A comparison between genetically humanized and chimeric liver humanized mouse models for studies in drug metabolism and toxicity

Drug Discovery Today  Volume 00, Number 00  July 2015

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A comparison between genetically humanized and chimeric liver humanized mouse models for studies in drug metabolism and toxicity Nico Scheer1 and Ian D. Wilson2 1 2

Independent Consultant, Cologne, Germany Imperial College London, South Kensington, London SW7 2AZ, UK

Mice that have been genetically humanized for proteins involved in drug metabolism and toxicity and mice engrafted with human hepatocytes are emerging and promising in vivo models for an improved prediction of the pharmacokinetic, drug–drug interaction and safety characteristics of compounds in humans. The specific advantages and disadvantages of these models should be carefully considered when using them for studies in drug discovery and development. Here, an overview on the corresponding genetically humanized and chimeric liver humanized mouse models described to date is provided and illustrated with examples of their utility in drug metabolism and toxicity studies. We compare the strength and weaknesses of the two different approaches, give guidance for the selection of the appropriate model for various applications and discuss future trends and perspectives. Introduction Q4 Two fundamentally different approaches of generating humanized mouse models have been

explored by various researchers: (i) the introduction of human genes into the mouse genome to generate genetically humanized mouse models; and (ii) the transplantation of human cells into competent recipients resulting in tissue humanized mouse models (Fig. 1). Both approaches have been used for a variety of applications. For example, genetically humanized mice have been described for components of the immune and haematopoietic system, as models for human aneuploidy and to reflect human diseases, for efficacy testing, for cancer research and to enable infections with human pathogens [1]. In a similar manner, tissue humanized mice were used for studies in human haematopoiesis, immune responses, autoimmunity, infectious diseases, cancer and regenerative medicine [2]. Another emerging and promising application of these models is in studies related to drug metabolism and toxicity. The prediction of human responses from traditional preclinical in vivo studies in this field is often limited by the significant species differences in the proteins Q5 involved in drug ADME [3–5]. Whereas the overall pathway of drug metabolism and disposition is highly conserved (Fig. 2), the substrate specificity, multiplicity and expression level of individual proteins mediating these processes can vary significantly between species. Such

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Nico Scheer received his PhD in developmental biology from the University of Cologne, Germany, in 2000, where he developed a gene expression system in zebrafish for studying the role of the notch signalling pathway in neural development. Until recently, he was Head of the tADMETTM portfolio at Taconic Biosciences, where he was responsible for a toolbox of innovative in vivo and in vitro tools to predict the pharmacokinetic, drug–drug interaction and safety characteristics of compounds in humans. Nico Scheer currently works as an independent consultant. He has published several papers and review articles in peer-reviewed scientific journals in the field of humanized mouse models. Ian Wilson trained as a biochemist at the University of Manchester Institute of Science and Technology, following with a PhD in the Chemistry Department at Keele University (1978) on insect moulting hormones. Subsequently, he worked in the pharmaceutical industry, most recently as a Senior Principal Scientist at AstraZeneca (Dept of Drug Metabolism and Pharmacokinetics). His research is directed towards the development of advanced analytical techniques, especially hyphenated methods that combine chromatography and spectroscopy, and their application to problems in drug metabolism, toxicology and metabonomics to facilitate the development of safer and more-effective drugs for human use.

Corresponding author: Wilson, I.D. ([email protected]) 1359-6446/ß 2015 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.drudis.2015.09.002

www.drugdiscoverytoday.com 1 Please cite this article in press as: Scheer, N., Wilson, D.T.D. A comparison between genetically humanized and chimeric liver humanized mouse models for studies in drug metabolism and toxicity, Drug Discov Today (2015), http://dx.doi.org/10.1016/j.drudis.2015.09.002

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Teaser The accurate use of genetically humanized and chimeric liver humanized mouse models has great potential to improve the prediction of clinical drug pharmacokinetics, drug–drug interaction and drug safety.

