1521-009X/43/8/1208–1217$25.00
DRUG METABOLISM AND DISPOSITION
Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/dmd.115.063479
Drug Metab Dispos 43:1208–1217, August 2015
Development of Murine Cyp3a Knockout Chimeric Mice with
Humanized Liver
Kota Kato,1 Masato Ohbuchi,1 Satoko Hamamura,4 Hiroki Ohshita,4 Yasuhiro Kazuki,2
Mitsuo Oshimura,2 Koya Sato,1 Naoyuki Nakada,1 Akio Kawamura, Takashi Usui,
Hidetaka Kamimura,3 and Chise Tateno4,5
Drug Metabolism Research Laboratories, Astellas Pharma Inc., Osaka, Japan (K.K., Ma.O., K.S., N.N., A.K., T.U.); PhoenixBio Co.,
Ltd., Hiroshima, Japan (S.H., H.O., C.T.); Liver Research Project Center, Hiroshima University, Hiroshima, Japan (C.T.); Department
of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science (Y.K., Mi.O.),
Chromosome Engineering Research Center (Y.K., Mi.O.), Tottori University, Tottori, Japan; ADME & Tox Research Institute,
Sekisui Medical Co., Ltd., Tokyo, Japan (H.K.)
Received January 22, 2015; accepted May 15, 2015
We developed murine Cyp3a knockout (KO) chimeric mice with
humanized liver expressing human P450s similar to those in humans
and whose livers and small intestines do not express murine Cyp3a.
This approach may overcome effects of residual mouse metabolic
enzymes like Cyp3a in conventional chimeric mice with humanized
liver, such as PXB-mice [urokinase plasminogen activator/severe
combined immunodeficiency (uPA/SCID) mice repopulated with over
70% human hepatocytes] to improve the prediction of drug metabolism and pharmacokinetics in humans. After human hepatocytes
were transplanted into Cyp3a KO/uPA/SCID host mice, human
albumin levels logarithmically increased until approximately 60 days
after transplantation, findings similar to those in PXB-mice. Quantitative real-time–polymerase chain reaction analyses showed that
hepatic human P450s, UGTs, SULTs, and transporters mRNA expression
levels in Cyp3a KO chimeric mice were also similar to those in PXBmice and confirmed the absence of Cyp3a11 mRNA expression in
mouse liver and intestine. Findings for midazolam and triazolam
metabolic activities in liver microsomes were comparable between
Cyp3a KO chimeric mice and PXB-mice. In contrast, these activities
in the intestine of Cyp3a KO chimeric mice were attenuated compared with PXB-mice. Owing to the knockout of murine Cyp3a,
hepatic Cyp2b10 and 2c55 mRNA levels in Cyp3a KO/uPA/SCID mice
(without hepatocyte transplants) were 8.4- and 61-fold upregulated
compared with PXB-mice, respectively. However, human hepatocyte
transplantation successfully restored Cyp2b10 level nearly fully and
Cyp2c55 level partly (still 13-fold upregulated) compared with those
in PXB-mice. Intestinal Cyp2b10 and 2c55 were also repressed by
human hepatocyte transplantation in Cyp3a KO chimeric mice.
Introduction
vast range of xenobiotic compounds, including medical drugs. Because
of the significant species differences in many hepatic enzymes, the
metabolism of drug candidates is normally evaluated in vitro using
human liver microsomes or isolated hepatocytes (Brandon et al., 2003;
Gómez-Lechón et al., 2003). Although significant advancements in
human biologic materials have been made, in vitro test systems have
only limited use in predicting human drug metabolism in vivo, particularly
for sequential metabolism, owing to the multiple drug-metabolizing
enzymes involved (Anderson et al., 2009; Dalvie et al., 2009). Thorough
evaluation of the safety and drug-drug interaction of these metabolites will
require more accurate methods of predicting human metabolites in the
early stages of drug development.
Several mouse models have been developed recently as in vivo test
systems that closely resemble clinical conditions. These models insert
and express human drug metabolism-related genes such as CYP1A1/2,
CYP2D6, CYP2E1, and CYP3A4 (Muruganandan and Sinal, 2008) or
the CYP3A cluster (Kazuki et al., 2013). While this transgenic approach has proven useful to a degree, a number of factors may induce
inappropriate expression of the transgene, and even if the transgene is
Liver is the principal site for drug metabolism by virtue of its high
expression of enzymes. Among these enzymes, cytochrome P450
enzymes (P450s) play central roles in the oxidative metabolism of a
Current affiliations:
1
Analysis and Pharmacokinetics Research Laboratories, Drug Discovery
Research, Astellas Pharma Inc. 2-1-6, Kashima, Yodogawa-ku, Osaka City,
Osaka 532-8514, Japan.
2
Department of Biomedical Science, Institute of Regenerative Medicine and
Biofunction, Graduate School of Medical Science, and Chromosome Engineering
Research Center, Tottori University, 86, Nishi-cho, Yonago City, Tottori 683-8503,
Japan.
3
ADME and Tox Research Institute, Sekisui Medical Co., Ltd., 13-5, Nihonbashi
3-chome, Chuo-ku, Tokyo 103-0027, Japan.
4
PhoenixBio Co., Ltd., 3-4-1, Kagamiyama, Higashi-Hiroshima City, Hiroshima
739-0046, Japan.
5
Liver Research Project Center, Hiroshima University, 1-2-3, Kasumi, Minami-ku,
Hiroshima City, Hiroshima 734-8551, Japan.
dx.doi.org/10.1124/dmd.115.063479.
ABBREVIATIONS: hAlb, human albumin; KO, knockout; LC-MS/MS, liquid chromatography–tandem mass spectrometry; MDR1, multidrug
resistance protein 1; MRP2, multidrug resistance-associated protein 2; P450, cytochrome P450; PMSF, phenylmethylsulfonyl fluoride; PXB-mice,
chimeric mice with humanized liver; qRT-PCR, quantitative real-time–polymerase chain reaction; RI, replacement index; SCID, severe combined
immunodeficiency; SULT, sulfotransferase; UGT, uridine 59-diphospho-glucuronosyl transferase; uPA, urokinase-type plasminogen activator.
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ABSTRACT
Development of Cyp3a KO Chimeric Mice with Humanized Liver
Materials and Methods
Chemicals. Midazolam, alprazolam, and triazolam were purchased from
Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 19- and 4-hydroxymidazolam were purchased from Toronto Research Chemicals Inc. (North York,
ON, Canada). 19-hydroxytriazolam and 19 -hydroxytriazolam-d4 were purchased from Cerilliant Corp. (Round Rock, TX). Gefitinib was purchased from
Selleckchem (Houston, TX). All other reagents and solvents used were
commercially available and of guaranteed purity.
