Troglitazone metabolism and transporter effects in chimeric mice: a comparison between chimeric humanized and chimeric murinized FRG mice.

http://informahealthcare.com/xen ISSN: 0049-8254 (print), 1366-5928 (electronic) Xenobiotica, 2014; 44(2): 186–195 ! 2014 Informa UK Ltd. DOI: 10.3109/00498254.2013.879237

RESEARCH ARTICLE

Troglitazone metabolism and transporter effects in chimeric mice: a comparison between chimeric humanized and chimeric murinized FRG mice Kristin Samuelsson1, Kathryn Pickup2, Sunil Sarda3, John R. Foster4, Kevin Randall4, Anna Abrahamsson1, Matt Jacobsen4, Lars Weidolf5, and Ian Wilson6* RIA iMED DMPK, AstraZeneca, Mo¨lndal, Sweden, 2Oncology iMED DMPK, AstraZeneca, Macclesfield, UK, 3Discovery Sciences, AstraZeneca, Macclesfield, UK, 4Drug Safety & Metabolism, AstraZeneca, Macclesfield, UK, 5CVMD iMED DMPK, AstraZeneca, Mo¨lndal, Sweden, and 6Department of Surgery and Cancer, Imperial College, London, UK Abstract

Keywords

1. The biotransformation, hepatic transporter and blood chemistry effects of troglitazone were investigated following 7 days of dosing at 600 mg/kg/day to chimeric murinized or humanized FRG mice, Mo-FRG and Hu-FRG mice, respectively. 2. Clinical chemistry and histopathology analysis revealed a significant drop in humanization over the time course of the study for the Hu-FRG mice but no significant changes associated with troglitazone treatment in either the Mo-FRG or the Hu-FRG models. No changes in transporter expression in livers of these mice were observed. Oxidative and conjugative metabolic pathways were identified with a 15- to 18-fold increase in formation of troglitazone sulfate in the Hu-FRG mice compared with the Mo-FRG mice in blood and bile, respectively. This resembles the troglitazone metabolism in human and these data are comparable with the formation of this metabolite in the chimeric uPA+/+/SCID mice. 3. However, larger amounts of troglitazone glucuronide were also observed in the Hu-FRG mouse compared with the Mo-FRG mouse which may be an effect of the drop in humanization of the Hu-FRG mouse during the study. 4. Highly humanized mice have a considerable potential in providing a useful first insight into circulating human metabolites of candidate drugs metabolized in the liver.

Chimeric mice, FRG mouse, human hepatocytes, PXB mouse, troglitazone

Introduction The ability to determine the likely metabolic fate of compounds in humans, before the first doses are given to patients, is highly desirable for the prediction of efficacy and safety, and would greatly help to ‘‘de-risk’’ the development of drug candidates. One potential route to obtaining this information during the preclinical phases of drug discovery may be through the use of ‘‘humanized’’ mouse models for studying drug metabolism. A potentially valuable in vivo model for studying human metabolism is provided by ‘‘chimeric’’ mice, in which the mouse hepatocytes have been replaced by human hepatocytes. This humanization process is performed via the infusion of human hepatocytes into immuno-compromised mice (Inoue et al., 2008, 2009; Katoh & Yokoi, 2007; Kamimura et al., 2010; Lootens et al., 2011; Okumura et al., 2007; Serres et al., 2011; Strom et al., *Formerly Department of Drug Metabolism and Pharmacokinetics iMED, AstraZeneca, Alderley Park, AstraZeneca UK Ltd., Macclesfield, Cheshire, UK. Address for correspondence: Kristin Samuelsson, RIA iMED DMPK, AstraZeneca, Pepparedsleden 1, 43183 Mo¨lndal, Sweden. Tel: +46-317761822. E-mail: [email protected]

History Received 11 November 2013 Revised 23 December 2013 Accepted 23 December 2013 Published online 13 January 2014

2010; Tateno et al., 2004). In the humanized livers the expression of Phase I and II enzymes, as well as transporter proteins, has been shown to demonstrate a similar profile to that present in the original human donor (Strom et al., 2010; Tateno et al., 2004; Yamazaki et al., 2010; Yoshizato & Tateno, 2009). The chimeric mouse model has been used for a number of in vivo metabolism studies and has demonstrated the ability to generate primary as well as secondary metabolites, including human specific metabolites (Aoki et al., 2006; Inoue et al., 2008, 2009; Kamimura et al., 2010; Kitamura et al., 2008; Schulz-Utermoehl et al., 2012; Serres et al., 2011). Currently only a limited number of differently derived chimeric mouse models are commercially available, and all possess two common denominators: (a) the mice are immune compromised to allow engraftment of human hepatocytes and (b) they have an induced liver failure that destroys the native mouse hepatocytes. The model investigated in this study is the FRG chimeric mouse, which has three genes knocked out. R is the recombination-activating gene 2 (Rag2) and G is the IL-2 receptor subunit g (Il2rg). By knocking out these two genes an immune-compromised mouse which lacks B- and T-cells and NK cells, respectively, is generated. The F in FRG