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(a)

Wild-type mouse

Humanized mouse

Genetic humanization

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(b)

Tissue (liver) humanization

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FIGURE 1

Concept of generating genetically and tissue humanized mouse models. The principle of genetic humanization (a) is illustrated by a homozygous replacement of a murine gene (white bars) with the corresponding human gene (grey bars). For the sake of simplicity other approaches of genetic humanization, such as random transgenesis or introduction of a freely segregating human chromosome, are not shown. Tissue humanization (b) is exemplified by the replacement of mouse hepatocytes (white circles) with human hepatocytes (grey circles) in chimeric liver humanized mice. Some residual mouse hepatocytes are usually maintained after transplantation with human hepatocytes, as indicated by the remaining white circles in the chimeric liver humanized mice.

species differences have been described for all major components of the pathway of drug metabolism and disposition: xenobiotic receptors [6], cytochrome P450s (CYPs) [4,7], phase 2 enzymes [8] and transporters [9].

One approach to minimise the impact of species differences is to replace single or multiple mouse genes with their human counterparts. A great variety of such genetically humanized mouse models expressing human instead of mouse receptors, drug metabolising enzymes and transporters has been described Q6 (see below) [10,11]. A second way to overcome the limitations associated with traditional animal models takes advantage of the predominant role of the liver in drug metabolism and detoxification. Hence, different mouse models with human hepatocytes engrafted in their livers have been created (see below) [12–15]. Varying degrees of engraftment have been achieved, with >95% repopulation with human hepatocytes reported in such liver humanized mouse models. Genetically and chimeric liver humanized mouse models have been shown to have value for various applications in drug metabolism and toxicity. However, to our knowledge no attempt has been made so far to compare the advantages and disadvantages of each approach directly for this type of application and to give guidance for the selection of the appropriate model for specific studies in this field. Here, we present an overview of the pros and cons and the promises and limitations of genetically and liver humanized mouse models for studies in drug metabolism and toxicity. The different models developed to date are described and an assessment comparing the two approaches in general, rather than evaluating specific models from each category, is made. Our analysis comes together with a comprehensive, but not exhaustive, description of proof-of-concept studies showing the applications of these mice, and recommendations on model selection for studies in drug metabolism and toxicity are provided. Finally, the authors’ personal perspectives on probable future developments and trends in this field are given.

Activation or inhibition by xenobiotic

Xenobiotic receptor (AHR, CAR, PPARα, PXR,…)

Induction or repression of expression

Phase 1 enzymes

Phase 2 enzymes

Transporters

(CYPs, monooxigenases,…)

(STs, UGTs, GSTs,…)

(SLCs, ABCs,…)

Introduction of reactive or polar groups into xenobiotic

Conjugation of xenobiotic to polar compounds

Transport of xenobiotic over biological membrane barriers Drug Discovery Today

FIGURE 2

Protein network defining the major pathways of drug metabolism and disposition. The boxes highlight the different protein families of the network with some of the major members listed in brackets. The arrows illustrate the sequence of events. For a given xenobiotic all or only parts of these events can occur. For simplicity Q13 certain factors, such as co-activators or repressors of xenobiotic receptors, as well as cellular compartments are not shown. Reprinted, with permission, from [11]. Q14 Abbreviations: AHR, aryl hydrocarbon receptor; CAR, constitutive androstane receptor; CYP, cytochrome P450; PPAR, peroxisome proliferator-activated receptor; PXR, pregnane X receptor; UGT, UDP glucuronosyltransferase. 2

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Genetically humanized mouse models for proteins involved in drug metabolism and disposition A large collection of genetically humanized mouse models of proteins involved in drug metabolism and disposition (i.e. xenobiotic receptors, drug metabolising enzymes and transporters) has been generated by various groups (Table 1) [10,11,16,17]. The methods that were applied by the different researchers to generate these mice varied significantly and included random insertions of human transgenes into the mouse genome, freely segregating mouse artificial chromosomes (MACs) or human artificial chromosomes (HACs), or targeted integrations at predefined positions. These approaches were combined, or not, with deletions of the corresponding murine genes with the human transgenes expressed off heterologous promoters, the corresponding mouse promoters or the cognate human promoters. The description of the technical details, advantages and disadvantages of each of these approaches is beyond the scope of this article and the reader is referred to other reviews on this subject [1,18]. Genetically humanized mice have been described for the four major xenobiotic receptors involved in drug metabolism, the aryl hydrocarbon receptor (AHR), the constitutive androstane receptor (CAR), the peroxisome proliferator-activated receptor (PPAR)a and the pregnane X receptor (PXR) [10]. Furthermore, genetically humanized mouse models expressing the key human phase 1 enzymes of the CYP family, such as CYP1A1/1A2, CYP2A6, TABLE 1