Generation of Cyp3a–/–/uPA+/+/SCID+/+ Mice. Cyp3a13–/– lines and
Cyp3a57-59–/– lines (lacking the rest of seven Cyp3a gene cluster) were
generated in a previous study, and the two lines were mated to generate
Cyp3a–/– mice lacking the mouse Cyp3a gene cluster (Kazuki et al., 2013).
The generation of uPA +/+ /SCID +/+ mice has been described previously
(Tateno et al., 2004). Sperm of Cyp3a–/– mice and the unfertilized ovum of
uPA+/+/SCID+/+ mice were combined and transferred into foster mothers for
artificial insemination. Litters with Cyp3a–/wt/uPA+/wt/SCID+/wt were backcrossed with uPA+/+/SCID+/+ mice via natural mating to obtain litters with
Cyp3a–/wt/uPA+/+/SCID+/+. Continuous backcrossing with uPA+/+/SCID+/+
mice was conducted twice via natural mating to obtain Cyp3a–/wt/uPA+/+/SCID+/+
mice. Subsequently, Cyp3a–/wt/uPA+/+/SCID+/+ mice were mated together
to obtain Cyp3a–/–/uPA+/+/SCID+/+ mice (designated Cyp3a KO/uPA/SCID
mice). Genotype analyses of Cyp3a–/– mice (designated Cyp3a KO mice) and
of uPA+/+/SCID+/+ mice (designated uPA/SCID mice) have been described
previously (Tateno et al., 2004; Kazuki et al., 2013). The genotypes of Cyp3a and
uPA were analyzed by the genomic polymerase chain reaction (PCR) method and
the genotypes of SCID by the PCR–restriction fragment length polymorphism
method.
Generation of Cyp3a KO Chimeric Mice with Humanized Liver. Cyp3a
KO chimeric mice transplanted with human hepatocytes were prepared by
PhoenixBio Co., Ltd. (Hiroshima, Japan). Human hepatocytes of two donors
(5-year-old African-American boy, Lot No. BD85, and 2-year-old Hispanic
girl, Lot No. BD195) were obtained from Corning Life Sciences K.K. (Tokyo,
Japan). The thawing and transplantation of human hepatocytes into CYP3a KO/
uPA/SCID mice has been described previously in the generation of PXB-mice
(Tateno et al., 2004). Briefly, these mice at 2–4 weeks of age were anesthetized
with ether and injected with 2.5–5.0 105 viable hepatocytes through a small
left-flank incision into the inferior splenic pole.
Generation of PXB-Mice. PXB-mice transplanted with human hepatocytes
of a donor (5-year-old African-American boy, Lot No. BD85) were prepared by
PhoenixBio Co., Ltd., as described previously (Tateno et al., 2004).
Measurement of Human Albumin. Blood samples (2 ml) were collected
periodically from the tail vein, and the human albumin (hAlb) level was measured using latex agglutination immunonephelometry (LX Reagent “Eiken”
Alb II; Eiken Chemical Co., Ltd., Tokyo, Japan) to predict the replacement
index (RI) of human hepatocytes in mouse liver (Tateno et al., 2004).
Immunohistochemistry. Cryosections prepared from the liver and intestine
(5-mm thick) were incubated with anti-human cytokeratin 8 and 18 (CK8/18)
mouse monoclonal antibodies (POG-10502; PROGEN Biotechnik GmbH,
Heidelberg, Germany) for the liver, or anti-mouse Cyp3a goat polyclonal antibodies (sc-30621; Santa Cruz Biotechnology, Inc., Dallas, TX) for the liver and
intestine. The primary antibodies were visualized with Alexa 488- or 594conjugated donkey anti-mouse-IgG or goat anti-rat IgG (Invitrogen, Carlsbad,
CA) as secondary antibodies. The sections prepared from the liver and intestine
were stained with Hoechst 33342 for nuclear staining.
Measurements of the Replacement Index. Measurements of the RI of
human hepatocytes to total human and mouse hepatocytes have been described
previously (Tateno et al., 2004). Briefly, RI was determined immunohistochemically using anti-human CK8/18 antibodies to calculate the ratio of area
occupied by anti-human CK8/18-positive hepatocytes to the entire area examined on immunohistochemical sections.
RNA Extraction and cDNA Synthesis. Total RNA was extracted from the
liver or intestine using the RNeasy minikit (Qiagen, Hilden, Germany), and
cDNA was generated from a random hexamer using the high-capacity cDNA
reverse transcription kit (Life Technologies, Grand Island, NY).
Quantitative Real-Time–Polymerase Chain Reaction. Quantitative realtime (qRT)-PCR was conducted using the ABI Prism 7900HT sequence
detection system with TaqMan gene expression assays (Life Technologies).
The primer and probe sets used were Mm00487224_m1 for Cyp1a2,
Mm00456591_m1 for Cyp2b10, Mm00725580_s1 for Cyp2c29, Mm00833845_m1
for Cyp2c37, Mm00472168_m1 for Cyp2c55, Mm00731567_m1 for Cyp3a11,
4652341E for b-Actin, Hs00167927_m1 for CYP1A2, Hs03044634_m1 for CYP2B6,
Hs00258314_m1 for CYP2C8, Hs00426397_m1 for CYP2C9, Hs00426380_m1 for
CYP2C19, Hs00164385_m1 for CYP2D6, Hs00430021_m1 for CYP3A4,
Hs00426592_m1 for UGT2B7, Hs00419411_m1 for SULT1A1, Hs00960941_m1
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expressed appropriately, the remaining cellular components—such as
inhibitors and activators and the corresponding cofactors—are all
derived from mice, resulting in failure of P450s to interact efficiently
or effectively with human genes/gene products.
An alternate approach involves producing chimeric mice with humanized
liver by transplanting human hepatocytes into liver-injured immunodeficient
mice that can accept xenografted human hepatocytes in their liver.
This process takes a few months to produce chimeric mice highly
repopulated with human hepatocytes. Several different methods of
inducing liver injury and different immunodeficiencies have been
investigated (Peltz, 2013), and chimeric mice with humanized liver
generated using urokinase-type plasminogen activator/severe combined
immunodeficiency (uPA/SCID) mice repopulated with human hepatocytes (PXB-mice; PhoenixBio Co., Ltd., Hiroshima, Japan) have been
reported (Tateno et al., 2004). These mice are repopulated with over
70% of human hepatocytes. Expression levels and activities of P450s
and non-P450 enzymes, such as uridine 59-diphospho-glucuronosyl
transferase (UGT) and sulfotransferase (SULT), in the liver of
PXB-mice are similar to those in humans (Katoh et al., 2004, 2005;
Nishimura et al., 2005; Katoh and Yokoi, 2007; Kitamura et al., 2008), and
human-specific metabolites are also formed in PXB-mice (Inoue et al.,
2009; Kamimura et al., 2010; Yamazaki et al., 2010; De Serres et al., 2011).