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denotes the lack of fumarylacetoacetate hydrolase (Fah) which accounts for a type of liver failure associated with Type 1 hereditary tyrosinemia (TH1) in humans. The lack of the FAH enzyme results in increased and toxic amounts of metabolites produced in the catabolic pathway of tyrosine which is fatal. However, this mouse phenotype, and also in humans with this disease, is ablated by the administration of an inhibitor of the 4-hydroxyphenylpyruvate oxidase enzyme that generates the toxic metabolites in this model, 2-(2-nitro-4-trifluoro-O-methylbenzolyl)-1,3-cyclohexanedione (NTBC), in the diet. Thus, the FRG mice transplanted with human hepatocytes are ‘‘cycled’’ on NTBC to ensure that the animals survive with engraftment of human hepatocytes and destruction of the mouse hepatocytes occurring during the periods of NTBC withdrawal. The aims of the current study were to investigate the metabolism and effects on hepatic transporters of troglitazone in the chimeric humanized FRG mice (Hu-FRG) and compare their profiles with chimeric murinized mice (Mo-FRG) as well as with data obtained from our previous study performed in the chimeric uPA+/+/SCID mouse (PXB mouse) (SchulzUtermoehl et al., 2012). In both studies, troglitazone was dosed orally at 600 mg/kg/day for 7 days, with serial blood sampling on the last day of dosing. Troglitazone, a peroxisome proliferator-activated receptor gamma agonist, was developed for treatment of type II diabetes mellitus, and showed good clinical efficacy in terms of lowering the glucose levels. However, in clinical use it was found to be a human selective hepatotoxin and was withdrawn from the market due to rare idiosyncratic hepatic effects and some deaths through liver failure in patients. There seem to be various possible mechanisms potentially responsible for the idiosyncratic hepatotoxicity observed, one of which may be related to reactive metabolites. Also, a number of reactive metabolites have been observed in human in vitro systems and it is unknown whether species differences in forming reactive metabolites could in part explain the human specific hepatotoxicity that was observed (Kassahun et al., 2001; Tettey et al., 2001). Troglitazone undergoes extensive hepatic metabolism and forms predominantly troglitazone sulfate in human, rat, dog, marmoset and cynomolgus monkey while troglitazone glucuronide was the predominant metabolite in mouse, thus making it an interesting compound to study in the chimeric humanized mouse model (Izumi et al., 1996; Kawai et al., 1997; Loi et al., 1999a,b). Consequently, we aimed to characterize the Hu-FRG mouse to see whether it was a useful animal model to study human metabolism and effects on hepatic transporters in vivo. We also investigated the hepatic immunohistopathology effects from repeated dosing of troglitazone and finally compared the Hu-FRG and PXB mouse models.

Materials and methods

Troglitazone metabolism in chimeric-humanized FRG mouse

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BSEP antibodies were raised in the rabbit and generated by the antibody generation group at AstraZeneca Ltd., UK (antibody clone H3310) and used at a dilution of 1:50. MRP2 antibodies were raised in rabbits and sourced from AbCam (Cambridge, UK, antibody clone ab58709) and used at a concentration of 1:50. Antibodies against Ki67 were obtained from Dako UK Ltd., Cambridge, UK) – clone MIB1 and were used at a concentration of 1:200, while those against human albumin were obtained from Sigma Aldrich, Gillingham, UK (Product Number 3293) and used at a concentration of 1:100. Animals The in-life phase animal study was approved by IACUC and conducted at Yecuris (Tualatin, OR). Eight male humanized FRG KO/NOD (Hu-FRG) mice transplanted with HepaCurTM hepatocytes (Yecuris, Tualatin, OR) from an 11 year old male human donor and eight male mice transplanted with normal mouse hepatocytes (Mo-FRG) were generated by Yecuris (Tualatin, OR). All Hu-FRG mice in the study were aged between 7 and 7.5 months and the Mo-FRG mice were between 5.5 and 6 months old. Mice of each type were divided into two groups, (n ¼ 4 each) and dosed orally with troglitazone (600 mg/kg/day) formulated in 0.5% HPMC in 0.1% aqueous polysorbate 80 or vehicle for 7 days. Prior to the first dose all mice were off CuRxTM Nitisone (NTBC) for 14 days and were given standard acidified drinking water. Blood samples (20 mL) for metabolism profiling were taken from the tail vein of the mice into EDTA-treated microtainer blood collection tubes (Franklin Lakes, NJ, reference number 365973) at day 1 prior to any dosing and on day 7 post-dosing at 0.5, 1, 2, 3, 4, 8 and 12 h. At the 12 h time point, the mice were terminated using a dose (1 mL/kg of body weight) of ketamine (7.5 mg/mL), xylazine (1.5 mg/mL), and acepromazine (0.25 mg/mL), and whole blood was collected by cardiac puncture. The terminal blood samples were centrifuged (7500 g for 5 min) to generate plasma. The degree of humanization of each mouse was estimated by measuring the amount of human albumin present in the mouse plasma by ELISA assay (Bethyl Laboratories, Montgomery, TX) taken from the humanized animals one week prior to the start of the study to ensure that the index of replacement had reached >90%. The degree of humanization was also measured in the terminal bleed for the Hu-FRG mice. All blood samples were stored at 80 C before shipment to AstraZeneca Ltd., UK. Livers from each animal were excised at the time of termination and gall bladders were removed before the liver weights were recorded. The livers were fixed as described in the section for preparation of liver samples for immunohistochemistry. Gall bladders were flash frozen in liquid nitrogen and stored at 80 C before shipment to AstraZeneca Ltd., UK.

Chemicals

Blood chemistry

Troglitazone (95%) was synthesized for research use only by AstraZeneca Sweden (Mo¨lndal, Sweden). Fisher Scientific (Loughborough, UK) supplied acetonitrile and all other chemicals or solvents were purchased from commercial suppliers and were of analytical grade or the best equivalent.