Genetically humanized mouse models for proteins involved in drug metabolism and disposition Human gene(s)

Refs

Xenobiotic receptors

AHR CAR CAR/PXR PPARa PXR

[67,68] [21,123] [21] [70,73] [21,63,124–128]

Drug metabolising enzymes

CYP1A1/1A2 CYP2A6 CYP2A13/2B6/2F1 CYP2C9 CYP2C18/2C19 CYP2D6 CYP2E1 CYP3A4 CYP3A4/2D6 CYP3A7 NAT2 UGT1A UGT2B7

[75,129,130] [131] [132] [45] [133] [44,134] [86,135] [24,41,64,65,136–138] [22] [139] [77,140] [141,142] [143]

Drug transporters

MRP2 OATP1A2 OATP1B1 OATP1B3 OATP1B1/1B3 PEPT1

[144] [79] [145] [145] [56] [19]

Composite models

PXR/CYP3A4 [23] CAR/PXR/CYP3A4/3A7 [24]

Updated, with permission, from [1].

Q16 Abbreviations: AHR, aryl hydrocarbon receptor; CAR, constitutive androstane receptor; CYP, cytochrome P450; MRP, multidrug resistance protein; NAT, N-acetyltransferase; OATP, organic anion-transporting polypeptides; PEPT, proton-coupled oligopeptide transporter; PPAR, peroxisome proliferator-activated receptor; PXR, pregnane X receptor; UGT, UDP glucuronosyltransferase.

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CYP2C9, CYP2C18/2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A7, and the phase 2 enzymes UDP glucuronosyltransferase (UGT)1A and UGT2B7 and arylamine N-acetyltransferase (NAT)2 have been generated [11]. By contrast, so far drug transporter humanized mice have only been described for the organic anion-transporting polypeptides (OATP)1A2, OATP1B1 and OATP1B3, the multidrug resistance protein (MRP)2 [11] and proton-coupled oligopeptide transporter (PEPT)1 [19]. The small number of transporter models described thus far probably reflects the fact that membrane transporters have been recognised only recently as key determinants of the pharmacokinetic, safety and efficacy profiles of drugs [20].

Complex, multiple humanized mouse models An interesting path forward aiming to overcome some of the limitations associated with single gene humanizations is the combination of individual genetic modifications into complex, multiple humanized mouse models. In this context, double humanized PXR/CAR [21], CYP2D6/CYP3A4 [22] and PXR/CYP3A4 [23] as well as quadruple humanized PXR/CAR/CYP3A4/3A7 [24] mice have been generated. Clinical drug–drug interactions (DDIs) of three different PXR-activators with triazolam, a fast clearance drug metabolised primarily by CYP3A4, were quantitatively predicted in the latter model [24], although the accuracy of such predictions over a wide range of compounds with different properties requires further assessment. Fortunately, from a humanization perspective, although many proteins can be involved in drug metabolism and disposition, only a small fraction of those are of key importance for the vast majority of drug reactions. For example, whereas there are 57 CYPs in humans [25], it has been estimated that 95% of human phase 1 drug metabolism is mediated by CYP3A4/5, CYP2D6, CYP2C9/ 2C19 or CYP1A1/1A2 [26]. Accordingly, many important aspects of human drug metabolism can be studied with the humanization of a few key proteins and the further combination of such multiple CYP humanized mice with appropriate humanizations of drug transporters could provide useful models for pharmacokinetic studies. As further discussed below, it should be noted however that drug metabolism and disposition can be complex and involve various nonhumanized proteins, so that extrapolations to humans should be made prudently and on a case-by-case basis only.