However, the utility of chimeric mice with humanized liver as
experimental models depends on the extent of replacement of host
hepatocytes with transplanted human hepatocytes, as the presence of
residual mouse hepatocytes hinders determination of the phenotypes
of human hepatocytes. Even in chimeric mice with high hepatic
replacement ratios, metabolism profiles are often similar to those in
the control mice depending on the compounds, a phenomenon that
occurs when examining compounds with metabolic rates in mice
higher than in humans, thereby suggesting some degree of metabolism
by the remaining mouse hepatocytes in the liver or host animal small
intestine, which acts as an extrahepatic metabolism organ (Kamimura
et al., 2010). Given the above, mice with their hepatocytes completely
replaced by human hepatocytes would be ideal for hepatic metabolism
studies, but no group has yet been able to successfully rear these mice.
In humans, CYP3A contributes to the oxidative/reductive metabolism of 50% of drugs used in the clinic and is the most abundant
P450 isozyme in human liver and small intestine (Guengerich, 1999;
Hall et al., 1999; Dresser et al., 2000). In mice, eight Cyp3a isozymes
(Cyp3a11, 3a13, 3a16, 3a25, 3a41, 3a44, 3a57, and 3a59) are known,
with Cyp3a13 being mainly expressed in small intestine and Cyp3a11,
3a25, and 3a41 in liver (Martignoni et al., 2006). The tissue distribution
and substrate specificities of these CYP3A and Cyp3a enzymes show very
extensive overlap. Therefore, as one approach to overcoming the issues
of concomitant mouse metabolic activities, we have developed murine
Cyp3a knockout (KO) chimeric mice with humanized liver by crossing
murine Cyp3a cluster-KO mice with uPA gene–introduced SCID mice.
Human hepatocytes were transplanted into the resulting liver-injured
Cyp3a KO immunodeficient mice to prepare Cyp3a KO chimeric mice.
Here, to investigate human-P450 and murine-P450 mRNA expressions as well as midazolam and triazolam metabolic activities in
liver and intestine, and human UGT, SULT, and transporter mRNA
expressions in liver, we describe initial characterizations of Cyp3a KO
chimeric mice and compare our findings with those in PXB-mice.
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Kato et al.
triazolam metabolism, microsomes were preincubated at 37C for 4 minutes
in the absence and presence of gefitinib (12.5 mM final concentration). Subsequently, triazolam (50 mM final concentration) was added and preincubated at
37C for 1 minute. The reaction was initiated by adding NADPH-regenerating
solution and allowed to proceed at 37C for 20 minutes for liver microsomes and
intestinal microsomes. The reaction was terminated by adding acetonitrile, and
19-hydroxytriazolam-d4 (internal standard) was added to the incubation mixtures.
The mixture was then centrifuged, and the supernatant was injected into LC-MS/MS
system.
LC-MS/MS Analysis for 19-Hydroxymidazolam and 4-Hydroxymidazolam. The concentrations of 19-hydroxymidazolam and 4-hydroxymidazolam in
microsomal samples were measured using a TSQ Quantum Ultra system
(Thermo Fisher Scientific Inc., San Jose, CA). The analytes were separated
with a Luna C18(2) column (100 2.1 mm, 3 mm; Phenomenex, Torrance,
CA) at 40C and a flow rate of 0.3 ml/min. The mobile phase consisted of 0.1%
acetic acid–acetonitrile (90:10 v/v) (A) and acetic acid–acetonitrile (0.1:100 v/v)
(B). The gradient condition was linearly increased from 5% to 17% B over
0.5 minutes, maintained at 17% B for 3.5 minutes, linearly increased to 80% B
over 1.5 minutes, maintained for 2.5 minutes, and then finally kept at 5% B
for 4.5 minutes. The mass spectrometry detection was performed using the
positive electrospray ionization selected reaction monitoring mode. The ion
spray voltage was set at 4500 V, and the transfer capillary temperature was
set at 350C. The mass spectrometer was set to monitor the transitions of
the precursors to the product ions as follows: m/z 342.0 to 324.0 for
19-hydroxymidazolam, m/z 342.0 to 325.0 for 4-hydroxymidazolam, and
m/z 309.0 to 205.0 for alprazolam. Data were processed using Xcalibur
software version 1.4.1 (Thermo Fisher Scientific Inc., San Jose, CA). The
calibration curves were linear over the range of 7.2 to 13314 nM (liver microsomes)
and 1.8 to 3328 nM (intestinal microsomes) for 19-hydroxymidazolam, and 0.5 to
953 nM (liver microsomes), and 1.2 to 238 nM (intestinal microsomes) for
4-hydroxymidazolam.
LC-MS/MS Analysis for 19-Hydroxytriazolam. The concentration of
19-hydroxytriazolam in microsomal samples was measured using a 4000 QTRAP
system (AB SCIEX, Framingham, MA). The analyte was separated with an
Inertsil ODS-4 (50 2.1 mm, 3 mm) (GL Sciences Inc., Tokyo, Japan) at 40C
and a flow rate of 0.2 ml/min. The mobile phase consisted of formic acid/water
(1:1000 v/v) (A) and methanol (B) at a ratio of 35:65 (A/B v/v). The mass
spectrometry detection was performed using the positive electrospray ionization
multiple reaction monitoring mode. The mass spectrometer was set to monitor
the transitions of the precursors to the product ions as follows: m/z 359.0 to
330.9 for 19-hydroxytriazolam, and m/z 365.2 to 337.1 for 19-hydroxytriazolam-d4.
Data were processed using Analyst software version 1.4.2 (AB SCIEX). The
calibration curve was linear over the range of 2.8 to 2784 nM (liver microsomes
and intestinal microsomes) for 19-hydroxytriazolam.
Statistical Analysis. Data in all tables and figures are shown as the means
and standard deviation. Statistical analyses were performed by one-way analysis
TABLE 1
Production of chimeric mice using two different donors and two different host mice
Data of body weight and hAlb at 12 weeks old are shown as mean 6 S.D.
Donor
Host Mouse
No. of Transplanted
Cells/Mouse
Sex
of Host
Mouse
No. of
Transplanted
Mice
No. of Dead Mice until
12 Weeks Old
Male
Female
Male
Female
Male
Female
Male
Female
Male
Female
Male
8
12
6
9
22
23
212
43
110
19
23
0
1
1
2
2
0
23
1
4
0
0
Body
No. of Mice
Weight
at 12
at 12 Weeks
Weeks
Old
Olda
105 cells
BD85
Cyp3aKO/uPA/SCID
2.5
3.5
5.0
uPA/SCID
BD195 Cyp3aKO/uPA/SCID
2.5
2.5
3.0
a
grams
Number of mice that were alive and unused for other studies at 12 weeks old.