Plasma samples for clinical chemistry were obtained 12 h post dose on day 7 and prepared as described above. Due to the low sample volume obtained, the plasma was diluted with isotonic saline to obtain sufficient volume to allow for a range of standard clinical chemistry analyses to be undertaken.

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The conducting laboratory had validated this method for a variety of analytes up to a maximum dilution of 1:4. Plasma samples were then analyzed for concentrations of albumin, alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), total bilirubin, total protein, glutamate dehydrogenase (GLDH), gamma glutamyl transferase (GGT), cholesterol and bile acids using a Roche P Modular analyser (Roche Diagnostics, Burgess Hill, UK) and standard Roche reagents. In all cases, the methods used did not differentiate between the human and murine forms of the analytes and the results obtained represented a total value or enzyme activity.


Preparation of liver samples for immunohistochemistry All tissues were fixed in 10% buffered formalin for 48–72 h before being dehydrated in graded ethanols, cleared in toluene and embedded in paraffin wax. Sections, 5-mm thick, were prepared and mounted onto glass slides before being stained with hematoxylin and eosin or by immunohistochemistry using the antibodies against BSEP and MRP2. All immunohistochemistry procedures were carried out on the Ventana Discovery immunostainer (Ventana Medical Systems, Tucson, AZ). Before the immunohistochemistry procedure, sections were dewaxed and subjected to an antigen retrieval methodology using citrate buffer on the Ventana immunostainer. Following incubation with the respective primary antibodies, the sections were washed and exposed to the rabbit Ventana OmniMAP DAB anti-rabbit visualization system (Ventana Medical Systems, Tucson, AZ). The sections were then counterstained with hematoxylin before being examined and photographed using a Leica DMLB light microscope. Sample preparation for metabolite identification Aliquots of blood (2 mL) from each mouse within a group were pooled according to time point. An equal volume of water was added before precipitation using six volumes of acetonitrile. The samples were mixed and centrifuged at 20 800g, 4 C, for 10 min and the supernatants were diluted with water (1:1) and 5 mL of each taken for analysis (see below). Gall bladders were pooled according to mouse group, and the weight of each pool was recorded. Eight volumes (w/v) of acetonitrile were added to pooled bile samples and were vigorously mixed before centrifugation at 20 800g, 4 C, for 10 min. Supernatants were removed and diluted with two volumes of water before analysis. Blood sample metabolite analysis, profiling and identification by UPLC-MSn, system 1 Extracts of blood samples were analyzed by UPLC-MS. Chromatographic separations were performed on an Acquity UPLC BEH C18 column 2.1 100 mm, 1.7 mm i.d. (Waters, Elstree, UK). The LC system consisted of an Acquity UPLC (Waters, Elstree, UK) with a binary solvent manager operating at a flow rate of 0.5 mL/min. The column was maintained at a temperature of 40 C and samples were kept at 10 C. Separation was achieved by gradient chromatography with an aqueous mobile phase (A) of 0.1% formic acid

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in water and acetonitrile as the organic mobile phase (B). The initial mobile phase composition was 10% solvent B which was increased to 70% over a period of 6 min. Solvent B was then instantly increased to 90% and maintained at this level for 0.7 min before returning to the initial conditions. The postcolumn eluent was directed to a Synapt G2 Q-TOF mass spectrometer (Waters, Elstree, UK) with an electrospray ionization (ESI) interface operating in negative ion mode. The specific mass spectrometer interface conditions were: cone voltage 35 kV, capillary voltage 2 kV, source temperature 120 C, desolvation temperature 550 C and desolvation gas flow 850 L/h. The instrument was calibrated using sodium formate in negative ion mode with a mass window of m/z 50– 1200 and leucine-enkephalin was used as the lock mass for the acquisition. The accuracy and sensitivity of the instrument was checked before analysis with a set of standard compounds. Negative ion mass spectra were acquired over a mass range m/z 80–1200 in centroid mode. MSMS scans of identified ions were acquired by ramping the collision energy from 15 to 35 V. The instrument control (UPLC system and mass spectrometer), data acquisition and evaluation was controlled via MassLynx version 4.1 software (Waters, Manchester, UK). Bile sample metabolite analysis, profiling and identification by HPLC-MSn, system 2 Troglitazone and its metabolites contained in bile extracts were injected (25 mL) onto and separated on a 2.7 mm particle size C18 reversed-phase HALOTM column, 150 4.6 mm i.d. (Advanced Materials Technologies, Wilmington, DE), using an Agilent 1200 binary pump HPLC system (Agilent Technologies, Stockport, UK) at a flow rate of 0.5 mL/min. The column was maintained at a temperature of 40 C using a column temperature control and autosampler unit (Agilent Technologies, Stockport, UK). Separation was achieved by gradient chromatography with an aqueous mobile phase (A) of 10 mM ammonium acetate (unadjusted, ca. pH 6.8) and acetonitrile as the organic mobile phase (B). The initial mobile phase consisted of 5% solvent B, which was held for 5 min before being increased to 35% over a period of 3 min, followed by an increase to 60% over 42 min. The final increase to 95% of solvent B was performed over a 2 min period, at which it was maintained for 5 min before returning to 5% solvent B for column equilibration for 3 min prior to the next injection. The post-column eluent flowed into a ThermoScientific LTQ Orbitrap XL hybrid linear ion trap instrument (ThermoScientific, Bremen, Germany) fitted with an ESI source, operating in negative ion mode. The instrument was operated under troglitazone-optimized conditions (source voltage of 3 kV, capillary voltage of 30 V, sheath and auxiliary gas (nitrogen) flow of 65 and 10 arbitrary units, respectively, a tube lens voltage of 102 V and capillary temperature at 300 C). The initial full MS scan ranged from m/z 200 to 800 in negative ion mode, followed by a datadependent MS2 product ion scan of the most abundant peak from a user-defined list of molecular ion masses representing the range of expected metabolites. Further molecular masses were added to this list in subsequent analyses following initial data processing to ensure any putative unexpected metabolites


