULTS
2
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,
11
CYP3A8 and NADPH-cytochrome P450 reductase (P450R) showed higher expression
12
in duodenum and jejunum than in ileum; their protein expression levels in duodenum
13
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
21
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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1
proteins that were not detected in microsomal membrane fractions of human small
2
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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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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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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Molecular Pharmaceutics
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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Molecular Pharmaceutics
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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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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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
29 ACS Paragon Plus Environment
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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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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;
31 ACS Paragon Plus Environment
LC/MS/MS,
liquid
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1
REFERENCES
2
(1)
3
transporters: an overview. Adv Drug Deliv Rev 2013, 65, (10), 1340-56.
4
(2)
5
seasonal allergic rhinitis and chronic idiopathic urticaria. Drugs 2000, 59, (2), 301-21.
6
(3)
7
Itraconazole, a P-glycoprotein and CYP3A4 inhibitor, markedly raises the plasma
8
concentrations and enhances the renin-inhibiting effect of aliskiren. J Clin Pharmacol
9
2011, 51, (3), 359-67.
Estudante, M.; Morais, J. G.; Soveral, G.; Benet, L. Z. Intestinal drug
Simpson, K.; Jarvis, B. Fexofenadine: a review of its use in the management of
Tapaninen, T.; Backman, J. T.; Kurkinen, K. J.; Neuvonen, P. J.; Niemi, M.
10
(4)
Delavenne, X.; Ollier, E.; Basset, T.; Bertoletti, L.; Accassat, S.; Garcin, A.;
11
Laporte, S.; Zufferey, P.; Mismetti, P. A semi-mechanistic absorption model to evaluate
12
drug-drug interaction with dabigatran: application with clarithromycin. Br J Clin
13
Pharmacol 2012, 76, (1), 107-13.
14
(5)
15
Fukizawa, S.; Morimoto, N.; Ieiri, I.; Morishita, M.; Sumita, K.; Mayahara, H.; Fujita,
16
T.; Maeda, K.; Sugiyama, Y. Pharmacokinetic interaction study of sulphasalazine in
17
healthy subjects and the impact of curcumin as an in vivo inhibitor of BCRP. Br J
18
Pharmacol 2012, 166, (6), 1793-803.
19
(6)
20
Niemi, M. ABCG2 polymorphism markedly affects the pharmacokinetics of atorvastatin
21
and rosuvastatin. Clin Pharmacol Ther 2009, 86, (2), 197-203.
22
(7)
23
Christian, W. V.; Ballatori, N. The heteromeric organic solute transporter alpha-beta,
24
Ostalpha-Ostbeta, is an ileal basolateral bile acid transporter. J Biol Chem 2005, 280, (8),
Kusuhara, H.; Furuie, H.; Inano, A.; Sunagawa, A.; Yamada, S.; Wu, C.;
Keskitalo, J. E.; Zolk, O.; Fromm, M. F.; Kurkinen, K. J.; Neuvonen, P. J.;
Dawson, P. A.; Hubbert, M.; Haywood, J.; Craddock, A. L.; Zerangue, N.;
32 ACS Paragon Plus Environment
Page 32 of 55
Page 33 of 55
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Molecular Pharmaceutics
1
6960-8.
2
(8)
3
Increasing systemic exposure of methotrexate by active efflux mediated by multidrug
4
resistance-associated protein 3 (mrp3/abcc3). J Pharmacol Exp Ther 2008, 327, (2),
5
465-73.
6
(9)
7
Oude Elferink, R. P. Oral availability of cefadroxil depends on ABCC3 and ABCC4.
8
Drug Metab Dispos 2012, 40, (3), 515-21.
9
(10)
Kitamura, Y.; Hirouchi, M.; Kusuhara, H.; Schuetz, J. D.; Sugiyama, Y.
de Waart, D. R.; van de Wetering, K.; Kunne, C.; Duijst, S.; Paulusma, C. C.;
Seward, D. J.; Koh, A. S.; Boyer, J. L.; Ballatori, N. Functional
10
complementation between a novel mammalian polygenic transport complex and an
11
evolutionarily ancient organic solute transporter, OSTalpha-OSTbeta. J. Biol. Chem.
12
2003, 278, (30), 27473-82.