Data of body weight and hAlb at 12 weeks old are shown as mean 6 S.D.
8
11
3
5
20
22
158
39
57
18
23
16.7
14.3
14.8
13.4
14.7
12.6
16.3
15.7
18.1
16.5
15.3
6
6
6
6
6
6
6
6
6
6
6
hAlb
at 12 Weeks
Old
mg/ml
3.6 4.0 6 3.1
2.3 3.2 6 2.7
0.8 8.5 6 6.3
2.2 8.5 6 3.7
2.9 11.7 6 2.7
2.5 8.9 6 5.4
3.1 10.0 6 3.0
2.5 8.6 6 2.7
2.6 8.1 6 4.0
1.9 6.9 6 3.0
2.7 10.6 6 2.8
Portion of Chimeric Mice
with RI . 70%
%
15.0
53.3
77.8
72.9
65.1
82.6
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for SULT1E1, Hs00234219_m1 for SULT2A1, Hs00104491_m1 for MDR1, and
4326315E for b-ACTIN. Other primers and probes used have been described
previously for UGT1A1 (Katoh et al., 2005) and UGT1A9 and MRP2 (Nishimura
et al., 2002). mRNA levels were normalized against those of b-ACTIN (human) or
b-Actin (mouse). The primers for human and murine mRNA were confirmed not to
crossreact.
Preparation of Liver and Intestinal Microsomes. Liver microsomes were
prepared as described previously (Sugihara et al., 2001). Briefly, liver samples
were homogenized in ice-cold 50 mM Tris-HCl buffer (pH 7.4) and centrifuged
at 9,000g for 20 minutes at 4C. The resulting supernatant was then centrifuged
at 105,000g for 60 minutes at 4C. The microsomal pellet was washed and
resuspended in 50 mM Tris-HCl buffer (pH 7.4) and then centrifuged at 105,000g
for 60 minutes at 4C. The final resulting pellet was then suspended in a small
amount of 250 mM sucrose and stored at 280C until analyses. Intestinal microsomes were prepared as described previously (Bonkovsky et al., 1985) with
some modifications. The inner space of small intestine samples was washed with
ice-cold solution A (pH 7.4, 1.5 mM KCl, 96 mM NaCl, 27 mM sodium citrate,
8 mM KH2PO4, 5.6 mM Na2HPO4 • 12H2O, 230 mM PMSF) and then cut into
three parts. These parts were placed in ice-cold solution B (pH 7.4, phosphatebuffered saline supplemented with 1.5 mM EDTA, 0.5 mM DDT, 3 IU/ml
heparin, and 230 mM PMSF) and cut longitudinally. Subsequently, the mucosal
cells were scraped off the cut samples with a spatula and centrifuged at 2,000g for
5 minutes at 4C. The centrifuged mucosal cells were added to ice-cold solution
C (pH 7.4, 5 mM histidine, 0.25 M sucrose, and 0.5 M EDTA), centrifuged at
2,000g for 10 minutes at 4C, and then homogenized. The homogenates were
centrifuged at 12,000g for 20 minutes at 4C, and the supernatant was centrifuged
at 104,000g for 60 minutes at 4C. The pellet was mixed with 150 mM Tris and
10 mM EDTA and then centrifuged at 104,000g for 60 minutes at 4C. The pellet
was then resuspended in a small amount of 250 mM sucrose and stored at 280C
until analyses. The protein concentration was determined using a Bradford protein
assay kit (Bio-Rad, Hercules, CA) with bovine serum albumin as the standard.
Midazolam Metabolism in Liver and Intestinal Microsomes. The
reaction mixture (200 ml) was composed of 100 mM Na-K phosphate buffer
(pH 7.4), 0.1 mM EDTA, 50 mM midazolam, and 0.1 mg protein/ml of liver
microsomes or 0.5 mg protein/ml of intestinal microsomes. After 5 minutes of
preincubation at 37C, the reaction was initiated by adding 1 mM NADPH to
the final concentration and allowed to proceed at 37C for 5 minutes for liver
microsomes and intestinal microsomes. The reaction was terminated by adding
formic acid–50% acetonitrile (10:90 v/v) containing alprazolam (internal
standard). The mixture was then centrifuged, and the supernatant was injected
into the liquid chromatography–tandem mass spectrometry (LC-MS/MS) system.
Triazolam Metabolism in Liver and Intestinal Microsomes. The incubations were conducted in a total volume of 200 ml containing 100 mM
potassium phosphate buffer (pH 7.4), and 0.5 mg protein/ml liver microsomes or
in a total volume of 100 ml containing 100 mM potassium phosphate buffer
(pH 7.4), and 1 mg protein/ml of intestinal microsomes. For the stimulation of
Development of Cyp3a KO Chimeric Mice with Humanized Liver
Fig. 1. Changes in hAlb levels in blood of Cyp3a KO chimeric mice and PXB-mice.
Results
Generation of Cyp3a KO Chimeric Mice with Humanized Liver.
Cyp3a KO mice were crossed with uPA/SCID mice to generate Cyp3a
KO/uPA/SCID mice. At 2–4 weeks of age, 2.5, 3.0, 3.5, or 5.0 105
viable human hepatocytes were transplanted into Cyp3a KO/uPA/SCID
mice to develop Cyp3a KO chimeric mice. In total, 80 transplantations
(donor: BD85) and 152 transplantations (donor: BD195) for Cyp3a KO
chimeric mice and 255 transplantations (donor: BD85) for PXB-mice
were performed, and the body weight and hAlb data of 12-week-old
mice are shown in Table 1. Changes in blood hAlb levels in Cyp3a KO
chimeric mice and PXB-mice (donor: BD85) are shown in Fig. 1. The
hAlb levels increased steadily after transplantation of human hepatocytes
and reached a steady state at around 9 weeks or more, depending on the
number of transplanted hepatocytes (Fig. 1). Engraftment of human
hepatocytes was inferior in the Cyp3a KO host mice compared with
the uPA/SCID host mice because hAlb levels of Cyp3a KO chimeric
mice were lower than those of uPA/SCID mice when the same numbers
of human hepatocytes (2.5 105) were transplanted into the host mice
(Fig. 1). Mean body weights and hAlb levels in males were higher than
those in 12-week-old females of both Cyp3a KO and uPA/SCID chimeric
mice, except for Cyp3a KO chimeric mice with 3.5 105 BD85 donor
cells (Table 1).