DOI: 10.3109/00498254.2013.879237

were also subjected to collisionally induced fragmentation. The final scan consisted of a data-dependent MS3 product ion scan (collision energy normalized to 40% for parent compound) following isolation of the most abundant fragment ion observed in the MS2 scan.


Metabolite profiling data analysis The metabolism of troglitazone was assessed by qualitative analysis of the chromatographic profiles. MS data obtained for the circulating metabolites were processed both manually and with MetaboLynx software (Waters, Elstree, UK) for metabolites by comparison with samples taken before dosing allowing elimination of false positives for potential metabolites due to the presence of endogenous compounds. MS data obtained for the bile metabolites were processed manually using Xcalibur v2.2 software (ThermoScientific, Hemel Hempstead, UK) for metabolites by comparison with samples taken from the vehicle control group to eliminate false positives for potential troglitazone metabolites.

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significant (p ¼ 0.015) decrease in mean body weight from day 1 to day 7, 30.0 g ± 0.4 to 27.5 g ± 0.5, respectively. Absolute liver weights at necropsy were also markedly larger in the Hu-FRG mice than in the Mo-FRG control group, a trend that applied between vehicle treated (p ¼ 0.029) and troglitazone treated mice (p ¼ 0.00002) (Table 1). Troglitazone dosing had no effect on the liver to body weight ratios in either of the mouse types. However, the difference in liver to body weight ratio between the two types of mice (Hu-FRG compared with Mo-FRG) was significant for both the troglitazone-treated and the vehicle-treated mice (p ¼ 0.0025 and p ¼ 0.000001, respectively) indicating that the livers in the Hu-FRG mice were larger than those in the Mo-FRG mice. The replacement index of mouse hepatocytes with human hepatocytes for the Hu-FRG mice was above 90% for all individuals one week before the study started (based upon the amount of human albumin present in the plasma) but was reduced to 41% and 45% after one week of treatment using vehicle and troglitazone, respectively (Table 1). Blood chemistry

Results General condition of the mice The mice were observed pre- and post-dosing and all were healthy throughout the study, with the exception of two animals. One mouse from the troglitazone treated Hu-FRG mouse group was euthanized on day 4 due to severe head and face infection and one Mo-FRG mouse died 1 h after dosing with troglitazone on day 7. No statistical differences were observed in the mean body weight on day 1 and day 7 of Hu-FRG and Mo-FRG mice treated with vehicle and for the Mo-FRG group dosed with troglitazone (Table 1). However, the Hu-FRG group dosed with troglitazone showed a

The clinical chemistry data generated from terminal plasma samples taken 12 h post-dose on day 7 from both types of mice are summarized in Table 2. The Hu-FRG mice results reflected value or total activity derived from both human and murine hepatocytes, as the analysis undertaken was based on colormetric methods that could not differentiate between mouse and human isoforms. The vehicle-treated Hu-FRG mice showed levels of GGT, cholesterol and total bilirubin which were broadly in line with human reference ranges. In the vehicle-treated Mo-FRG mice, albumin, ALT, cholesterol, GGT, total protein, total bilirubin and bile acids were similar to the expected range for mouse strains commonly

Table 1. Summary of Hu-FRG and Mo-FRG mice conditions.

Dose N Estimated replacement indexa Body weight (g)b Body weight (g)c Liver weight (g)d Liver to body ratio (%)e

Hu-FRG

Hu-FRG

Mo-FRG

Mo-FRG

Vehicle 4 41 ± 22 30.0 ± 2.4 28.7 ± 4.9 2.49 ± 0.34 8.50 ± 1.39

600 mg/kg 3 45 ± 10 30.0 ± 0.4 27.5 ± 0.5 2.54 ± 0.10 9.24 ± 0.29

Vehicle 4 NA 29.2 ± 3.0 28.9 ± 2.6 1.34 ± 0.33 4.59 ± 0.74

600 mg/kg 4 NA 28.0 ± 3.6 26.7 ± 3.2 1.23 ± 0.12 4.60 ± 0.14

Results are expressed as the mean ± SD. NA, not applicable. Determined post dose on day 7. Body weights on day 1. c Body weights on day 7. d Determined post necropsy on day 7. e The body weight recorded prior dosing on day 7 was used. a

b

Table 2. Summary of clinical chemistry results of plasma samples.

Strain

Dose (mg/kg) n

ALB (g/l)

ALT (IU/l)

ALP (IU/l)

AST (IU/l)

Hu-FRG Vehicle 4 30.8 ± 6.2 154.3 ± 17.3 29.0 ± 26.5 908.3 ± 812.7 Mo-FRG Vehicle 4 33.0 ± 8.8 56.5 ± 5.9 14.0 ± 22.4 301.5 ± 128.3 Hu-FRG 600 3 36.3 ± 3.5 188.3 ± 62.3 36.7 ± 36.7 1053.3 ± 657.1 Mo-FRG 600 4 27.0 ± 7.4 73.0 ± 26.4 4.0 ± 4.6 279.5 ± 84.6 Data are given as mean ± SD.