Chimeric liver humanized mouse models Different groups have successfully generated chimeric liver humanized mouse models. Although the technical strategies differ for each model, the underlying principle in order to achieve efficient repopulation of the mouse liver with human hepatocytes is the same in all cases. Namely, these mice carry deficiencies in certain components of the immune system to avoid rejection of the human cells, and the mouse hepatocytes are ablated by genetic modifications that result in toxicity within the murine liver cells. A detailed description of each liver humanized mouse model is beyond the scope of this manuscript and we refer the reader to previous reviews for further information [13,27,28], with the main features of the three most intensively studied models to date summarised below. The first of these was the urokinase-type plasminogen activator (uPA)/severe combined immunodeficient (SCID) mouse model, which was developed in the 1990s [29–31]

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and, because of its pioneering position, has been most widely published so far. Mouse hepatocyte ablation in the uPA/SCID model is achieved through the constitutive hepatic expression of the liver-toxic serine protease uPA. Q7 The so-called FRG model was first described in 2007 and combines immune-deficiency-mediating mutations, in the recombination activating gene (Rag)2 and the gamma chain of the Q8 interleukin 2 receptor (Il2rg), with a functional knockout of the fumarylacetoacetate hydrolase (Fah) gene [32]. The latter gene codes for an enzyme in the tyrosine catabolic pathway and its mutation leads to an intracellular accumulation of a toxic intermediate in hepatocytes. Unlike the uPA/SCID model, the onset and severity of hepatocellular injury in FRG mice is controllable through the administration and withdrawal of the protective drug 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), which blocks an upstream enzyme in the tyrosine pathway and thereby prevents accumulation of the toxic intermediate. More recently, another chimeric liver humanized mouse model with inducible liver injury was described [33]. Mouse hepatocyte ablation in this TK-NOG model was achieved through the liverspecific expression of the herpes simplex virus 1 thymidine kinase (HSVtk) in severely immunodeficient NOG mice and administration of ganciclovir (GCV), utilising the fact that HSVtk converts the otherwise nontoxic GCV into a toxic intermediate. Two further examples of less-extensively studied chimeric liver Q9 humanized mice are the recently described AFC8 [34] and AlbTRECK/SCID [35] models. A brief comparison of the major features of the different liver humanized models is given in Table 2. Fig. 3 compares the process of liver reconstitution in the most frequently used models to date: uPA/SCID, FRG and TK-NOG. The replacement index (RI) is the percentage of human hepatocytes in the liver of transplanted chimeric mice, and this can be determined by measuring the concentrations of human serum albumin levels in the chimeric mice. RIs of up to 95% have been reported in most of these models.

Dual chimeric liver and immune system humanized mice Analogous to the combination of single genetic modifications into more-complex, multiple humanized mouse models, described above, it is also of interest to merge different cellular-based humanization approaches in a single organism. This was recently achieved by dual humanization of liver and haematopoiesis in an FRG model combined with the signal regulatory protein (Sirp)a

allele of the nonobese diabetic (NOD) mouse strain [36]. Owing to the suitability of the FRG model for reconstitution with human hepatocytes (see above) and the NOD background for generation of human haematopoietic chimeras, human liver repopulation of >80% and haematopoietic chimerism of 40–80% in bone marrow was obtained in the combined FRG/NOD model. The authors speculated that these double-chimeric mice might serve as a new model for disease processes that involve interactions between hepatocytes and haematolymphoid cells. Dual liver and immune system humanization was also described in a uPA-based mouse model [37]. Given the involvement of the immune system in the downstream sequelae of the biotransformation of drugs leading to reactive metabolites [38] it is not difficult to envisage applications in this area as well.

Advantages and disadvantages of genetically and chimeric liver humanized mouse models Genetically and chimeric liver humanized mouse models have immanent advantages and disadvantages, and the pros and cons of these approaches are summarised in Table 3 and further explained below.

Advantages of genetically humanized mice A clear advantage of the genetically humanized compared to chimeric mice is the ease of maintaining the models by simple breeding or cryopreservation (of embryos or spermatozoa) without the need for surgical procedures. By contrast, every chimeric liver humanized mouse needs to be individually transplanted and the humanization cannot be propagated to the next generation. This differentiator inevitably affects two other distinguishing features between genetically and chimeric liver humanized mice, namely the difference in cost of production and the variability between individual mice. With regards to the former, the generation of chimeric liver humanized mice is significantly more expensive compared with genetically humanized animals. Apart from the necessity of individual surgery, the high procurement costs of human hepatocytes and the overproduction of animals that do not meet the required quality standards (e.g. regarding the degree of humanization) contribute to this expense. Prices from typical vendors are therefore usually about a few hundred dollars per genetically humanized mouse, but more in the range of a few thousand dollars for a chimeric liver humanized animal. Furthermore, chimeric liver humanized mice are more heterogeneous

