1521-009X/43/8/1208–1217$25.00 http://dx.doi.org/10.1124/dmd.115.063479DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 43:1208–1217, August 2015Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

Development of Murine Cyp3a Knockout Chimeric Mice withHumanized 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.); Departmentof 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

ABSTRACT

We developed murine Cyp3a knockout (KO) chimeric mice withhumanized liver expressing human P450s similar to those in humansand whose livers and small intestines do not express murine Cyp3a.This approach may overcome effects of residual mouse metabolicenzymes like Cyp3a in conventional chimeric mice with humanizedliver, such as PXB-mice [urokinase plasminogen activator/severecombined immunodeficiency (uPA/SCID) mice repopulated with over70% human hepatocytes] to improve the prediction of drug metab-olism and pharmacokinetics in humans. After human hepatocyteswere transplanted into Cyp3a KO/uPA/SCID host mice, humanalbumin levels logarithmically increased until approximately 60 daysafter transplantation, findings similar to those in PXB-mice. Quanti-tative real-time–polymerase chain reaction analyses showed thathepatic human P450s, UGTs, SULTs, and transportersmRNA expression

levels in Cyp3a KO chimeric mice were also similar to those in PXB-mice and confirmed the absence of Cyp3a11 mRNA expression inmouse liver and intestine. Findings for midazolam and triazolammetabolic activities in liver microsomes were comparable betweenCyp3a KO chimeric mice and PXB-mice. In contrast, these activitiesin the intestine of Cyp3a KO chimeric mice were attenuated com-pared with PXB-mice. Owing to the knockout of murine Cyp3a,hepatic Cyp2b10 and 2c55mRNA levels in Cyp3a KO/uPA/SCID mice(without hepatocyte transplants) were 8.4- and 61-fold upregulatedcompared with PXB-mice, respectively. However, human hepatocytetransplantation successfully restored Cyp2b10 level nearly fully andCyp2c55 level partly (still 13-fold upregulated) compared with thosein PXB-mice. Intestinal Cyp2b10 and 2c55 were also repressed byhuman hepatocyte transplantation in Cyp3a KO chimeric mice.

Introduction

Liver is the principal site for drug metabolism by virtue of its highexpression of enzymes. Among these enzymes, cytochrome P450enzymes (P450s) play central roles in the oxidative metabolism of a

vast range of xenobiotic compounds, including medical drugs. Becauseof the significant species differences in many hepatic enzymes, themetabolism of drug candidates is normally evaluated in vitro usinghuman liver microsomes or isolated hepatocytes (Brandon et al., 2003;Gómez-Lechón et al., 2003). Although significant advancements inhuman biologic materials have been made, in vitro test systems haveonly limited use in predicting human drug metabolism in vivo, particularlyfor sequential metabolism, owing to the multiple drug-metabolizingenzymes involved (Anderson et al., 2009; Dalvie et al., 2009). Thoroughevaluation of the safety and drug-drug interaction of these metabolites willrequire more accurate methods of predicting human metabolites in theearly stages of drug development.Several mouse models have been developed recently as in vivo test

systems that closely resemble clinical conditions. These models insertand express human drug metabolism-related genes such as CYP1A1/2,CYP2D6, CYP2E1, and CYP3A4 (Muruganandan and Sinal, 2008) orthe CYP3A cluster (Kazuki et al., 2013). While this transgenic ap-proach has proven useful to a degree, a number of factors may induceinappropriate expression of the transgene, and even if the transgene is

Current affiliations:1Analysis and Pharmacokinetics Research Laboratories, Drug Discovery

Research, Astellas Pharma Inc. 2-1-6, Kashima, Yodogawa-ku, Osaka City,Osaka 532-8514, Japan.

2Department of Biomedical Science, Institute of Regenerative Medicine andBiofunction, Graduate School of Medical Science, and Chromosome EngineeringResearch Center, Tottori University, 86, Nishi-cho, Yonago City, Tottori 683-8503,Japan.

3ADME and Tox Research Institute, Sekisui Medical Co., Ltd., 13-5, Nihonbashi3-chome, Chuo-ku, Tokyo 103-0027, Japan.

4PhoenixBio Co., Ltd., 3-4-1, Kagamiyama, Higashi-Hiroshima City, Hiroshima739-0046, Japan.

5Liver 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, multidrugresistance 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 combinedimmunodeficiency; SULT, sulfotransferase; UGT, uridine 59-diphospho-glucuronosyl transferase; uPA, urokinase-type plasminogen activator.

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expressed appropriately, the remaining cellular components—such asinhibitors and activators and the corresponding cofactors—are allderived from mice, resulting in failure of P450s to interact efficientlyor effectively with human genes/gene products.An alternate approach involves producing chimeric mice with humanized