CHOL (mmol/l)

GGT (IU/l)

GLDH (mmol/l)

TBIL (IU/l)

3.9 ± 1.5 77.8 ± 63.8 217.0 ± 87.4 2.8 ± 2.1 2.8 ± 0.7 1.5 ± 3.0 31.3 ± 11.9 0.5 ± 1.0 3.7 ± 0.4 75.0 ± 22.6 157.7 ± 95.7 6.3 ± 4.9 2.3 ± 0.6 2.0 ± 4.0 68.5 ± 49.1 1.5 ± 1.9

TP (g/l)

BA (IU/l)

42.2 ± 7.1 111.5 ± 33.8 48.1 ± 8.7 8.5 ± 6.6 47.5 ± 4.5 175.3 ± 66.6 37.6 ± 6.5 26.5 ± 18.0

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used in toxicology studies. Significant differences in ALT (p ¼ 0.00004), GLDH (p ¼ 0.0056) and bile acids (p ¼ 0.0010) levels were observed when comparing vehicletreated Hu-FRG and Mo-FRG mice. ALT (p ¼ 0.019), cholesterol (p ¼ 0.022), GGT (p ¼ 0.0013) and bile acids (p ¼ 0.0071) levels were significantly lower in the troglitazone-treated Mo-FRG group compared with troglitazonetreated Hu-FRG mice. For the remainder of the analytes in both Hu-FRG and Mo-FRG mice, there was considerable variation between animals. The Mo-FRG mice showed no differences between the dosed and control animals. For the Hu-FRG mice there may have been a trend toward increased ALT, total bilirubin and total bile acids following troglitazone administration, but the large inter-individual variability in these parameters between animals meant that these were not statistically significant. Histology and immunocytochemistry In the Hu-FRG mice repopulated with human hepatocytes, the latter showed highly vacuolated cytoplasm due to the accumulation of glycogen (Figures 1A and 2A). The residual mouse liver that persisted in the Hu-FRG mice appeared to be mainly retained in the area of the portal regions while the human liver occupied the bulk of the remainder of the liver. The murine liver areas showed the presence of yellow pigmentation within the Kupffer cells while the hepatocytes

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themselves were more deeply eosinophilic than were the surrounding, vacuolated, human hepatocytes. The residual atrophying mouse hepatocytes also had occasional cells with large nuclei with inclusions of various types. In some, but not in all areas of the mouse hepatocyte blocks, there was an increase in cells possessing oval, to oblong, nuclei and these were considered to be proliferating bile duct (BD) epithelial cells. A third hepatocyte population was present in the HuFRG livers and was made up of focal areas of small, deeply eosinophilic, non-vacuolated hepatocytes with uniform nuclei and without the presence of BD proliferation or the pigmented Kupffer cells (Figure 4A). In sections stained with antibodies against a human Ki67 (Mib1), the nuclei in these cells failed to stain even though they were clearly proliferating from their morphological appearance (Figure 4B). This supported the conclusion that these were not human hepatocytes even though they showed the morphology of hepatocytes. In addition, this population failed to immunostain with antibodies raised against human albumin (Figure 4C) Their fully differentiated morphology was consistent with their being of hepatocyte origin but did not support them being of stem cell origin. The precise characterization of these cells will be carried out in future work. The multidrug resistance associated protein transporter, MRP2, was strongly expressed in the human hepatocytes but was frequently lost, or decreased in intensity, from the degenerating areas of mouse hepatocytes (Figure 1B). By

Figure 1. Liver sections of chimeric livers from the Hu-FRG mouse. (A) Hematoxylin and eosin stained. (B) The MRP2 expression by immunohistochemistry in the Hu-FRG mouse liver with high expression in human liver (H) and low expression in the residual mouse liver (M) that shows brown pigment in the Kupffer cells (K). (C) The expression of BSEP by immunohistochemistry of the Hu-FRG mouse in the human liver (H) and the residual mouse liver (M). CV, Central vein; PV, portal vein. All three figures are 20 microscope magnifications.

Figure 2. Liver sections of chimeric livers from the Hu-FRG mouse. (A) Hematoxylin and eosin stained liver section showing focal eosinophilic hepatocytes (E). (B) Equivalent section to A showing high expression of BSEP in the focal eosinophilic hepatocytes (E) while the adjacent human hepatocytes (H) show low expression. (C) MRP2 protein expression in the focal areas. The expression levels are high and similar in the human (H) and the eosinophilic foci (E). All three figures are 20 microscope magnifications.