TABLE 2

Selected chimeric liver humanized mice Model

Genetic cause of mouse hepatocyte ablation

Control of mouse hepatocyte ablation

Immune deficient background

Refs

uPA-SCID

Uroplasminogen activator (uPA)

None

SCID

[29,146]

FRG

Fumarylacetoacetate hydrolase deficiency

 NTBC  Low tyrosine diet

Il2rg Rag2

/

[32]

/

TK-NOG

Herpes simplex virus thymidine kinase

 Ganciclovir

Il2rg SCID

/

[33]

AFC8

FK508-caspase 8 fusion

 AP20187

Il2rg Rag2

/

[34]

Alb-TRECK/SCID

Human heparin-binding EGF-like receptor

 Diphtheria toxin

SCID

/

[35]

Q17 Abbreviations: EGF, epidermal growth factor; Il2rg, common g-chain of the interleukin 2 receptor; NTBC, 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexanedione; Rag2, recombination activating gene 2; SCID, severe combined immunodeficiency.

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Transplant human hepatocytes

(a)

(b) Maintain on NTBC

Remove NTBC

Transplant human hepatocytes

FRG

(c) Add ganciclovir

Transplant human hepatocytes

TK-NOG

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FIGURE 3

Comparison of mouse hepatocyte ablation and transplantation with human cells between urokinase-type plasminogen activator (uPA)/severe combined Q15 immunodeficient (SCID), FRG and TK-NOG models. Ablation of mouse hepatocytes (white circles) in uPA-SCID (a) is constitutive and noncontrollable. Mouse hepatocyte ablation in FRG (b) is prevented by 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) and triggered by the removal of the compound. In TK-NOG (c) the mouse hepatocytes are healthy by default and their ablation is induced via treatment with ganciclovir. The dying mouse hepatocytes are indicated by the dashed white circles. The remaining solid white circles indicate surviving residual mouse hepatocytes. After transplantation human hepatocytes (grey circles) repopulate the mouse livers.

TABLE 3

Advantages and disadvantages of genetically and chimeric liver humanized mice Genetically humanized mice

Liver chimeric mice

Advantages – Permanent model without recreation – Low cost of production – High consistency between individual mice – Human gene expressed in various organs – Human gene expressed in all liver cells – Availability of knockout controls – Usually healthy

Disadvantages – Continuous recreation required – High cost of production – Varying degree of humanization – Humanization restricted to the liver – Residual mouse hepatocytes express murine gene – Knockout controls usually not available – Immune compromised

Disadvantages – Expression of selected human genes only – Different genes of interest require different mouse lines – High effort to generate donor variability – No infection with human specific pathogens – Nontransplantable with human cells – More challenging human extrapolation through in vitro–in vivo correlations – Potential compensatory gene expression changes

Advantages – Human hepatocytes express all human genes – One mouse line fits different purposes – Ease of generating donor variability – Susceptible to human-specific pathogens – Transplantable with various human cells – Combined use with human hepatocytes supports extrapolation to humans – No compensatory gene expression changes reported