liver by transplanting human hepatocytes into liver-injured immunodeficientmice that can accept xenografted human hepatocytes in their liver.This process takes a few months to produce chimeric mice highlyrepopulated with human hepatocytes. Several different methods ofinducing liver injury and different immunodeficiencies have beeninvestigated (Peltz, 2013), and chimeric mice with humanized livergenerated using urokinase-type plasminogen activator/severe combinedimmunodeficiency (uPA/SCID) mice repopulated with human hepato-cytes (PXB-mice; PhoenixBio Co., Ltd., Hiroshima, Japan) have beenreported (Tateno et al., 2004). These mice are repopulated with over70% of human hepatocytes. Expression levels and activities of P450sand non-P450 enzymes, such as uridine 59-diphospho-glucuronosyltransferase (UGT) and sulfotransferase (SULT), in the liver ofPXB-mice are similar to those in humans (Katoh et al., 2004, 2005;Nishimura et al., 2005; Katoh and Yokoi, 2007; Kitamura et al., 2008), andhuman-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 hosthepatocytes with transplanted human hepatocytes, as the presence ofresidual mouse hepatocytes hinders determination of the phenotypesof human hepatocytes. Even in chimeric mice with high hepaticreplacement ratios, metabolism profiles are often similar to those inthe control mice depending on the compounds, a phenomenon thatoccurs when examining compounds with metabolic rates in micehigher than in humans, thereby suggesting some degree of metabolismby the remaining mouse hepatocytes in the liver or host animal smallintestine, which acts as an extrahepatic metabolism organ (Kamimuraet al., 2010). Given the above, mice with their hepatocytes completelyreplaced by human hepatocytes would be ideal for hepatic metabolismstudies, but no group has yet been able to successfully rear these mice.In humans, CYP3A contributes to the oxidative/reductive metab-

olism of 50% of drugs used in the clinic and is the most abundantP450 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 distributionand substrate specificities of these CYP3A and Cyp3a enzymes show veryextensive overlap. Therefore, as one approach to overcoming the issuesof concomitant mouse metabolic activities, we have developed murineCyp3a knockout (KO) chimeric mice with humanized liver by crossingmurine Cyp3a cluster-KO mice with uPA gene–introduced SCID mice.Human hepatocytes were transplanted into the resulting liver-injuredCyp3a KO immunodeficient mice to prepare Cyp3a KO chimeric mice.Here, to investigate human-P450 and murine-P450 mRNA ex-

pressions as well as midazolam and triazolam metabolic activities inliver and intestine, and human UGT, SULT, and transporter mRNAexpressions in liver, we describe initial characterizations of Cyp3a KOchimeric mice and compare our findings with those in PXB-mice.

Materials and Methods

Chemicals. Midazolam, alprazolam, and triazolam were purchased fromWako Pure Chemical Industries, Ltd. (Osaka, Japan). 19- and 4-hydroxymi-dazolam were purchased from Toronto Research Chemicals Inc. (North York,ON, Canada). 19-hydroxytriazolam and 19 -hydroxytriazolam-d4 were pur-chased from Cerilliant Corp. (Round Rock, TX). Gefitinib was purchased from

Selleckchem (Houston, TX). All other reagents and solvents used werecommercially available and of guaranteed purity.

Generation of Cyp3a–/–/uPA+/+/SCID+/+ Mice. Cyp3a13–/– lines andCyp3a57-59–/– lines (lacking the rest of seven Cyp3a gene cluster) weregenerated in a previous study, and the two lines were mated to generateCyp3a–/– 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 ofuPA+/+/SCID+/+ mice were combined and transferred into foster mothers forartificial insemination. Litters with Cyp3a–/wt/uPA+/wt/SCID+/wt were back-crossed with uPA+/+/SCID+/+ mice via natural mating to obtain litters withCyp3a–/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 togetherto obtain Cyp3a–/–/uPA+/+/SCID+/+ mice (designated Cyp3a KO/uPA/SCIDmice). Genotype analyses of Cyp3a–/– mice (designated Cyp3a KO mice) andof uPA+/+/SCID+/+ mice (designated uPA/SCID mice) have been describedpreviously (Tateno et al., 2004; Kazuki et al., 2013). The genotypes of Cyp3a anduPA were analyzed by the genomic polymerase chain reaction (PCR) method andthe genotypes of SCID by the PCR–restriction fragment length polymorphismmethod.

Generation of Cyp3a KO Chimeric Mice with Humanized Liver. Cyp3aKO chimeric mice transplanted with human hepatocytes were prepared byPhoenixBio Co., Ltd. (Hiroshima, Japan). Human hepatocytes of two donors(5-year-old African-American boy, Lot No. BD85, and 2-year-old Hispanicgirl, 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 anesthetizedwith ether and injected with 2.5–5.0 � 105 viable hepatocytes through a smallleft-flank incision into the inferior splenic pole.

Generation of PXB-Mice. PXB-mice transplanted with human hepatocytesof a donor (5-year-old African-American boy, Lot No. BD85) were prepared byPhoenixBio Co., Ltd., as described previously (Tateno et al., 2004).

Measurement of Human Albumin. Blood samples (2 ml) were collectedperiodically from the tail vein, and the human albumin (hAlb) level was mea-sured using latex agglutination immunonephelometry (LX Reagent “Eiken”Alb II; Eiken Chemical Co., Ltd., Tokyo, Japan) to predict the replacementindex (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 anti-bodies (sc-30621; Santa Cruz Biotechnology, Inc., Dallas, TX) for the liver andintestine. The primary antibodies were visualized with Alexa 488- or 594-conjugated donkey anti-mouse-IgG or goat anti-rat IgG (Invitrogen, Carlsbad,CA) as secondary antibodies. The sections prepared from the liver and intestinewere stained with Hoechst 33342 for nuclear staining.