Correlation of hAlb levels and the RI of PXB-mice (donor: BD85)
is shown in Fig. 2. hAlb levels were exponentially correlated with RI
(y = 733927e0.0314x, r2 = 0.912), indicating that a level of more than
6.6 mg/ml hAlb represents RI exceeding 70%. hAlb and RI of Cyp3a
KO mice were determined and plotted on the correlation curve
between hAlb and RI of uPA/SCID mice, resulting an almost identical
correlation (Fig. 2). The yield of chimeric mice was calculated as the
portion of chimeric mice with RI . 70% (hAlb . 6.6 mg/ml) among
mice transplanted with human hepatocytes. The portion was more
than 70% when 2.5 105 and 5.0 105 BD85 donor cells were
transplanted into the uPA/SCID and the Cyp3a KO host mice, respectively, and 3.0 105 BD195 donor cells were transplanted into
the Cyp3a KO host mice (Table 1).
For further animal studies of human P450s and murine P450s
expressions (in males and females), human UGTs, SULTs, MDR1,
MRP2 expressions (in males), midazolam and triazolam metabolic
activities (in males), Cyp3a KO chimeric mice, and PXB-mice (donor:
BD85) with 6.3–15.7 mg/ml hAlb (68–97% RI) in males and 5.4–
13.2 mg/ml hAlb (64–92% RI) in females were used. Detailed information
on chimeric mice used is shown in Table 2.
Immunohistochemistry. Immunohistochemistry of liver transplanted with human hepatocytes and that of the intestine from Cyp3a
KO chimeric mice and PXB-mice are shown in Fig. 3. Liver sections
prepared from the left lateral lobe in Cyp3a KO chimeric mice (at 10–
11 weeks after transplantation) and PXB-mice (at 10–11 weeks after
transplantation) were stained with anti-human CK8/18 antibodies and
anti-mouse Cyp3a antibodies. The areas of anti-human CK8/18-negative
mouse hepatocytes correspond to those of anti-mouse Cyp3apositive mouse hepatocytes in PXB-mice. In contrast, the areas of
anti-human CK8/18-negative mouse hepatocytes correspond to those
of anti-mouse Cyp3a-negative mouse hepatocytes in Cyp3a KO chimeric
mice, suggesting that mouse Cyp3a enzymes are not expressed in Cyp3a
KO chimeric mice and that human hepatocytes are replaced in the
host liver.
Intestinal sections prepared from the same mice were stained with
anti-mouse Cyp3a antibodies. Although intestinal section areas in
PXB-mice were anti-mouse Cyp3a-positive, those in Cyp3a KO chimeric
mice were anti-mouse Cyp3a-negative, suggesting that mouse Cyp3a enzymes are not expressed in the host intestine.
Human and Murine P450s Expression. Human P450s (CYP1A2,
2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and murine P450s (Cyp1a2, 2b10,
2c29, 2c37, 2c55, and 3a11) mRNA expression levels in the liver from
PXB-mice, Cyp3a KO chimeric mice, and Cyp3a KO/uPA/SCID mice
Fig. 2. Correlation between hAlb levels and RI of Cyp3a
KO chimeric mice and PXB-mice.
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
of variance followed by Tukey’s test using PRISM 5.0 software (GraphPad
Software Inc., San Diego, CA) for murine P450 mRNA expressions and
midazolam and triazolam metabolic activities, and by t test using WinNonlin
6.1 software (Pharsight Corporation, St. Louis, MO) for triazolam metabolic
activity in the presence or absence of gefitinib.
1211
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Kato et al.
TABLE 2
Chimeric mice used for analyses in the present study
Animal Number
Cyp3aKO 2-3
Cyp3aKO 3-4
Cyp3aKO 3-6
Cyp3aKO 2-5
Cyp3aKO 2-10
Cyp3aKO 3-10
PXB 194-4
PXB 194-22
PXB 195-21
PXB 196-24
PXB 196-26
PXB 196-29
a
Sex
of host mouse
Male
Female
Male
Female
No. of Transplanted Cells
Age
at Sacrifice
Body Weight
at Sacrifice
hAlb
at Sacrifice
Expected RI
from hAlb Levela
105 cells/mouse
weeks
grams
mg/ml
%
2.5
5.0
5.0
2.5
2.5
5.0
2.5
2.5
2.5
2.5
2.5
2.5
14
14
14
14
14
14
15
15
14
14
14
14
17.1
12.9
12.9
14.8
14.0
16.8
21.8
20.1
17.6
14.0
17.9
14.8
11.2
11.2
15.7
5.4
11.0
6.5
11.5
8.7
6.3
12.2
9.1
13.2
87
87
97
64
86
69
88
79
68
89
80
92
Expected RI from hAlb level was calculated by the formula: y = 733927e0.0314x, where x = expected RI, y = hAlb.
The representative isozyme of murine Cyp3a, Cyp3a11 mRNA
level was not detected in any liver or intestine samples from Cyp3a
KO chimeric mice or Cyp3a KO/uPA/SCID host mice. Hepatic
Cyp2b10, 2c29, 2c37, and 2c55 mRNA levels in Cyp3a KO/uPA/
SCID mice (without hepatocyte transplant) were 8.4-, 35-, 5.6-, and
61-fold upregulated in males compared with PXB-mice, respectively,
owing to the knockout of murine Cyp3a. In Cyp3a KO chimeric mice,
hepatic Cyp2b10 and 2c37 mRNA levels were nearly fully restored
to those in PXB-mice, but Cyp2c29 and 2c55 levels were 6.1- and
13-fold upregulated in males, respectively, following human hepatocyte transplantation. Intestinal Cyp2b10 and 2c55 mRNA levels in Cyp3a
KO/uPA/SCID mice were 2.3- and 51-fold upregulated in males compared
Fig. 3. Immunohistochemistry of liver transplanted with
human hepatocytes and intestine from Cyp3a KO chimeric
mice and PXB-mice. Sections (5-mm thick) of the liver
from PXB-mice (A–C) and Cyp3a KO chimeric mice (D–F),
and the intestine from PXB-mice (G–I), and Cyp3a KO
chimeric mice (J–L) were stained with anti-mouse Cyp3a
goat polyclonal antibody for A, D, G, and J in green, antihuman cytokeratin 8 and 18 (CK8/18) mouse monoclonal
antibody for B and E in red, and Hoechst 33342 for H and
K in blue. In addition, sections A and B or D and E were
merged with Hoechst 33342 for C or F, and sections G and
H or J and K were merged for I or L, respectively.
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
are shown in Figs. 4 and 5, respectively. In addition, murine P450s
(Cyp2b10, 2c55, and 3a11) mRNA expression levels in the intestine
are shown in Fig. 6. Liver and intestine samples from Cyp3a KO
chimeric mice and PXB-mice at 10–11 weeks after hepatocyte
transplantation and those from Cyp3a KO/uPA/SCID mice without
hepatocyte transplantation were used from three male and three
female mice. mRNA expression levels were determined via qRTPCR and normalized using human b-ACTIN for human P450s or
murine b-actin for murine P450s. In the liver, human P450s expression levels in Cyp3a KO chimeric mice were comparable (approximately 70–117% in males and 79–122% in females) to those
in male PXB-mice.