13
(11)
14
human gut wall. J Pharm Pharmacol 2009, 61, (5), 541-58.
15
(12)
16
biological responses to drugs and xenobiotics. Expert Opin Drug Metab Toxicol 2007, 3,
17
(1), 93-107.
18
(13)
19
D. C. The human intestinal cytochrome P450 "pie". Drug Metab Dispos 2006, 34, (5),
20
880-6.
21
(14)
22
Drozdzik, M.; Siegmund, W.; Oswald, S. Absolute protein quantification of clinically
23
relevant cytochrome P450 enzymes and UDP-glucuronosyltransferases by mass
24
spectrometry-based targeted proteomics. J Pharm Biomed Anal 2014, 100, 393-401.
Thelen, K.; Dressman, J. B. Cytochrome P450-mediated metabolism in the
Ritter, J. K. Intestinal UGTs as potential modifiers of pharmacokinetics and
Paine, M. F.; Hart, H. L.; Ludington, S. S.; Haining, R. L.; Rettie, A. E.; Zeldin,
Groer, C.; Busch, D.; Patrzyk, M.; Beyer, K.; Busemann, A.; Heidecke, C. D.;
33 ACS Paragon Plus Environment
Molecular Pharmaceutics
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
1
(15)
Miyauchi, E.; Tachikawa, M.; Decleves, X.; Uchida, Y.; Bouillot, J. L.; Poitou,
2
C.; Oppert, J. M.; Mouly, S.; Bergmann, J. F.; Terasaki, T.; Scherrmann, J. M.;
3
Lloret-Linares, C. Quantitative atlas of cytochrome P450, UDP-glucuronosyltransferase,
4
and transporter proteins in jejunum of morbidly obese subjects. Mol. Pharmaceutics
5
2016, 13, (8), 2631-40.
6
(16)
7
J. P.; Barr, D. M.; Gillies, B. S.; Thummel, K. E. First-pass metabolism of midazolam
8
by the human intestine. Clin Pharmacol Ther 1996, 60, (1), 14-24.
9
(17)
Paine, M. F.; Shen, D. D.; Kunze, K. L.; Perkins, J. D.; Marsh, C. L.; McVicar,
Rashid, T. J.; Martin, U.; Clarke, H.; Waller, D. G.; Renwick, A. G.; George, C.
10
F. Factors affecting the absolute bioavailability of nifedipine. Br J Clin Pharmacol 1995,
11
40, (1), 51-8.
12
(18)
13
R. H.; Manns, M. P. Polymorphic gene regulation and interindividual variation of
14
UDP-glucuronosyltransferase activity in human small intestine. J Biol Chem 2000, 275,
15
(46), 36164-71.
16
(19)
17
P. C. Quantification of human uridine-diphosphate glucuronosyl transferase 1A isoforms
18
in liver, intestine, and kidney using nanobore liquid chromatography-tandem mass
19
spectrometry. Anal. Chem. 2012, 84, (1), 98-105.
20
(20)
21
CYPs. Xenobiotica 2009, 39, (8), 578-81.
22
(21)
23
evaluation of extrapolation of intravenous pharmacokinetic parameters from rat, dog,
24
and monkey to humans. I. Clearance. Drug Metab. Dispos. 2004, 32, (6), 603-11.
Strassburg, C. P.; Kneip, S.; Topp, J.; Obermayer-Straub, P.; Barut, A.; Tukey,
Harbourt, D. E.; Fallon, J. K.; Ito, S.; Baba, T.; Ritter, J. K.; Glish, G. L.; Smith,
Iwasaki, K.; Uno, Y. Cynomolgus monkey CYPs: a comparison with human
Ward, K. W.; Smith, B. R. A comprehensive quantitative and qualitative
34 ACS Paragon Plus Environment
Page 34 of 55
Page 35 of 55
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Molecular Pharmaceutics
1
(22)
Takahashi, M.; Washio, T.; Suzuki, N.; Igeta, K.; Yamashita, S. The species
2
differences of intestinal drug absorption and first-pass metabolism between cynomolgus
3
monkeys and humans. J. Pharm. Sci. 2009, 98, (11), 4343-53.
4
(23)
5
Animal versus human oral drug bioavailability: do they correlate? Eur. J. Pharm. Sci.
6
2014, 57, 280-91.