Measurements of the Replacement Index. Measurements of the RI ofhuman hepatocytes to total human and mouse hepatocytes have been describedpreviously (Tateno et al., 2004). Briefly, RI was determined immunohisto-chemically using anti-human CK8/18 antibodies to calculate the ratio of areaoccupied by anti-human CK8/18-positive hepatocytes to the entire area exa-mined on immunohistochemical sections.

RNA Extraction and cDNA Synthesis. Total RNA was extracted from theliver or intestine using the RNeasy minikit (Qiagen, Hilden, Germany), andcDNA was generated from a random hexamer using the high-capacity cDNAreverse transcription kit (Life Technologies, Grand Island, NY).

Quantitative Real-Time–Polymerase Chain Reaction. Quantitative real-time (qRT)-PCR was conducted using the ABI Prism 7900HT sequencedetection 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_m1for 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 forCYP2C19, Hs00164385_m1 for CYP2D6, Hs00430021_m1 for CYP3A4,Hs00426592_m1 for UGT2B7, Hs00419411_m1 for SULT1A1, Hs00960941_m1

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for SULT1E1, Hs00234219_m1 for SULT2A1, Hs00104491_m1 for MDR1, and4326315E for b-ACTIN. Other primers and probes used have been describedpreviously for UGT1A1 (Katoh et al., 2005) and UGT1A9 and MRP2 (Nishimuraet al., 2002). mRNA levels were normalized against those of b-ACTIN (human) orb-Actin (mouse). The primers for human and murine mRNA were confirmed not tocrossreact.

Preparation of Liver and Intestinal Microsomes. Liver microsomes wereprepared as described previously (Sugihara et al., 2001). Briefly, liver sampleswere homogenized in ice-cold 50 mM Tris-HCl buffer (pH 7.4) and centrifugedat 9,000g for 20 minutes at 4�C. The resulting supernatant was then centrifugedat 105,000g for 60 minutes at 4�C. The microsomal pellet was washed andresuspended in 50 mM Tris-HCl buffer (pH 7.4) and then centrifuged at 105,000gfor 60 minutes at 4�C. The final resulting pellet was then suspended in a smallamount of 250 mM sucrose and stored at 280�C until analyses. Intestinal mi-crosomes were prepared as described previously (Bonkovsky et al., 1985) withsome modifications. The inner space of small intestine samples was washed withice-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 intothree parts. These parts were placed in ice-cold solution B (pH 7.4, phosphate-buffered saline supplemented with 1.5 mM EDTA, 0.5 mM DDT, 3 IU/mlheparin, and 230 mM PMSF) and cut longitudinally. Subsequently, the mucosalcells were scraped off the cut samples with a spatula and centrifuged at 2,000g for5 minutes at 4�C. The centrifuged mucosal cells were added to ice-cold solutionC (pH 7.4, 5 mM histidine, 0.25 M sucrose, and 0.5 M EDTA), centrifuged at2,000g for 10 minutes at 4�C, and then homogenized. The homogenates werecentrifuged at 12,000g for 20 minutes at 4�C, and the supernatant was centrifugedat 104,000g for 60 minutes at 4�C. The pellet was mixed with 150 mM Tris and10 mM EDTA and then centrifuged at 104,000g for 60 minutes at 4�C. The pelletwas then resuspended in a small amount of 250 mM sucrose and stored at280�Cuntil analyses. The protein concentration was determined using a Bradford proteinassay kit (Bio-Rad, Hercules, CA) with bovine serum albumin as the standard.

Midazolam Metabolism in Liver and Intestinal Microsomes. Thereaction 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 livermicrosomes or 0.5 mg protein/ml of intestinal microsomes. After 5 minutes ofpreincubation at 37�C, the reaction was initiated by adding 1 mM NADPH tothe final concentration and allowed to proceed at 37�C for 5 minutes for livermicrosomes and intestinal microsomes. The reaction was terminated by addingformic acid–50% acetonitrile (10:90 v/v) containing alprazolam (internalstandard). The mixture was then centrifuged, and the supernatant was injectedinto the liquid chromatography–tandem mass spectrometry (LC-MS/MS) system.

Triazolam Metabolism in Liver and Intestinal Microsomes. The incu-bations were conducted in a total volume of 200 ml containing 100 mMpotassium phosphate buffer (pH 7.4), and 0.5 mg protein/ml liver microsomes orin 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

triazolam metabolism, microsomes were preincubated at 37�C for 4 minutesin the absence and presence of gefitinib (12.5 mM final concentration). Sub-sequently, triazolam (50 mM final concentration) was added and preincubated at37�C for 1 minute. The reaction was initiated by adding NADPH-regeneratingsolution and allowed to proceed at 37�C for 20 minutes for liver microsomes andintestinal microsomes. The reaction was terminated by adding acetonitrile, and19-hydroxytriazolam-d4 (internal standard) was added to the incubation mixtures.The mixture was then centrifuged, and the supernatant was injected into LC-MS/MSsystem.