Development of Cyp3a KO Chimeric Mice with Humanized Liver
1213
Discussion
Fig. 4. Human P450 mRNA expression in liver of PXB-mice, Cyp3a KO
chimeric mice, and Cyp3a KO/uPA/SCID mice. Human P450 mRNA expression
was determined via qRT-PCR and normalized using the mRNA expression of
human b-ACTIN. The primer for human P450s and b-ACTIN used in the present
study was confirmed to not crossreact with mouse mRNA. Data are shown as mean +
S.D. obtained from three male mice (A–G) and three female mice (H–N). ND, not
detected.
We successfully developed Cyp3a KO chimeric mice with humanized
liver via transplantation of human hepatocytes into liver-injured Cyp3a
KO immunodeficient mice, which were generated through the conventional approach of crossing Cyp3a cluster KO mice with uPA
gene–introduced SCID mice. The initial characterization of Cyp3a KO
chimeric mice was conducted to investigate human P450s and murine
P450 mRNA expression levels, as well as midazolam and triazolam
metabolic activities in liver and intestine, and human UGT, SULT, and
transporter mRNA expression levels in liver, and then these findings
were compared with PXB-mice.
In our initial attempt to generate Cyp3a KO chimeric mice, the
replacement of mouse hepatocytes with human hepatocytes after
transplantation was much less successful than in PXB-mice (data not
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
with PXB-mice, respectively, owing to the knockout of murine Cyp3a.
In Cyp3a KO chimeric mice, intestinal Cyp2b10 and 2c55 mRNA
levels were repressed in the same manner as hepatic Cyp2b10 and 2c55
levels by hepatocyte transplantation. Hepatic Cyp1a2, 2b10, and 2c37
mRNA levels did not differ significantly between Cyp3a KO chimeric
mice and PXB-mice.
In females, comparable levels of hepatic and intestinal P450 mRNA
were detected without any remarkable sex differences.
Midazolam 19 - and 4-Hydroxylation Activities. Midazolam 19and 4-hydroxylation activities in liver and intestinal microsomes
prepared from PXB-mice, Cyp3a KO chimeric mice, and Cyp3a KO/
uPA/SCID mice are shown in Table 3. In liver microsomes, both
midazolam 19- and 4-hydroxylation activities in Cyp3a KO chimeric
mice were comparable to those in PXB-mice, although those in Cyp3a
KO/uPA/SCID mice (without hepatocyte transplant) were lower owing
to the knockout of murine Cyp3a. In intestinal microsomes, both
midazolam 19- and 4-hydroxylation activities in Cyp3a KO chimeric mice
were much lower than those in PXB-mice and nearly equal to those in
Cyp3a KO/uPA/SCID host mice.
Triazolam 19-Hydroxylation Activity. Triazolam 19-hydroxylation
activity in liver and intestinal microsomes prepared from PXB-mice,
Cyp3a KO chimeric mice, Cyp3a KO/uPA/SCID mice, and uPA/SCID
mice as well as humans (liver microsomes only) is shown in Table 4. In
addition, triazolam was coincubated with gefitinib to investigate whether
triazolam 19-hydroxylation activity was stimulated in both liver and
intestinal microsomes. In liver microsomes, triazolam 19-hydroxylation
activity in Cyp3a KO chimeric mice was comparable to that in PXB-mice
and humans, whereas the lower activity in Cyp3a KO/uPA/SCID mice
(without hepatocyte transplant) was attributed to the knockout of murine
Cyp3a. After coincubation with gefitinib, triazolam 19-hydroxylation
activity was significantly stimulated in PXB-mice and humans, and
tended to increase in Cyp3a KO chimeric mice, and significantly
inhibited in uPA/SCID mice. In intestinal microsomes, triazolam
19-hydroxylation activity was not detected in either Cyp3a KO chimeric
mice or Cyp3a KO/uPA/SCID host mice.
Human UGTs and SULTs Expression. Human UGTs (UGT1A1,
1A9, and 2B7) and SULTs (SULT1A1, 1E1, and 2A1) mRNA
expression levels in the liver from PXB-mice, Cyp3a KO chimeric
mice, and Cyp3a KO/uPA/SCID mice are shown in Fig. 7. Hepatic
UGT and SULT mRNA levels did not differ significantly between
Cyp3a KO chimeric mice and PXB-mice.
Human MDR1 and MRP2 Expression. Human MDR1 and MRP2
mRNA expression levels in the liver from PXB-mice, Cyp3a KO
chimeric mice, and Cyp3a KO/uPA/SCID mice are shown in Fig. 8.
Hepatic MDR1 and MRP2 mRNA levels did not differ significantly
between Cyp3a KO chimeric mice and PXB-mice.
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Kato et al.
Fig. 5. Murine P450 mRNA expression in liver of PXB-mice, Cyp3a KO chimeric
mice, and Cyp3a KO/uPA/SCID mice. Murine P450 mRNA expression was determined
via qRT-PCR and normalized using the mRNA expression of murine b-actin. The primer
for murine P450s and b-actin used in the present study was confirmed not to crossreact
with human mRNA. Data are shown as mean + S.D. obtained from three male mice
(A–F) and three female mice (G–L). Statistical differences in three mice groups were
determined using one-way analysis of variance followed by Tukey’s test. *P , 0.05;
**P , 0.01; ***P , 0.001 versus Cyp3a KO/uPA/SCID mice; ND, not detected.
shown), probably owing to differences in background genes between
Cyp3a KO mice and uPA/SCID mice. Therefore, several continuous
backcrosses with uPA/SCID mice were applied to increase the genetic
background of the uPA/SCID mice. While engraftment and/or growth
of human hepatocytes in the Cyp3a KO host mice were inferior to the
PXB-host mice, hAlb levels of Cyp3a KO chimeric mice transplanted
with 5.0 105 human hepatocytes were similar to those of PXB-mice
transplanted with 2.5 105 human hepatocytes. In addition, engraftment
and/or growth of human hepatocytes were different between BD85- and
BD195-transplanted Cyp3a KO chimeric mice. The chimeric mice
transplanted with BD195 donor cells showed higher hAlb levels in the
blood than those transplanted with BD85 donor cells.
The RI, which was calculated by immunostaining using anti-human
CK8/18 antibodies, was correlated with hAlb concentrations in the
blood of PXB-mice from a previous study (Tateno et al., 2004) and
also those of Cyp3a KO chimeric mice in the present study. Of note,
the higher cell number for transplantation resulted in higher hAlb
levels in Cyp3a KO chimeric mice.