7
(24)
8
comparison of pharmacokinetics between humans and monkeys. Drug Metab. Dispos.
9
2010, 38, (2), 308-16.
Musther, H.; Olivares-Morales, A.; Hatley, O. J.; Liu, B.; Rostami Hodjegan, A.
Akabane, T.; Tabata, K.; Kadono, K.; Sakuda, S.; Terashita, S.; Teramura, T. A
10
(25)
Zhang, Y.; Benet, L. Z. The gut as a barrier to drug absorption: combined role
11
of cytochrome P450 3A and P-glycoprotein. Clin. Pharmacokinet. 2001, 40, (3),
12
159-68.
13
(26)
14
UDP-glucuronosyltransferases. Methods Enzymol. 2005, 400, 104-16.
15
(27)
16
Quantitative evaluation of the impact of active efflux by p-glycoprotein and breast
17
cancer resistance protein at the blood-brain barrier on the predictability of the unbound
18
concentrations of drugs in the brain using cerebrospinal fluid concentration as a
19
surrogate. J. Pharmacol. Exp. Ther. 2011, 339, (3), 935-44.
20
(28).
21
C. The need for human breast cancer resistance protein substrate and inhibition
22
evaluation in drug discovery and development: why, when, and how? Drug Metab.
23
Dispos. 2014, 42, (9), 1466-77.
24
(29)
Court, M. H. Isoform-selective probe substrates for in vitro studies of human
Kodaira, H.; Kusuhara, H.; Fujita, T.; Ushiki, J.; Fuse, E.; Sugiyama, Y.
Poirier, A.; Portmann, R.; Cascais, A. C.; Bader, U.; Walter, I.; Ullah, M.; Funk,
Nishimura, M.; Koeda, A.; Morikawa, H.; Satoh, T.; Narimatsu, S.; Naito, S. 35 ACS Paragon Plus Environment
Molecular Pharmaceutics
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
1
Tissue-specific mRNA expression profiles of drug-metabolizing enzymes and
2
transporters in the cynomolgus monkey. Drug Metab. Pharmacokinet. 2009, 24, (2),
3
139-44.
4
(30)
5
distribution of mRNA expression of human cytochrome P450 isoforms assessed by
6
high-sensitivity real-time reverse transcription PCR. Yakugaku Zasshi 2003, 123, (5),
7
369-75.
8
(31)
9
phase I metabolizing enzymes except for cytochrome P450 and phase II metabolizing
Nishimura, M.; Yaguti, H.; Yoshitsugu, H.; Naito, S.; Satoh, T. Tissue
Nishimura, M.; Naito, S. Tissue-specific mRNA expression profiles of human
10
enzymes. Drug Metab. Pharmacokinet. 2006, 21, (5), 357-74.
11
(32)
12
ATP-binding cassette and solute carrier transporter superfamilies. Drug Metab.
13
Pharmacokinet. 2005, 20, (6), 452-77.
14
(33)
15
Kulkarni, A. V.; Hafey, M. J.; Evers, R.; Johnson, J. M.; Ulrich, R. G.; Slatter, J. G.
16
Expression profiles of 50 xenobiotic transporter genes in humans and pre-clinical
17
species: a resource for investigations into drug disposition. Xenobiotica 2006, 36,
18
(10-11), 963-88.
19
(34)
20
Schrenk, D.; Lennernas, H. Gene and protein expression of P-glycoprotein, MRP1,
21
MRP2, and CYP3A4 in the small and large human intestine. Mol. Pharmaceutics 2007,
22
4, (2), 252-7.
23
(35)
24
Modeling of human hepatic CYP3A4 enzyme kinetics, protein, and mRNA indicates
Nishimura, M.; Naito, S. Tissue-specific mRNA expression profiles of human
Bleasby, K.; Castle, J. C.; Roberts, C. J.; Cheng, C.; Bailey, W. J.; Sina, J. F.;
Berggren, S.; Gall, C.; Wollnitz, N.; Ekelund, M.; Karlbom, U.; Hoogstraate, J.;
Sy, S. K.; Ciaccia, A.; Li, W.; Roberts, E. A.; Okey, A.; Kalow, W.; Tang, B. K.