LC-MS/MS Analysis for 19-Hydroxymidazolam and 4-Hydroxymidazo-lam. The concentrations of 19-hydroxymidazolam and 4-hydroxymidazolam inmicrosomal samples were measured using a TSQ Quantum Ultra system(Thermo Fisher Scientific Inc., San Jose, CA). The analytes were separatedwith a Luna C18(2) column (100 � 2.1 mm, 3 mm; Phenomenex, Torrance,CA) at 40�C 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 over0.5 minutes, maintained at 17% B for 3.5 minutes, linearly increased to 80% Bover 1.5 minutes, maintained for 2.5 minutes, and then finally kept at 5% Bfor 4.5 minutes. The mass spectrometry detection was performed using thepositive electrospray ionization selected reaction monitoring mode. The ionspray voltage was set at 4500 V, and the transfer capillary temperature wasset at 350�C. The mass spectrometer was set to monitor the transitions ofthe precursors to the product ions as follows: m/z 342.0 to 324.0 for19-hydroxymidazolam, m/z 342.0 to 325.0 for 4-hydroxymidazolam, andm/z 309.0 to 205.0 for alprazolam. Data were processed using Xcalibursoftware version 1.4.1 (Thermo Fisher Scientific Inc., San Jose, CA). Thecalibration 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 to953 nM (liver microsomes), and 1.2 to 238 nM (intestinal microsomes) for4-hydroxymidazolam.

LC-MS/MS Analysis for 19-Hydroxytriazolam. The concentration of19-hydroxytriazolam in microsomal samples was measured using a 4000 QTRAPsystem (AB SCIEX, Framingham, MA). The analyte was separated with anInertsil ODS-4 (50 � 2.1 mm, 3 mm) (GL Sciences Inc., Tokyo, Japan) at 40�Cand 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 massspectrometry detection was performed using the positive electrospray ionizationmultiple reaction monitoring mode. The mass spectrometer was set to monitorthe transitions of the precursors to the product ions as follows: m/z 359.0 to330.9 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). Thecalibration curve was linear over the range of 2.8 to 2784 nM (liver microsomesand intestinal microsomes) for 19-hydroxytriazolam.

Statistical Analysis. Data in all tables and figures are shown as the meansand 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 MouseNo. of Transplanted

Cells/Mouse

Sexof HostMouse

No. ofTransplanted

Mice

No. of Dead Mice until12 Weeks Old

No. of Miceat 12WeeksOlda

BodyWeight

at 12 WeeksOld

hAlbat 12 Weeks

Old

Portion of Chimeric Micewith RI . 70%

�105 cells grams mg/ml %

BD85 Cyp3aKO/uPA/SCID 2.5 Male 8 0 8 16.7 6 3.6 4.0 6 3.1 15.0Female 12 1 11 14.3 6 2.3 3.2 6 2.7

3.5 Male 6 1 3 14.8 6 0.8 8.5 6 6.3 53.3Female 9 2 5 13.4 6 2.2 8.5 6 3.7

5.0 Male 22 2 20 14.7 6 2.9 11.7 6 2.7 77.8Female 23 0 22 12.6 6 2.5 8.9 6 5.4

uPA/SCID 2.5 Male 212 23 158 16.3 6 3.1 10.0 6 3.0 72.9Female 43 1 39 15.7 6 2.5 8.6 6 2.7

BD195 Cyp3aKO/uPA/SCID 2.5 Male 110 4 57 18.1 6 2.6 8.1 6 4.0 65.1Female 19 0 18 16.5 6 1.9 6.9 6 3.0

3.0 Male 23 0 23 15.3 6 2.7 10.6 6 2.8 82.6

aNumber 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.

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of variance followed by Tukey’s test using PRISM 5.0 software (GraphPadSoftware Inc., San Diego, CA) for murine P450 mRNA expressions andmidazolam and triazolam metabolic activities, and by t test using WinNonlin6.1 software (Pharsight Corporation, St. Louis, MO) for triazolam metabolicactivity in the presence or absence of gefitinib.

Results

Generation of Cyp3a KO Chimeric Mice with Humanized Liver.Cyp3a KO mice were crossed with uPA/SCID mice to generate Cyp3aKO/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/SCIDmice to develop Cyp3a KO chimeric mice. In total, 80 transplantations(donor: BD85) and 152 transplantations (donor: BD195) for Cyp3a KOchimeric mice and 255 transplantations (donor: BD85) for PXB-micewere performed, and the body weight and hAlb data of 12-week-oldmice are shown in Table 1. Changes in blood hAlb levels in Cyp3a KOchimeric mice and PXB-mice (donor: BD85) are shown in Fig. 1. ThehAlb levels increased steadily after transplantation of human hepatocytesand reached a steady state at around 9 weeks or more, depending on thenumber of transplanted hepatocytes (Fig. 1). Engraftment of humanhepatocytes was inferior in the Cyp3a KO host mice compared withthe uPA/SCID host mice because hAlb levels of Cyp3a KO chimericmice were lower than those of uPA/SCID mice when the same numbersof human hepatocytes (2.5 � 105) were transplanted into the host mice(Fig. 1). Mean body weights and hAlb levels in males were higher thanthose in 12-week-old females of both Cyp3a KO and uPA/SCID chimericmice, except for Cyp3a KO chimeric mice with 3.5 � 105 BD85 donorcells (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 than6.6 mg/ml hAlb represents RI exceeding 70%. hAlb and RI of Cyp3aKO mice were determined and plotted on the correlation curvebetween hAlb and RI of uPA/SCID mice, resulting an almost identicalcorrelation (Fig. 2). The yield of chimeric mice was calculated as theportion of chimeric mice with RI . 70% (hAlb . 6.6 mg/ml) amongmice transplanted with human hepatocytes. The portion was morethan 70% when 2.5 � 105 and 5.0 � 105 BD85 donor cells weretransplanted into the uPA/SCID and the Cyp3a KO host mice, re-spectively, and 3.0 � 105 BD195 donor cells were transplanted intothe 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 metabolicactivities (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 informationon chimeric mice used is shown in Table 2.Immunohistochemistry. Immunohistochemistry of liver trans-