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contrast, expression levels for the bile salt export pump transporter, BSEP, were generally very low in the human and mouse hepatocytes from the Hu-FRG mouse (Figure 1C). The exception to this was found in small foci of hepatocytes where expression levels of BSEP were high (Figure 2C) and consistent with what can be seen from control mouse liver. This expression was present in the small, non-vacuolated, hepatocytes with the uniformly eosinophilic cytoplasm (Figure 2A). MRP2 showed a similar expression pattern in these cells to that present in the human hepatocytes (Figure 2B). The exact identity of these cells remains unknown. There was no effect of troglitazone treatment on the distribution or expression levels of either BSEP or MRP2 in the Hu-FRG mouse livers. In contrast to the Hu-FRG mice, the Mo-FRG mouse livers showed the repopulating mouse populations as focal proliferations of deeply eosinophilic, homogeneous aggregates of hepatocytes without the presence of proliferating BD epithelial cells prominent in the atrophying areas of the recipient mouse liver (Figure 3A). MRP2 was strongly expressed, in a courser appearance in the recipient liver while in the donor liver, the expression was generally finer and at low to very low levels (Figure 3B) suggesting an earlier recannulation of the biliary system. In contrast to the situation with MRP2, those sections stained for BSEP showed a higher expression level in the donor liver while the recipient liver generally showed much lower levels (Figure 3C). There was no effect of troglitazone on the distribution or expression levels of either BSEP or MRP2 in the mouse donor-mouse recipient livers. Metabolite profiling UPLC-MS analysis revealed the presence of 20 metabolites in blood samples from the Hu-FRG mice while only 8 (M1, M11, M13–M16 and M19–M20) of those were observed as circulating metabolites in the Mo-FRG mice (Table 3). The mass spectrometric data for troglitazone and its metabolites are summarized in Table 4. Not only were there fewer metabolites observed in the Mo-FRG mouse blood samples but these were also found at relatively lower abundances compared with those observed in the Hu-FRG samples. Most metabolites were observed in the 1 h blood sample from both


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groups of mice and the MS peak areas of these are represented in Table 3. The metabolites presented in this table were also observed in other blood samples obtained at different time points, although no additional metabolites were detected other than metabolite M3 which was detected in the 8 h blood sample of the Hu-FRG mice. The biliary contents of the excised gall bladders were also analyzed by UPLC-MS, where the parent compound was detected in both types of mice. In the bile of Mo-FRG mice, three further metabolites (M2, M17 and M18) were observed in the bile extracts of the Mo-FRG mice when compared with those present in the circulation (Table 3). However, no additional metabolites were detected in bile samples compared with those circulating in the Hu-FRG mice (Table 3). Nine circulating metabolites identified in the Hu-FRG mouse (M3-M10 and M12) seem to be specific to this model as they were not present in the Mo-FRG mouse samples. These metabolites were assigned to various combinations of hydroxylated, glucuronidated and sulfated troglitazone products. The observed masses and the major MSMS fragments for all metabolites are shown in Table 4. Quantitative comparisons could not be made between circulating and biliary metabolites since different MS instruments were employed for each matrix, although the two major peaks observed corresponded to direct glucuronide (M11) and sulfate (M13) conjugations, respectively. Both of these metabolites were observed in relatively large amounts in the Hu-FRG mice when compared with the Mo-FRG mice. Metabolite identification in blood and bile The extracted ion mass chromatogram for unchanged parent compound at m/z 440, showed a characteristic product ion spectrum with the loss of 43.0075 u, corresponding to a CONH moiety, resulting in a fragment ion at m/z 397. The structure of troglitazone and the fragment as well as the MSMS spectra are shown in Figure 5A. Other cleavages of the molecule which were characteristic of the remaining structure are shown in Table 4. The majority of troglitazone metabolism was via hydroxylation, sulfation and glucuronidation. Various combinations of these three biotransformations constituted the greater proportion of the metabolite profiles of troglitazone in either Hu-FRG or Mo-FRG mice. Seven metabolites in

Figure 3. Liver sections from a mouse given mouse donor hepatocytes, Mo-FRG mouse. (A) Hematoxylin and eosin stained liver section, showing a focus of donor hepatocytes (DM) surrounded by atrophying host mouse hepatocytes (HM) with large numbers of proliferating BD cells. (B) Course expression of MRP2 protein in atrophying HM hepatocytes and finer and lower expression levels in the DM hepatocytes. (C) Higher expression of BSEP in DM liver and very low expression levels of BSEP in the atrophying HM liver. All three figures are 10 microscope magnifications.

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Table 3. Metabolite profiles in Hu-FRG and Mo-FRG mice following 7 days of dosing of troglitazone (600 mg/kg).


Peak ID

Blood extracts

Gall bladders

Analysis system 1

Analysis system 2a

Rt (min)

Hu-FRG

5.66 2.89 3.01 3.17 3.30 3.51 3.87 3.92 3.93 4.05 4.12 4.18 4.24 4.36 4.64 4.72 4.87 4.91 4.94 5.03 5.39

347 275 36 16 56 12 65 32 50 14 7195 28 6265 30 86 343 25 10 1504 17

Parent M1 M2 M3b M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20

Mo-FRG 31

Hu-FRG

Mo-FRG

Rt (min)

4064 28586

480 4323 84

24.96 8.57 8.92

585

1725

72572 111 39461 43 52 48 696c 374c

415 21 30 88

9.16

8475

9.38 10.37 10.51 11.91 12.15 12.57 17.53 18.52

2169 17 24 29 216c 9c

532 14

Data are presented as MS peak areas. Semi-quantification assuming a linear MS response for each metabolite for each analysis systems used. The MS areas for metabolites observed using analysis system 2 are divided by 10 000. b Metabolite identified in 8 h sample, all the others were present in the 1 h sample. c These metabolites may correspond to any of the mono-oxygenated metabolites, M17–M19. a

Table 4. Mass spectrometric data for troglitazone and its metabolites.