36 ACS Paragon Plus Environment
Page 36 of 55
Page 37 of 55
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Molecular Pharmaceutics
1
deviation from log-normal distribution in CYP3A4 gene expression. Eur. J. Clin.
2
Pharmacol. 2002, 58, (5), 357-65.
3
(36)
4
Ishiguro, N.; Kishimoto, W.; Ludwig-Schwellinger, E.; Ebner, T.; Terasaki, T.
5
Simultaneous absolute protein quantification of transporters, cytochromes P450, and
6
UDP-glucuronosyltransferases as a novel approach for the characterization of individual
7
human liver: comparison with mRNA levels and activities. Drug Metab Dispos 2012,
8
40, (1), 83-92.
9
(37)
Ohtsuki, S.; Schaefer, O.; Kawakami, H.; Inoue, T.; Liehner, S.; Saito, A.;
Hoffmeyer, S.; Burk, O.; von Richter, O.; Arnold, H. P.; Brockmoller, J.; Johne,
10
A.; Cascorbi, I.; Gerloff, T.; Roots, I.; Eichelbaum, M.; Brinkmann, U. Functional
11
polymorphisms of the human multidrug-resistance gene: multiple sequence variations
12
and correlation of one allele with P-glycoprotein expression and activity in vivo. Proc.
13
Natl. Acad. Sci. U S A 2000, 97, (7), 3473-8.
14
(38)
15
levels on the concentration-dependent permeability of drugs to the cell membrane. J.
16
Pharm. Sci. 2008, 97, (1), 553-65.
17
(39)
18
Sekine, Y.; Uchida, Y.; Ito, S.; Terasaki, T. Quantitative atlas of membrane transporter
19
proteins: development and application of a highly sensitive simultaneous LC/MS/MS
20
method combined with novel in-silico peptide selection criteria. Pharm. Res. 2008, 25,
21
(6), 1469-83.
22
(40)
23
Oppert, J. M.; Laplanche, J. L.; Mouly, S.; Scherrmann, J. M.; Uchida, Y.; Tachikawa,
24
M.; Terasaki, T.; Bergmann, J. F.; Decleves, X. Oral morphine pharmacokinetics in
Shirasaka, Y.; Sakane, T.; Yamashita, S. Effect of P-glycoprotein expression
Kamiie, J.; Ohtsuki, S.; Iwase, R.; Ohmine, K.; Katsukura, Y.; Yanai, K.;
Lloret-Linares, C.; Miyauchi, E.; Luo, H.; Labat, L.; Bouillot, J. L.; Poitou, C.;
37 ACS Paragon Plus Environment
Molecular Pharmaceutics
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 38 of 55
1
obesity: The role of P-glycoprotein, MRP2, MRP3, UGT2B7, and CYP3A4 jejunal
2
contents and obesity-associated biomarkers. Mol. Pharmaceutics 2016, 13, (3), 766-73.
3
(41)
4
Siegmund, W.; Oswald, S. LC-MS/MS-based quantification of clinically relevant
5
intestinal uptake and efflux transporter proteins. J. Pharm. Biomed. Anal. 2013, 85,
6
253-61.
7
(42)
8
Rostami-Hodjegan, A. Application of an LC-MS/MS method for the simultaneous
9
quantification of human intestinal transporter proteins absolute abundance using a
Groer, C.; Bruck, S.; Lai, Y.; Paulick, A.; Busemann, A.; Heidecke, C. D.;
Harwood, M. D.; Achour, B.; Russell, M. R.; Carlson, G. L.; Warhurst, G.;
10
QconCAT technique. J. Pharm. Biomed. Anal. 2015, 110, 27-33.
11
(43)
12
B.; Unadkat, J. D.; Siegmund, W.; Oswald, S. Protein abundance of clinically relevant
13
multidrug transporters along the entire length of the human intestine. Mol Pharm 2014,
14
11, (10), 3547-55.
15
(44)
16
W.; Terasaki, T.; Ohtsuki, S. Large-scale multiplex absolute protein quantification of
17
drug-metabolizing enzymes and transporters in human intestine, liver, and kidney
18
microsomes by SWATH-MS: Comparison with MRM/SRM and HR-MRM/PRM.
19
Proteomics 2016, 16, (15-16), 2106-17.
20
(45)
21
targeted
22
reconstruction of P-glycoprotein function in mouse small intestine. Mol. Pharm. 2016,
23
13, (7), 2443-56.