planted with human hepatocytes and that of the intestine from Cyp3aKO chimeric mice and PXB-mice are shown in Fig. 3. Liver sectionsprepared from the left lateral lobe in Cyp3a KO chimeric mice (at 10–11 weeks after transplantation) and PXB-mice (at 10–11 weeks aftertransplantation) were stained with anti-human CK8/18 antibodies andanti-mouse Cyp3a antibodies. The areas of anti-human CK8/18-negativemouse hepatocytes correspond to those of anti-mouse Cyp3a-positive mouse hepatocytes in PXB-mice. In contrast, the areas ofanti-human CK8/18-negative mouse hepatocytes correspond to thoseof anti-mouse Cyp3a-negative mouse hepatocytes in Cyp3a KO chimericmice, suggesting that mouse Cyp3a enzymes are not expressed in Cyp3aKO chimeric mice and that human hepatocytes are replaced in thehost liver.Intestinal sections prepared from the same mice were stained with

anti-mouse Cyp3a antibodies. Although intestinal section areas inPXB-mice were anti-mouse Cyp3a-positive, those in Cyp3a KO chimericmice were anti-mouse Cyp3a-negative, suggesting that mouse Cyp3a en-zymes 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 fromPXB-mice, Cyp3a KO chimeric mice, and Cyp3a KO/uPA/SCID mice

Fig. 2. Correlation between hAlb levels and RI of Cyp3aKO chimeric mice and PXB-mice.

Fig. 1. Changes in hAlb levels in blood of Cyp3a KO chimeric mice and PXB-mice.

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are shown in Figs. 4 and 5, respectively. In addition, murine P450s(Cyp2b10, 2c55, and 3a11) mRNA expression levels in the intestineare shown in Fig. 6. Liver and intestine samples from Cyp3a KOchimeric mice and PXB-mice at 10–11 weeks after hepatocytetransplantation and those from Cyp3a KO/uPA/SCID mice withouthepatocyte transplantation were used from three male and threefemale mice. mRNA expression levels were determined via qRT-PCR and normalized using human b-ACTIN for human P450s ormurine b-actin for murine P450s. In the liver, human P450s ex-pression levels in Cyp3a KO chimeric mice were comparable (ap-proximately 70–117% in males and 79–122% in females) to thosein male PXB-mice.

The representative isozyme of murine Cyp3a, Cyp3a11 mRNAlevel was not detected in any liver or intestine samples from Cyp3aKO chimeric mice or Cyp3a KO/uPA/SCID host mice. HepaticCyp2b10, 2c29, 2c37, and 2c55 mRNA levels in Cyp3a KO/uPA/SCID mice (without hepatocyte transplant) were 8.4-, 35-, 5.6-, and61-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 restoredto those in PXB-mice, but Cyp2c29 and 2c55 levels were 6.1- and13-fold upregulated in males, respectively, following human hepato-cyte transplantation. Intestinal Cyp2b10 and 2c55 mRNA levels in Cyp3aKO/uPA/SCID mice were 2.3- and 51-fold upregulated in males compared

Fig. 3. Immunohistochemistry of liver transplanted withhuman hepatocytes and intestine from Cyp3a KO chimericmice and PXB-mice. Sections (5-mm thick) of the liverfrom PXB-mice (A–C) and Cyp3a KO chimeric mice (D–F),and the intestine from PXB-mice (G–I), and Cyp3a KOchimeric mice (J–L) were stained with anti-mouse Cyp3agoat polyclonal antibody for A, D, G, and J in green, anti-human cytokeratin 8 and 18 (CK8/18) mouse monoclonalantibody for B and E in red, and Hoechst 33342 for H andK in blue. In addition, sections A and B or D and E weremerged with Hoechst 33342 for C or F, and sections G andH or J and K were merged for I or L, respectively.

TABLE 2

Chimeric mice used for analyses in the present study

Animal NumberSex

of host mouseNo. of Transplanted Cells

Ageat Sacrifice

Body Weightat Sacrifice

hAlbat Sacrifice

Expected RIfrom hAlb Levela

�105 cells/mouse weeks grams mg/ml %

Cyp3aKO 2-3 Male 2.5 14 17.1 11.2 87Cyp3aKO 3-4 5.0 14 12.9 11.2 87Cyp3aKO 3-6 5.0 14 12.9 15.7 97Cyp3aKO 2-5 Female 2.5 14 14.8 5.4 64Cyp3aKO 2-10 2.5 14 14.0 11.0 86Cyp3aKO 3-10 5.0 14 16.8 6.5 69PXB 194-4 Male 2.5 15 21.8 11.5 88PXB 194-22 2.5 15 20.1 8.7 79PXB 195-21 2.5 14 17.6 6.3 68PXB 196-24 Female 2.5 14 14.0 12.2 89PXB 196-26 2.5 14 17.9 9.1 80PXB 196-29 2.5 14 14.8 13.2 92

aExpected RI from hAlb level was calculated by the formula: y = 733927e0.0314x, where x = expected RI, y = hAlb.