Peak ID Troglitazone M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20

Rt (min)

Observed [M-H](m/z)

Dppm

5.66 2.89 3.01 3.17 3.3 3.51 3.87 3.92 3.93 4.05 4.12 4.18 4.24 4.36 4.64 4.72 4.78 4.91 4.94 5.03 5.39

440.1537 634.196 538.1202 554.1186 632.1798 536.1007 632.1793 632.1805 688.2075 536.1022 614.1666 616.1854 472.1428 520.1099 472.1466 472.1396 472.1425 456.1462 456.1451 456.1475 458.1653

0.2 0.7 4.7 0.6 7.9 1.4 0.5 1.6 5.1 4.9 0.2 0.5 0.2 7.6 7.2 1.1 4.2 6.6 1.3 3.4

Major MSMS fragment 397.1482, 458.1625, 458.1654, 536.1050, 589.1656, 456.1444, 456.1510, 456.1428, 645.1976, 456.1620, 438.1449, 440.1533, MS only 440.1542, MS only 179.0216, 179.0216, MS only MS only 413.1379, MS only

179.0220, 415.1634, 415.1611, 474.1462, 456.1524, 413.1476, 438.1213, 385.1332, 512.1818, 438.1445, 222.0099, 397.1486,

163.0731, 222.0238, 222.0174, 456.1451, 413.1405, 161.0490 413.1674 150.0155 469.1741 412.1572 151.0178 369.1592,

150.0151, 145.0306, 117.0355 151.0218 151.0249 179.0185, 151.0247 150.0206

150.0158

397.1410, 163.0773, 145.0266, 119.0509, 117.0325 151.0226, 117.0334 151.0226, 117.0334 385.1464, 179.0179, 145.0285, 117.0400

total were assigned as glucuronides (M1, M4, M6–M8 and M10–M11) and all displayed the characteristic loss of 176.0321 u to yield the aglycone, whereas a total of five metabolites (M2, M3, M5, M9 and M13) were conjugated with a sulfate group, as assigned by the characteristic loss of 79.9568 u. The most likely location of both glucuronic acid (M11) and sulfate (M13) conjugation was on the chromane ring moiety as reported previously (Schulz-Utermoehl et al., 2012), and are denoted with an R-group in Figure 4. The MSMS spectra of these two metabolites are shown in

Assignment Parent Hydrated glucuronide Hydrated sulfate Hydrated hydroxyl sulfate Hydroxy glucuronide Hydroxy sulfate Hydroxy glucuronide Hydroxy glucuronide Unassigned glucuronide Hydroxy sulfate Dehydrogenated glucuronide Glucuronide Di-hydroxy Sulfate Di-hydroxy Di-hydroxy Di-hydroxy Mono-hydroxy Mono-hydroxy Mono-hydroxy Hydrated

Figure 5(B) and 5(C), respectively. The mass accuracy of all the fragments was within 5 ppm and is shown in Figure 5. The product ion mass spectra of metabolites M17–M19 indicated mono-oxygenation while metabolites M12, M14– M16 were assigned as di-oxygenated metabolites. It was not possible to assign the positions of these oxygenations based on fragmentation data. It is also possible that one or more of the mono-oxygenated and di-oxygenated metabolites corresponded to quinoid structures but it was not possible to distinguish between these functionalities on the basis of the

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Figure 4. Liver sections from a mouse given human donor hepatocytes, Hu-FRG mouse. (A) Hematoxylin and eosin stained liver section, showing a focus of donor human hepatocytes with the ‘‘third population’’ of hepatocytes in the top left hand corner. (B) The presence of Ki67 staining cells in the human liver (shown by round brown nuclear staining) and the absence of staining in the ‘‘third population’’. (C) The results of immunostaining with an antibody against human albumin. The product of granular in the human hepatocytes but is absent from the ‘‘third population’’ of hepatocyte-like cells. All three figures are 10 microscope magnification.

Figure 5. Structure of troglitazone (R¼H) and the fragment showing the characteristic loss of CONH (43.0075). The MSMS spectra of troglitazone (R ¼ H), troglitazone sulfate (R ¼ SO3H) and troglitazone glucuronide (R ¼ C6H9O6) are shown in (A), (B) and (C), respectively.

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detected fragment ions. Additionally, metabolites M1–M3 and M20 were assigned as hydrated or hydroquinone-derived metabolites due to their +18 u increments. Metabolite M8 corresponded to an unassigned glucuronide conjugate and was considered drug-related as it was not observed in vehicle control samples.


Discussion The present study aimed to investigate the metabolism, hepatic and transporter effects of troglitazone in the chimeric mouse models investigated, and also to compare the data obtained in the Hu-FRG model with that obtained previously for the PXB mouse. Hence, in both studies metabolite profiles and liver histopathology were investigated following 7 days of oral dosing of troglitazone (600 mg/kg/day). A possible indication of hepatic effects of the drug may be evident in the fact that the body weights of the Hu-FRG mice decreased significantly upon 7 days of troglitazone dosing but no difference in body weights were seen for the Mo-FRG group. A similar pattern was observed for the troglitazone exposed PXB mice for which the mean body weight decreased from 17.4 g ± 1.2 to 16.5 g ± 0.5 (Schulz-Utermoehl et al., 2012). The size of the livers, as well as the liver to body weight ratios, in the Hu-FRG mice was significantly larger than that of the Mo-FRG mice even though there was no effect of troglitazone treatment. The mean liver to body weight ratio was lower for the Hu-FRG mice, being 8.50–9.24% compared with the PXB mouse ratios of 13.6–13.0% in vehicle exposed and troglitazone-treated mice, respectively. However, the liver weights in the chimeric PXB mice and the Hu-FRG mice were of the same magnitude, but the body weights of the PXB mice were lower than those of the Hu-FRG mice. Surprisingly, a dramatic drop in the replacement index of human hepatocytes in the livers of Hu-FRG mice occurred during the course of the study. The humanization was 95% one week before first dose given and decreased to 41–45% after 7 days of vehicle and troglitazone treatment, respectively. This was unexpected and may well have impacted the troglitazone metabolism results, as well as the transporter evaluation, compared with the higher levels of humanization present at the outset of the study. With respect to hepatotoxicity, blood chemistry analysis showed no significant changes in either type of mouse upon 7 days of troglitazone dosing. There was, however, considerable inter-animal variation in many of the analytes measured in both treated and untreated animals from both the PXB and Hu-FRG mice (e.g. see Table 2 for ALT, AST and total bile acids etc. for the Hu-FRG mice used in this study and Schulz-Utermoehl et al., 2012 for the equivalent data from PXB mice), making it difficult to interpret any changes in troglitazone-treated animals. In more recent studies in the PXB mouse a significant increase in ALT levels was reported for animals administered troglitazone at 1000 mg/kg for 14 and 23 days of treatment but no significant differences were seen from PXB mice at lower doses for a treatment period of 7–28 days (Kakuni et al., 2012). This suggests that the dose level and number of days of dosing are important to detect a positive response in standard blood chemistry parameters in these animals. In this respect, the loss