24
(46)
Drozdzik, M.; Groer, C.; Penski, J.; Lapczuk, J.; Ostrowski, M.; Lai, Y.; Prasad,
Nakamura, K.; Hirayama-Kurogi, M.; Ito, S.; Kuno, T.; Yoneyama, T.; Obuchi,
Akazawa, T.; Uchida, Y.; Tachikawa, M.; Ohtsuki, S.; Terasaki, T. Quantitative absolute
proteomics
of
transporters
and
pharmacoproteomics-based
Wegler, C.; Gaugaz, F. Z.; Andersson, T. B.; Wisniewski, J. R.; Busch, D.; 38 ACS Paragon Plus Environment
Page 39 of 55
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Molecular Pharmaceutics
1
Groer, C.; Oswald, S.; Noren, A.; Weiss, F.; Hammer, H. S.; Joos, T. O.; Poetz, O.;
2
Achour, B.; Rostami-Hodjegan, A.; van de Steeg, E.; Wortelboer, H. M.; Artursson, P.
3
Variability in Mass Spectrometry-based Quantification of Clinically Relevant Drug
4
Transporters and Drug Metabolizing Enzymes. Mol Pharm 2017, 14, (9), 3142-3151.
5
(47)
6
Kamiie, J.; Terasaki, T. Quantitative membrane protein expression at the blood-brain
7
barrier of adult and younger cynomolgus monkeys. J Pharm Sci 2011, 100, (9),
8
3939-50.
9
(48)
Ito, K.; Uchida, Y.; Ohtsuki, S.; Aizawa, S.; Kawakami, H.; Katsukura, Y.;
Uchida, Y.; Ohtsuki, S.; Katsukura, Y.; Ikeda, C.; Suzuki, T.; Kamiie, J.;
10
Terasaki, T. Quantitative targeted absolute proteomics of human blood-brain barrier
11
transporters and receptors. J. Neurochem. 2011, 117, (2), 333-45.
12
(49)
13
absolute proteomics of rat blood-cerebrospinal fluid barrier transporters: comparison
14
with a human specimen. J. Neurochem. 2015, 134, (6), 1104-15.
15
(50)
16
Fortlage, L. A.; Brown, M. B.; Guo, W.; Watkins, P. B. Grapefruit juice increases
17
felodipine oral availability in humans by decreasing intestinal CYP3A protein
18
expression. J Clin Invest 1997, 99, (10), 2545-53.
19
(51)
20
K.; Terashita, S.; Tabata, K.; Teramura, T. Species differences in intestinal
21
glucuronidation activities between humans, rats, dogs and monkeys. Xenobiotica 2014,
22
44, (3), 205-16.
23
(52)
24
Miners, J. O.; Mackenzie, P. I. Interindividual variability in acetaminophen
Uchida, Y.; Zhang, Z.; Tachikawa, M.; Terasaki, T. Quantitative targeted
Lown, K. S.; Bailey, D. G.; Fontana, R. J.; Janardan, S. K.; Adair, C. H.;
Furukawa, T.; Naritomi, Y.; Tetsuka, K.; Nakamori, F.; Moriguchi, H.; Yamano,
Court, M. H.; Duan, S. X.; von Moltke, L. L.; Greenblatt, D. J.; Patten, C. J.;
39 ACS Paragon Plus Environment
Molecular Pharmaceutics
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
1
glucuronidation by human liver microsomes: identification of relevant acetaminophen
2
UDP-glucuronosyltransferase isoforms. J Pharmacol Exp Ther 2001, 299, (3),
3
998-1006.
4
(53)
5
intestinal first-pass metabolism of CYP3A substrates in humans using cynomolgus
6
monkeys. Drug Metab Dispos 2010, 38, (11), 1967-75.
7
(54)
8
T.; Booth-Genthe, C. Characterization of the rhesus monkey CYP3A64 enzyme: species
9
comparisons of CYP3A substrate specificity and kinetics using baculovirus-expressed
Nishimuta, H.; Sato, K.; Mizuki, Y.; Yabuki, M.; Komuro, S. Prediction of the
Carr, B.; Norcross, R.; Fang, Y.; Lu, P.; Rodrigues, A. D.; Shou, M.; Rushmore,
10
recombinant enzymes. Drug Metab Dispos 2006, 34, (10), 1703-12.