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with PXB-mice, respectively, owing to the knockout of murine Cyp3a.In Cyp3a KO chimeric mice, intestinal Cyp2b10 and 2c55 mRNAlevels were repressed in the same manner as hepatic Cyp2b10 and 2c55levels by hepatocyte transplantation. Hepatic Cyp1a2, 2b10, and 2c37mRNA levels did not differ significantly between Cyp3a KO chimericmice 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 19-

and 4-hydroxylation activities in liver and intestinal microsomesprepared from PXB-mice, Cyp3a KO chimeric mice, and Cyp3a KO/uPA/SCID mice are shown in Table 3. In liver microsomes, bothmidazolam 19- and 4-hydroxylation activities in Cyp3a KO chimericmice were comparable to those in PXB-mice, although those in Cyp3aKO/uPA/SCID mice (without hepatocyte transplant) were lower owingto the knockout of murine Cyp3a. In intestinal microsomes, bothmidazolam 19- and 4-hydroxylation activities in Cyp3a KO chimeric micewere much lower than those in PXB-mice and nearly equal to those inCyp3a 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/SCIDmice as well as humans (liver microsomes only) is shown in Table 4. Inaddition, triazolam was coincubated with gefitinib to investigate whethertriazolam 19-hydroxylation activity was stimulated in both liver andintestinal microsomes. In liver microsomes, triazolam 19-hydroxylationactivity in Cyp3a KO chimeric mice was comparable to that in PXB-miceand humans, whereas the lower activity in Cyp3a KO/uPA/SCID mice(without hepatocyte transplant) was attributed to the knockout of murineCyp3a. After coincubation with gefitinib, triazolam 19-hydroxylationactivity was significantly stimulated in PXB-mice and humans, andtended to increase in Cyp3a KO chimeric mice, and significantlyinhibited in uPA/SCID mice. In intestinal microsomes, triazolam19-hydroxylation activity was not detected in either Cyp3a KO chimericmice or Cyp3a KO/uPA/SCID host mice.Human UGTs and SULTs Expression. Human UGTs (UGT1A1,

1A9, and 2B7) and SULTs (SULT1A1, 1E1, and 2A1) mRNAexpression levels in the liver from PXB-mice, Cyp3a KO chimericmice, and Cyp3a KO/uPA/SCID mice are shown in Fig. 7. HepaticUGT and SULT mRNA levels did not differ significantly betweenCyp3a 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 KOchimeric mice, and Cyp3a KO/uPA/SCID mice are shown in Fig. 8.Hepatic MDR1 and MRP2 mRNA levels did not differ significantlybetween Cyp3a KO chimeric mice and PXB-mice.

Discussion

We successfully developed Cyp3a KO chimeric mice with humanizedliver via transplantation of human hepatocytes into liver-injured Cyp3aKO immunodeficient mice, which were generated through the con-ventional approach of crossing Cyp3a cluster KO mice with uPAgene–introduced SCID mice. The initial characterization of Cyp3a KOchimeric mice was conducted to investigate human P450s and murineP450 mRNA expression levels, as well as midazolam and triazolammetabolic activities in liver and intestine, and human UGT, SULT, andtransporter mRNA expression levels in liver, and then these findingswere compared with PXB-mice.In our initial attempt to generate Cyp3a KO chimeric mice, the

replacement of mouse hepatocytes with human hepatocytes aftertransplantation was much less successful than in PXB-mice (data not

Fig. 4. Human P450 mRNA expression in liver of PXB-mice, Cyp3a KOchimeric mice, and Cyp3a KO/uPA/SCID mice. Human P450 mRNA expressionwas determined via qRT-PCR and normalized using the mRNA expression ofhuman b-ACTIN. The primer for human P450s and b-ACTIN used in the presentstudy 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, notdetected.

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shown), probably owing to differences in background genes betweenCyp3a KO mice and uPA/SCID mice. Therefore, several continuousbackcrosses with uPA/SCID mice were applied to increase the geneticbackground of the uPA/SCID mice. While engraftment and/or growthof human hepatocytes in the Cyp3a KO host mice were inferior to thePXB-host mice, hAlb levels of Cyp3a KO chimeric mice transplantedwith 5.0 � 105 human hepatocytes were similar to those of PXB-micetransplanted with 2.5� 105 human hepatocytes. In addition, engraftmentand/or growth of human hepatocytes were different between BD85- andBD195-transplanted Cyp3a KO chimeric mice. The chimeric micetransplanted with BD195 donor cells showed higher hAlb levels in theblood 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 theblood of PXB-mice from a previous study (Tateno et al., 2004) andalso those of Cyp3a KO chimeric mice in the present study. Of note,the higher cell number for transplantation resulted in higher hAlblevels in Cyp3a KO chimeric mice.One of the major limitations inherent to current PXB-mice is the

incomplete replacement of mouse hepatocytes with human hepato-cytes (Yoshizato et al., 2012). One example of problematic cases isthat of chimeric mice administered a drug candidate that is wellmetabolized by mouse hepatocytes but metabolized only to a limitedextent by human hepatocytes, and all its human metabolites aredetected as its mouse metabolites (Kamimura et al., 2010). In sucha case, the plasma metabolic profile is similar to that of control mice,and no significant increase in the peaks of human metabolites isfound. To overcome the metabolic activity caused by residual mousehepatocytes, we eliminated the contribution of residual host mouse

Fig. 6. Murine P450 mRNA expression in the intestine of PXB-mice, Cyp3a KOchimeric mice, and Cyp3a KO/uPA/SCID mice. Murine P450 mRNA expressionwas determined via qRT-PCR and normalized using the mRNA expression ofmurine 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 weredetermined using one-way analysis of variance followed by Tukey’s test. *P , 0.05versus Cyp3a KO/uPA/SCID mice. ND, not detected.