One of the major limitations inherent to current PXB-mice is the
incomplete replacement of mouse hepatocytes with human hepatocytes (Yoshizato et al., 2012). One example of problematic cases is
that of chimeric mice administered a drug candidate that is well
metabolized by mouse hepatocytes but metabolized only to a limited
extent by human hepatocytes, and all its human metabolites are
detected as its mouse metabolites (Kamimura et al., 2010). In such
a case, the plasma metabolic profile is similar to that of control mice,
and no significant increase in the peaks of human metabolites is
found. To overcome the metabolic activity caused by residual mouse
hepatocytes, we eliminated the contribution of residual host mouse
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
Fig. 6. Murine P450 mRNA expression in the intestine of PXB-mice, Cyp3a KO
chimeric mice, and Cyp3a KO/uPA/SCID mice. Murine P450 mRNA expression
was determined via qRT-PCR and normalized using the mRNA expression of
murine b-actin. Data are shown as mean + S.D. obtained from three male mice (A–C)
and three female mice (D–F). Statistical differences in three mouse groups were
determined using one-way analysis of variance followed by Tukey’s test. *P , 0.05
versus Cyp3a KO/uPA/SCID mice. ND, not detected.
1215
Development of Cyp3a KO Chimeric Mice with Humanized Liver
TABLE 3
Metabolic activities of 19- and 4- hydroxymidazolam in liver and intestinal microsomes
Metabolic Activity
19-OH Midazolam
4-OH Midazolam
pmol/min per milligram of protein
Liver microsomes
Intestinal microsomes
Cyp3a KO/uPA/SCID mice
Cyp3a KO chimeric mice
PXB-mice
Cyp3a KO/uPA/SCID mice
Cyp3a KO chimeric mice
PXB-mice
281.5
846.2
611.7
0.6
1.3
14.9
6
6
6
6
6
6
21.1
372.3*
32.0
0.4
0.7
15.5
63.9
420.9
306.1
0.7
0.9
9.8
6
6
6
6
6
6
14.0
194.8*
6.5
0.1
0.0
9.5
*P , 0.05 versus Cyp3a KO/uPA/SCID mice.
Midazolam 19- and 4-hydroxylation in liver and intestinal microsomes were determined using LC-MS/MS. Midazolam (50 mM) was
incubated in liver (0.1 mg/ml) and intestinal (0.5 mg/ml) microsomes at 37C for 5 minutes. Data are shown as mean 6 S.D. obtained from
three male mice. Statistical differences in three mice groups were determined using one-way analysis of variance followed by Tukey’s test.
been investigated, our results suggest that the Cyp3a KO chimeric
mouse is a viable model for drug metabolism and pharmacokinetics
research in a manner similar to PXB-mice.
In contrast, significant increases in the expression levels of such
murine P450s as Cyp2b10, 2c29, 2c37, and 2c55 in the liver and
Cyp2c55 in the intestine were observed in Cyp3a KO/uPA/SCID mice.
These findings agree with those of previous reports that found the
deletion of the murine Cyp3a cluster to be associated with considerable compensatory changes in other gene families involved in drug
metabolism and pharmacokinetics (van Waterschoot et al., 2009b).
Interestingly, the upregulated hepatic and intestinal Cyp2b10 expression levels observed in Cyp3a KO/uPA/SCID mice (without human
hepatocytes) were restored to near-normal levels in Cyp3a KO
chimeric mice that received transplanted human hepatocytes, and
remarkable reductions in the expression of hepatic Cyp2c29 and 2c37,
and hepatic and intestinal Cyp2c55 from upregulated levels, were also
observed, suggesting that transplanted human hepatocytes were able to
compensate in part for the loss of murine Cyp3a enzymes not only in
the liver but also in the intestine. Although further investigations are
needed, the metabolism of dietary phytochemicals of P450 inducers
and/or the synthesis or biotransformation of endogenous substances,
such as bile acids, might be involved in this compensatory mechanism
because these can directly or indirectly regulate P450 expressions
(Zhu et al., 2014).
TABLE 4
Metabolic activity of 19-hydroxytriazolam without or with gefitinib in liver and intestinal microsomes
Metabolic Activity 19-OH Triazolam
Without Gefitinib
With Gefitinib
pmol/min per milligram of protein
Liver microsomes
Intestinal microsomes
uPA/SCID mice
Cyp3a KO/uPA/SCID mice
Cyp3a KO chimeric mice
PXB-mice
Humans
uPA/SCID mice
Cyp3a KO/uPA/SCID mice
Cyp3a KO chimeric mice
PXB-mice
6 151
6 3.49†††
6 120†††, ‡
6 30.3†††, ‡‡
6 5.29
6 11.9
ND
ND
5.94 6 10.1
1670
20.7
293
413
385
11.5
6 142*
6 6.23
6 285
6 39.0**
6 10.2***
6 11.7
ND
ND
5.71 6 9.69
1250
23.5
699
617
946
11.6
No mark, not significant; ND, not detected.
*P , 0.05, **P , 0.01, ***P , 0.001 versus the corresponding in the absence of gefitinib. Statistical differences in four mice groups in
the absence of gefitinib were determined using one-way analysis of variance followed by Tukey’s test. †††P , 0.001 versus uPA/SCID
mice; ‡P , 0.05 and ‡‡P , 0.01 versus Cyp3a KO/uPA/SCID mice.
Triazolam 19 -hydroxylation in liver and intestinal microsomes were determined using LC-MS/MS. Triazolam (50 mM) was incubated in
liver (0.5 mg/ml) and intestinal (1 mg/ml) microsomes without or with gefitinib (12.5 mM) at 37C for 20 minutes. Data are shown as mean 6
S.D. obtained from three male mice or triplicate incubations in human liver microsomes. Statistical differences in the presence or absence of
gefitinib were determined by t test.
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
hepatocytes from the currently available PXB-mice by generating
Cyp3a KO chimeric mice.
To date, the usefulness of PXB-mice has been recognized in studies
for the prediction of human metabolism of drugs (Yoshizato and
Tateno, 2009a, b). In the liver of PXB-mice, the human enzymes
that play important roles in drug metabolisms include several P450
enzymes, such as CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6,
3A4, and 3A5 and non-P450 enzymes such as UGT, SULT, glutathione S-transferase, N-acetyltransferase, and aldehyde oxidase
(Katoh et al., 2004, 2005; Nishimura et al., 2005; Katoh and Yokoi,
2007; Kitamura et al., 2008). In the present study, mRNA levels of
selected P450s (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4),
UGTs (UGT1A1, 1A9, and 2B7), and SULTs (SULT1A1, 1E1, and
2A1) were measured in the liver to determine whether Cyp3a KO mice
are as useful as PXB-mice in drug metabolism and pharmacokinetic
research. In addition, as the murine Cyp3a cluster was knocked out,
expression levels of several murine P450s (Cyp1a2, 2b10, 2c29, 2c37,
2c55, and 3a11) were measured in the liver and intestine to investigate
the impact of the expression of other murine P450s. Given the correlation between hAlb levels and the RI, we concluded that the
expression levels of human P450s, UGTs, and SULTs after the
transplantation of human hepatocytes into mice did not differ significantly between Cyp3a KO chimeric mice and PXB-mice. Although
the expression levels of limited human metabolizing enzymes have
1216
Kato et al.