11
(55)
12
S.; Hirouchi, M.; Watanabe, N.; Okudaira, N.; Izumi, T. Evaluation of the usefulness of
13
breast cancer resistance protein (BCRP) knockout mice and BCRP inhibitor-treated
14
monkeys to estimate the clinical impact of BCRP modulation on the pharmacokinetics
15
of BCRP substrates. Pharm. Res. 2015, 32, (5), 1634-47.
16
(56)
17
El-Kattan, A.; Troutman, M. D. Physicochemical space for optimum oral
18
bioavailability: contribution of human intestinal absorption and first-pass elimination. J
19
Med Chem 2010, 53, (3), 1098-108.
20
(57)
21
first-pass effect of midazolam and fexofenadine in cynomolgus monkeys. Drug Metab.
22
Dispos. 2007, 35, (3), 410-8.
23
(58)
24
McGrath, J. S.; Jolicoeur, E.; Lee, W.; Leake, B. F.; Tirona, R. G.; Kim, R. B. Intestinal
Karibe, T.; Hagihara-Nakagomi, R.; Abe, K.; Imaoka, T.; Mikkaichi, T.; Yasuda,
Varma, M. V.; Obach, R. S.; Rotter, C.; Miller, H. R.; Chang, G.; Steyn, S. J.;
Ogasawara, A.; Kume, T.; Kazama, E. Effect of oral ketoconazole on intestinal
Glaeser, H.; Bailey, D. G.; Dresser, G. K.; Gregor, J. C.; Schwarz, U. I.;
40 ACS Paragon Plus Environment
Page 40 of 55
Page 41 of 55
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Molecular Pharmaceutics
1
drug transporter expression and the impact of grapefruit juice in humans. Clin
2
Pharmacol Ther 2007, 81, (3), 362-70.
3
(59)
4
biochemistry of humans and commonly used laboratory animals. Biopharm. Drug
5
Dispos. 1995, 16, (5), 351-80.
6
(60)
7
Tsuji, A. Species difference in intestinal absorption mechanism of etoposide and digoxin
8
between cynomolgus monkey and rat. Pharm. Res. 2008, 25, (11), 2467-76.
9
(61)
Kararli, T. T. Comparison of the gastrointestinal anatomy, physiology, and
Nishimura, T.; Kato, Y.; Amano, N.; Ono, M.; Kubo, Y.; Kimura, Y.; Fujita, H.;
Nakanishi, Y.; Matsushita, A.; Matsuno, K.; Iwasaki, K.; Utoh, M.; Nakamura,
10
C.; Uno, Y. Regional distribution of drug-metabolizing enzyme activities in the liver and
11
small intestine of cynomolgus monkeys. Drug Metab. Pharmacokinet. 2011, 25, (3),
12
290-7.
13
(62)
14
K.; Kashiyama, E. Characterization of intestinal and hepatic P450 enzymes in
15
cynomolgus monkeys with typical substrates and inhibitors for human P450 enzymes.
16
Xenobiotica 2012, 42, (8), 719-30.
17
(63)
18
L.; Perkins, J. D.; Thummel, K. E. Characterization of interintestinal and intraintestinal
19
variations in human CYP3A-dependent metabolism. J Pharmacol Exp Ther 1997, 283,
20
(3), 1552-62.
21
(64)
22
Characterization of human small intestinal cytochromes P-450. Drug Metab Dispos
23
1999, 27, (7), 804-9.
24
(65)
Yoda, N.; Emoto, C.; Date, S.; Kondo, S.; Miyake, M.; Nakazato, S.; Umehara,
Paine, M. F.; Khalighi, M.; Fisher, J. M.; Shen, D. D.; Kunze, K. L.; Marsh, C.
Zhang, Q. Y.; Dunbar, D.; Ostrowska, A.; Zeisloft, S.; Yang, J.; Kaminsky, L. S.
Obach, R. S.; Zhang, Q. Y.; Dunbar, D.; Kaminsky, L. S. Metabolic 41 ACS Paragon Plus Environment
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
1
characterization of the major human small intestinal cytochrome p450s. Drug Metab
2
Dispos 2001, 29, (3), 347-52.
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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 (