Fig. 5. Murine P450 mRNA expression in liver of PXB-mice, Cyp3a KO chimericmice, and Cyp3a KO/uPA/SCID mice. Murine P450 mRNA expression was determinedvia qRT-PCR and normalized using the mRNA expression of murine b-actin. The primerfor murine P450s and b-actin used in the present study was confirmed not to crossreactwith 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 weredetermined 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.

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hepatocytes from the currently available PXB-mice by generatingCyp3a KO chimeric mice.To date, the usefulness of PXB-mice has been recognized in studies

for the prediction of human metabolism of drugs (Yoshizato andTateno, 2009a, b). In the liver of PXB-mice, the human enzymesthat play important roles in drug metabolisms include several P450enzymes, such as CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6,3A4, and 3A5 and non-P450 enzymes such as UGT, SULT, glu-tathione 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 ofselected P450s (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4),UGTs (UGT1A1, 1A9, and 2B7), and SULTs (SULT1A1, 1E1, and2A1) were measured in the liver to determine whether Cyp3a KO miceare as useful as PXB-mice in drug metabolism and pharmacokineticresearch. 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 investigatethe impact of the expression of other murine P450s. Given the cor-relation between hAlb levels and the RI, we concluded that theexpression levels of human P450s, UGTs, and SULTs after thetransplantation of human hepatocytes into mice did not differ sig-nificantly between Cyp3a KO chimeric mice and PXB-mice. Althoughthe expression levels of limited human metabolizing enzymes have

been investigated, our results suggest that the Cyp3a KO chimericmouse is a viable model for drug metabolism and pharmacokineticsresearch 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 andCyp2c55 in the intestine were observed in Cyp3a KO/uPA/SCID mice.These findings agree with those of previous reports that found thedeletion of the murine Cyp3a cluster to be associated with consider-able compensatory changes in other gene families involved in drugmetabolism and pharmacokinetics (van Waterschoot et al., 2009b).Interestingly, the upregulated hepatic and intestinal Cyp2b10 expres-sion levels observed in Cyp3a KO/uPA/SCID mice (without humanhepatocytes) were restored to near-normal levels in Cyp3a KOchimeric mice that received transplanted human hepatocytes, andremarkable reductions in the expression of hepatic Cyp2c29 and 2c37,and hepatic and intestinal Cyp2c55 from upregulated levels, were alsoobserved, suggesting that transplanted human hepatocytes were able tocompensate in part for the loss of murine Cyp3a enzymes not only inthe liver but also in the intestine. Although further investigations areneeded, the metabolism of dietary phytochemicals of P450 inducersand/or the synthesis or biotransformation of endogenous substances,such as bile acids, might be involved in this compensatory mechanismbecause 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 uPA/SCID mice 1670 6 151 1250 6 142*Cyp3a KO/uPA/SCID mice 20.7 6 3.49††† 23.5 6 6.23Cyp3a KO chimeric mice 293 6 120†††, ‡ 699 6 285PXB-mice 413 6 30.3†††, ‡‡ 617 6 39.0**Humans 385 6 5.29 946 6 10.2***

Intestinal microsomes uPA/SCID mice 11.5 6 11.9 11.6 6 11.7Cyp3a KO/uPA/SCID mice ND NDCyp3a KO chimeric mice ND NDPXB-mice 5.94 6 10.1 5.71 6 9.69

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/SCIDmice; ‡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 inliver (0.5 mg/ml) and intestinal (1 mg/ml) microsomes without or with gefitinib (12.5 mM) at 37�C for 20 minutes. Data are shown as mean 6S.D. obtained from three male mice or triplicate incubations in human liver microsomes. Statistical differences in the presence or absence ofgefitinib were determined by t test.

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 Cyp3a KO/uPA/SCID mice 281.5 6 21.1 63.9 6 14.0Cyp3a KO chimeric mice 846.2 6 372.3* 420.9 6 194.8*PXB-mice 611.7 6 32.0 306.1 6 6.5

Intestinal microsomes Cyp3a KO/uPA/SCID mice 0.6 6 0.4 0.7 6 0.1Cyp3a KO chimeric mice 1.3 6 0.7 0.9 6 0.0PXB-mice 14.9 6 15.5 9.8 6 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 37�C for 5 minutes. Data are shown as mean 6 S.D. obtained fromthree male mice. Statistical differences in three mice groups were determined using one-way analysis of variance followed by Tukey’s test.