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uPA-SCID

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than genetically humanized models, owing to the varying degree of liver humanization. Although it is possible to set a threshold for a reconstitution level (e.g. >90%) it might be necessary to use larger groups per treatment to obtain statistical significance with chimeric liver humanized mice. Another benefit of genetically humanized mice is the possibility to express the human transgene in a variety of organs (e.g. where they are naturally expressed). Although the liver is the dominant organ of drug metabolism and disposition for many compounds, extrahepatic metabolism and transport can make major contributions to the fate of a drug in the human body [39]. A limitation of the chimeric liver humanized mice in this regard is the liverrestricted humanization and the potential contribution of hepatic human and nonhepatic mouse components to drug metabolism and disposition. Accordingly, chimeric liver humanized mice have little value where extrahepatic factors of drug metabolism and disposition require investigation, such as, for example, the role of transporters expressed in the intestine, kidney, testis or blood– brain barrier in the tissue distribution and clearance of drugs [20]. Furthermore, chimeric mice need to be used with caution where extrahepatic tissues make major contributions to the metabolism and disposition of a particular compound, as exemplified by the important role of the intestine in CYP3A4-mediated metabolism of various drugs, such as docetaxel, triazolam and lopinavir [40–42]. For a given humanized gene, the genetically humanized mice also have the advantage of its expression in all liver cells, whereas the residual mouse hepatocytes in the chimeric liver humanized mice will express the murine version of this particular gene. This can have important consequences if, for example, the metabolism of a compound is dominated by one particular drug-metabolising enzyme and if the rate of metabolism of this compound differs between mouse and human hepatocytes. This potential drawback of chimeric mice can be illustrated by the hypothetical situation where, if the intrinsic clearance of a drug in mouse liver was tenfold higher than for human, even in situations where the liver was 90% humanized, there would be an equal contribution of mouse and human hepatocytes to its clearance. A prime example is the antiarrhythmic drug propafenone, which is metabolised by Q10 human CYP2D6 and mouse Cyp2d enzymes at different turnover rates and to distinct metabolites. Owing to the significantly preferred metabolism of propafenone by mouse over human enzymes, the residual mouse hepatocytes in a highly chimeric liver humanized mouse model obscured the effect of the human cells to the extent that the human-specific metabolites could not be detected [43]. Such an obscuration is unlikely to occur in a recently described genetically humanized mouse model with a replacement of the mouse Cyp2d genes with human CYP2D6 [44]. For some applications the availability of genetic knockout controls for a given human gene are beneficial (e.g. to assess the relevance of the corresponding human gene product in the metabolism, disposition or toxicity of a drug). Such controls, which only lack the human gene but are otherwise genetically identical to the humanized model, are available for most genetically humanized mice. By contrast, for chimeric liver humanized mice the sourcing of human hepatocytes with a complete functional deletion of a particular gene of interest will often be difficult, costly or even unfeasible. However, one possibility to overcome this limitation of chimeric liver humanized mice is 6

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the use of chemical inhibitors that specifically block the activity of the corresponding gene product. Finally, it should be noted that genetically humanized mice for proteins involved in drug metabolism and disposition are usually healthy and phenotypically indistinguishable from the corresponding wild-type controls, with the rare exception of a phenotypic alteration caused by mutation of the mouse gene(s) and lack of compensation by the human counterpart(s), for example see [45]. By contrast, the mandatory immune deficiency of chimeric liver humanized mice is a deviation from a normal health status by definition, which might or might not be an issue, depending on the purpose of the study.

Advantages of chimeric liver humanized mice Genetically humanized mouse models are clearly outcompeted by chimeric liver humanized mice when it comes to the degree of humanization on the single hepatocyte level. As in this case, humanization is achieved by the transplantation of human hepatocytes, all genes, proteins and pathways are of human origin per se, whereas even in the most complex, multiple genetically humanized mouse model only a few genes have so far been modified. On the single cell level, genetically humanized mouse models therefore suffer from the contribution of potentially mouse-specific pathways of drug metabolism and disposition. In a similar manner to the aforementioned obscuration of human-specific responses by residual mouse hepatocytes or extrahepatic organs in the chimeric liver humanized mice, genetically humanized mice bear the risk of such an undesirable contribution of mouse components in every single cell of the transgenic organism. Model selection of chimeric liver humanized mice is also considerably more straightforward compared with genetically humanized mouse models. Apart from the choice of models from different vendors (Table 2) and potential subgroups of models (e.g. FRG vs FRG/NOD), one model in principle fits all purposes. In the case of genetically humanized mice the experimenter has to select the appropriate model from a large collection of different strains (Table 1) and, for a given compound, this selection usually requires certain information or a hypothesis about the involved pathways of drug metabolism and disposition. A particularly useful feature of chimeric liver humanized mouse models is the relative ease of generating donor variability. Hepatocytes from donors with specific features, such as natural polymorphisms in drug metabolising enzymes or transporters, or from diseased patients, can be selected for transplantation and, if desired, compared to animals reconstituted with control hepatocytes. An interesting approach in this regard is the recently described transplantation of hepatocytes derived from human induced pluripotent stem cells (iPSCs) to generate chimeric liver humanized mice [46– 48], because this potentially provides access to a very large pool of donors and enables the deliberate introduction of targeted mutations. It should be noted, however, that the level of reconstitution achieved with human iPSCs is still low compared with primary human hepatocytes and needs to be improved for wider application of this approach. Although it is in principle possible to mimic donor variability by genetic humanization, as previously demonstrated in genetically humanized mouse models expressing different polymorphic variants of CYP2D6 [44], the effort to generate and maintain such mice is very high.