It has been reported that the biotransformation of midazolam is
comparable in both humans and mice, yielding 19-hydroxymidazolam
as the major metabolite and 4-hydroxymidazolam as a minor metabolite,
and that 19-hydroxymidazolam formation in mice is not only dependent
on murine Cyp3a but also imbued with significant murine Cyp2c
components, whereas 4-hydroxymidazolam formation is considered
specific to murine Cyp3a (van Waterschoot et al., 2008). Consistent
with that report, the present study demonstrated that midazolam
19-hydroxylation in the liver microsomes of Cyp3a KO/uPA/SCID
mice was detectable with partial compensation, probably via induced
murine Cyp2c enzymes following murine Cyp3a knockout, and that
4-hydroxylation in the liver microsomes of Cyp3a KO/uPA/SCID
mice was reduced. Further, relatively little midazolam metabolism was
detected in the intestinal microsomes of either Cyp3a KO chimeric
mice or Cyp3a KO/uPA/SCID mice, indicating that the expression
level of induced murine Cyp2c enzymes is too low to significantly
contribute to the midazolam metabolism in the intestine after the
knockout of murine Cyp3a. Midazolam 19- and 4-hydroxylations in
Cyp3a KO chimeric mice were comparable with findings in liver
microsomes of PXB-mice after human hepatocyte transplantation,
regardless of murine Cyp3a knockout.
In contrast to midazolam, the structurally related drug triazolam is
a more specific substrate for CYP3A4 compared with other murine
P450 enzymes (Perloff et al., 2000). Although some residual metabolism of triazolam was still mediated by murine Cyp2c enzymes in
Cyp3a KO mice, the compensatory triazolam metabolism was much
lower compared with midazolam metabolism (van Waterschoot et al.,
2009a). From the results in this study, triazolam was metabolized in
liver microsomes of uPA/SCID mice but scarcely metabolized in
Cyp3a KO/uPA/SCID mice, suggesting involvement of murine Cyp3a
in triazolam metabolism of 19-hydroxylation. Triazolam 19-hydroxylation
Fig. 8. Human MDR1 and MRP2 mRNA expression in liver of PXB-mice, Cyp3a
KO chimeric mice, and Cyp3a KO/uPA/SCID mice. Human MDR1 and MRP2
mRNA expression was determined via qRT-PCR and normalized using the mRNA
expression of human b-ACTIN. Primers for human MDR1, MRP2, and b-ACTIN
used in the present study were confirmed not to crossreact with mouse mRNA. Data
are shown as mean + S.D. obtained from three male mice (A, B). ND, not detected.
in liver microsomes of Cyp3a KO chimeric mice was more than
10-fold higher than that of Cyp3a KO/uPA/SCID mice. The formation
of 19-hydroxylation triazolam was increased by more than twice with
gefitinib in liver microsomes of Cyp3a KO chimeric mice, although
findings have no significance between those without and with gefitinib
in liver microsomes of Cyp3a KO chimeric mice, probably owing to
intervariability within the limited number of the mice tested. As it was
reported that the anticancer drug gefitinib significantly stimulated
triazolam metabolism in human liver microsomes (van Waterschoot
et al., 2009a), these results suggest that the triazolam metabolism in
Cyp3a KO chimeric mice is attributable to human liver. Triazolam
19-hydroxylation in Cyp3a KO chimeric mice was comparable to the
finding in liver microsomes of PXB-mice after human hepatocyte
transplantation, regardless of murine Cyp3a knockout. In intestinal
microsomes, 19-hydroxytriazolam was scarcely formed in any microsomes of murine Cyp3a knock-out mice.
Given the importance of metabolizing enzymes in the intestine to
limit hepatic and systemic exposure to orally administered drugs in
humanized animal models, the present findings suggest that Cyp3a
KO chimeric mice could prove more useful than PXB-mice, because
their lack of significant metabolism in the intestine improves predictability of human metabolites. The present study also demonstrated
a similarity in the mRNA expression levels of the transporters human
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
Fig. 7. Human UGT and SULT mRNA expression in liver of PXB-mice, Cyp3a KO
chimeric mice, and Cyp3a KO/uPA/SCID mice. Human UGT and SULT mRNA
expression was determined via qRT-PCR and normalized using the mRNA
expression of human b-ACTIN. Primers for human UGTs, SULTs, and b-ACTIN
used in the present study were confirmed not to crossreact with mouse mRNA. Data
are shown as mean + S.D. obtained from three male mice (A–F). ND, not detected.
Development of Cyp3a KO Chimeric Mice with Humanized Liver
MDR1 and MRP2, which both contribute to the distribution and excretion of drugs in both Cyp3a KO chimeric mice and PXB-mice.
In conclusion, we successfully developed Cyp3a KO chimeric mice
with humanized liver expressing human P450s similar to PXB-mice
but with liver and intestine not expressing murine Cyp3a, a property
which may prove useful in drug metabolism and pharmacokinetics
research. Further, the method of generating Cyp3a KO chimeric mice
described here may be useful for generating humanized animal models
with knockout of other murine P450s, non-P450s, and transporters,
although the possibility of unexpected side effects (upregulation or
downregulation) after knockout of these genes cannot be excluded; as
such, any future attempts at generating new models should be conducted
with caution.
Acknowledgments
The authors thank Hiroki Ebine, Satoko Yoshida, Miyoko Ono, and
Toshihiro Sasaki in Sekisui Medical Co., Ltd. for triazolam metabolism study
using liver and intestinal microsomes.
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Address correspondence to: Dr. Chise Tateno, PhoenixBio Co., Ltd, 3-4-1,
Kagamiyama, Higashi-Hiroshima City, Hiroshima 739-0046, Japan. E-mail: chise.
[email protected]
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
Authorship Contributions
Participated in research design: Kato, Kawamura, Kazuki, Oshimura,
Kamimura, Tateno.
Conducted experiments: Ohbuchi, Hamamura, Ohshita.
Performed data analysis: Kato, Ohbuchi, Hamamura, Ohshita.
Wrote or contributed to the writing of the manuscript: Kato, Ohbuchi,
Nakada, Sato, Kawamura, Usui, Kazuki, Oshimura, Kamimura, Tateno.
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