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It has been reported that the biotransformation of midazolam iscomparable in both humans and mice, yielding 19-hydroxymidazolamas the major metabolite and 4-hydroxymidazolam as a minor metabolite,and that 19-hydroxymidazolam formation in mice is not only dependenton murine Cyp3a but also imbued with significant murine Cyp2ccomponents, whereas 4-hydroxymidazolam formation is consideredspecific to murine Cyp3a (van Waterschoot et al., 2008). Consistentwith that report, the present study demonstrated that midazolam19-hydroxylation in the liver microsomes of Cyp3a KO/uPA/SCIDmice was detectable with partial compensation, probably via inducedmurine Cyp2c enzymes following murine Cyp3a knockout, and that4-hydroxylation in the liver microsomes of Cyp3a KO/uPA/SCIDmice was reduced. Further, relatively little midazolam metabolism wasdetected in the intestinal microsomes of either Cyp3a KO chimericmice or Cyp3a KO/uPA/SCID mice, indicating that the expressionlevel of induced murine Cyp2c enzymes is too low to significantlycontribute to the midazolam metabolism in the intestine after theknockout of murine Cyp3a. Midazolam 19- and 4-hydroxylations inCyp3a KO chimeric mice were comparable with findings in livermicrosomes 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 murineP450 enzymes (Perloff et al., 2000). Although some residual metab-olism of triazolam was still mediated by murine Cyp2c enzymes inCyp3a KO mice, the compensatory triazolam metabolism was muchlower compared with midazolam metabolism (van Waterschoot et al.,2009a). From the results in this study, triazolam was metabolized inliver microsomes of uPA/SCID mice but scarcely metabolized inCyp3a KO/uPA/SCID mice, suggesting involvement of murine Cyp3ain triazolam metabolism of 19-hydroxylation. Triazolam 19-hydroxylation

in liver microsomes of Cyp3a KO chimeric mice was more than10-fold higher than that of Cyp3a KO/uPA/SCID mice. The formationof 19-hydroxylation triazolam was increased by more than twice withgefitinib in liver microsomes of Cyp3a KO chimeric mice, althoughfindings have no significance between those without and with gefitinibin liver microsomes of Cyp3a KO chimeric mice, probably owing tointervariability within the limited number of the mice tested. As it wasreported that the anticancer drug gefitinib significantly stimulatedtriazolam metabolism in human liver microsomes (van Waterschootet al., 2009a), these results suggest that the triazolam metabolism inCyp3a KO chimeric mice is attributable to human liver. Triazolam19-hydroxylation in Cyp3a KO chimeric mice was comparable to thefinding in liver microsomes of PXB-mice after human hepatocytetransplantation, regardless of murine Cyp3a knockout. In intestinalmicrosomes, 19-hydroxytriazolam was scarcely formed in any micro-somes 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 inhumanized animal models, the present findings suggest that Cyp3aKO chimeric mice could prove more useful than PXB-mice, becausetheir lack of significant metabolism in the intestine improves pre-dictability of human metabolites. The present study also demonstrateda similarity in the mRNA expression levels of the transporters human

Fig. 8. Human MDR1 and MRP2 mRNA expression in liver of PXB-mice, Cyp3aKO chimeric mice, and Cyp3a KO/uPA/SCID mice. Human MDR1 and MRP2mRNA expression was determined via qRT-PCR and normalized using the mRNAexpression of human b-ACTIN. Primers for human MDR1, MRP2, and b-ACTINused in the present study were confirmed not to crossreact with mouse mRNA. Dataare shown as mean + S.D. obtained from three male mice (A, B). ND, not detected.

Fig. 7. Human UGT and SULT mRNA expression in liver of PXB-mice, Cyp3a KOchimeric mice, and Cyp3a KO/uPA/SCID mice. Human UGT and SULT mRNAexpression was determined via qRT-PCR and normalized using the mRNAexpression of human b-ACTIN. Primers for human UGTs, SULTs, and b-ACTINused in the present study were confirmed not to crossreact with mouse mRNA. Dataare shown as mean + S.D. obtained from three male mice (A–F). ND, not detected.

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MDR1 and MRP2, which both contribute to the distribution and ex-cretion 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-micebut with liver and intestine not expressing murine Cyp3a, a propertywhich may prove useful in drug metabolism and pharmacokineticsresearch. Further, the method of generating Cyp3a KO chimeric micedescribed here may be useful for generating humanized animal modelswith knockout of other murine P450s, non-P450s, and transporters,although the possibility of unexpected side effects (upregulation ordownregulation) after knockout of these genes cannot be excluded; assuch, any future attempts at generating new models should be conductedwith caution.

Acknowledgments

The authors thank Hiroki Ebine, Satoko Yoshida, Miyoko Ono, andToshihiro Sasaki in Sekisui Medical Co., Ltd. for triazolam metabolism studyusing liver and intestinal microsomes.

Authorship ContributionsParticipated 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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Yoshizato K and Tateno C (2009a) A human hepatocyte-bearing mouse: an animal model topredict drug metabolism and effectiveness in humans. PPAR Res 2009:476217.

Yoshizato K and Tateno C (2009b) In vivo modeling of human liver for pharmacological studyusing humanized mouse. Expert Opin Drug Metab Toxicol 5:1435–1446.

Yoshizato K, Tateno C, and Utoh R (2012) Mice with liver composed of human hepatocytes as ananimal model for drug testing. Curr Drug Discov Technol 9:63–76.

Zhu Y, Ding X, Fang C, and Zhang QY (2014) Regulation of intestinal cytochrome P450expression by hepatic cytochrome P450: possible involvement of fibroblast growth factor 15and impact on systemic drug exposure. Mol Pharmacol 85:139–147.

Address correspondence to: Dr. Chise Tateno, PhoenixBio Co., Ltd, 3-4-1,Kagamiyama, Higashi-Hiroshima City, Hiroshima 739-0046, Japan. E-mail: chise.mukaidani@phoenixbio.co.jp

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