Xenobiotica, 2014; 44(2): 186–195

in humanization of the Hu-FRG mice from 90% to ca. 40% over the course of the present study represents a limitation in the use of Hu-FRG mice for this multi-dose type of evaluation. Histopathological examination showed that, as detected in the humanized areas of the PXB mouse liver and as reported by (Meuleman et al., 2005), accumulation of cytoplasmic glycogen was observed in the human parts of the livers of Hu-FRG mice. Immunocytochemistry of the canalicular transporters, BSEP and MRP2, revealed no differences between any of the troglitazone treated or vehicle-treated Hu-FRG or Mo-FRG mice. In contrast troglitazone has been shown to down regulate the MRP2 and BSEP expression in the humanized, but not residual mouse, parts of PXB mouse livers (Schulz-Utermoehl et al., 2012). Metabolite profiling and the identification of putative metabolites revealed the presence of 20 metabolites in total in Hu-FRG and Mo-FRG mouse samples. All metabolites observed in the Mo-FRG mice were present in the Hu-FRG mice, although nine were unique to the Hu-FRG mice alone. Only two of these nine metabolites were detected in bile whereas all nine were found in blood. Of the metabolites observed in the Hu-FRG mouse blood samples, metabolites M8 and M10 have not previously been reported as troglitazone products in chimeric mice. We observed that the sulfate (M13) was predominant in the Hu-FRG mice compared with the Mo-FRG mice at all time points, with the MS response area ca. 15-fold higher in the 1 h blood sample and 18-fold greater in the 12 h bile sample (Table 3). Although the degree of humanization dropped significantly during the study a clear and interesting observation of the relative abundance of the sulfate (M13) metabolite was made. The sulfate metabolite of troglitazone (M13) has been shown to be the predominant circulating metabolite in clinical samples of human subjects dosed with troglitazone, with the glucuronide conjugate (M11) present in minor amounts (Loi et al., 1999a,b). The relative amount of the sulfate conjugate in the Hu-FRG, mice compared with that in Mo-FRG mice revealed clear differences between them with a higher concentration being present in the Hu-FRG mice. The sulfate was also the major metabolite in PXB mice compared with the control group reported by Schultz-Utermoehl et al. (2011). Consequently, in this respect the FRG and PXB mice appear somewhat similar as models for the human metabolism of troglitazone. The glucuronide conjugate (M11) has been reported to be present in higher amounts than the sulfate (M13) in mouse plasma (Izumi et al., 1996; Kawai et al., 1997). No quantitative comparison could be made between the two metabolites in the present study but the relatively higher abundance of the glucuronide metabolite (M11) in the Hu-FRG mice compared with that formed in the Mo-FRG mice does not reflect the human situation but rather that of a mouse and might be explained by the drop in humanization by the end of the study (although the rate of this decline in humanization was not established here).

Conclusion In conclusion, this study with the Hu-FRG chimeric mouse has shown that for the model compound, troglitazone, the


DOI: 10.3109/00498254.2013.879237

mice demonstrate some similarity to human metabolism in terms of troglitazone sulfate formation. This result together with the blood chemistry results makes this model comparable to another chimeric mouse type, the PXB mouse. However, the two models were not equivalent in terms metabolism, as evidenced by the amounts of the major glucuronide metabolite formed, and the differences in effects on hepatic transporters. It cannot be excluded that these differences may be related to differences in donor characteristics. Further metabolism studies need to be performed to validate the Hu-FRG mouse and perhaps metabolism studies utilizing a single dose, to avoid the potentially confounding effects of the loss of humanization of the chimeric livers, may yield a better understanding of these phenomena. If the level of liver humanization cannot be maintained the use of the model may be constrained. However, if this loss in human hepatocytes can be prevented the Hu-FRG model may have considerable potential for bridging the current gap between in vitro human cell systems and human clinical trials and should permit the early evaluation of human hepatic metabolism of drugs, in the discovery phase of drug development, prior to entering human clinical trials.

Declaration of interest All authors were employed and funded by AstraZeneca Pharmaceuticals during the conduct of these studies and all authors were employed and funded by AstraZeneca Pharmaceuticals during the preparation of the manuscript apart from IDW who is employed by Department of Surgery and Cancer, Imperial College, London, UK.

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