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It should also be noted that the chimeric liver humanized mouse models do have unique uses beyond drug metabolism and toxicity studies. For example, chimeric liver humanized mice have been used extensively to study infectious diseases involving the liver, such as malaria [49,50] or hepatitis [51–53]. Owing to their host specificities, the corresponding pathogens are unable to infect mouse hepatocytes, but the transplantation with human cells renders the chimeric liver humanized mice susceptible. Accordingly, genetically engineered mouse models for proteins involved in drug metabolism and disposition are usually resistant to infections with these pathogens, although the transgenic expression of certain human factors can confer susceptibility to the mouse cells [54]. Furthermore, whereas the immune-deficiency of the chimeric liver humanized mice can be disadvantageous for certain applications, it can have its benefits for other studies. As described above for the dual liver and immune system reconstitution [36], chimeric liver humanized mice are generally transplantable with various human cell types, including human cancer cells. Transplantation of human liver cancer cells into chimeric humanized liver mice enables the study of these cells in a contextual and physiological environment [55]. The loop between studies in infectious diseases and oncology on the one hand and drug metabolism on the other is thus closed enabling drug efficacy testing in such liver chimeric mouse models. In addition to the susceptibility of these mice to liver pathogens and cancer cells, they also provide a human-like drug metabolism profile, at least as far as the liver is concerned. The anti-infective and anticancer characteristics of a test compound can therefore be studied in the context of human (liver) metabolism. An attractive concept related to the chimeric liver humanized mice is the possibility to use the same human hepatocytes for in vitro and in vivo applications and thereby to maximise the consistency between these studies. In fact, it has been shown that primary human hepatocytes can be serially transplanted into the FRG model and it is thereby possible to expand the pool of available human cells for in vitro applications [32]. The combined use of human hepatocytes and chimeric liver humanized mice can help to establish more accurate in vitro–in vivo correlations and to improve the extrapolation to the clinical situation. Equivalent to the use of human hepatocytes derived from chimeric liver humanized mice, it is possible to extract mouse hepatocytes derived from genetically humanized mouse models for in vitro studies [56]. Human extrapolations from the concomitant use of genetically humanized mice and derived hepatocytes might be possible as well, provided that there is a reasonable agreement between results obtained from the humanized mouse hepatocytes and human liver cells. A limiting factor that applies to the genetically humanized but not chimeric liver humanized mice is the risk of compensatory gene expression changes in alternative mouse pathways as a consequence of the genetic modification. An example is the profound upregulation of mouse Cyp2c enzymes in a Cyp3a gene cluster knockout mouse model [57]. Although it was subsequently found that other detoxifying systems were induced in these knockout mice as well, the transgenic expression of human CYP3A4 in the liver or intestine normalised the expression of most of these genes [58]. When a knockout is used as a control for a genetically humanized mouse model, it is therefore recommended to take potential changes in the

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expression of mouse pathways into account, specifically if these pathways can contribute to the metabolism and disposition of the compound under investigation. Changes in the expression of proteins involved in drug metabolism and disposition, in particular genetically humanized mouse models or even in the mouse or human hepatocytes of chimeric liver humanized mice, cannot be ruled out, but all published data available so far indicate that this might not be of general concern. The rigorous application of mRNA and protein quantification approaches in the validation of these models will undoubtedly shed further light on the relevance of such changes.

Applications of genetically and chimeric liver humanized mouse models in drug metabolism, disposition and toxicity As discussed earlier, genetically and chimeric liver humanized mice do have various applications in drug metabolism, disposition and toxicity. There was no intent to give an exhaustive summary of the numerous reports describing such applications, see refs [1,10,11,16,17] for reviews on genetically modified and [13,27,28] for chimeric liver humanized mouse models, but instead a concise description of the general principles and concepts is provided. A list of selected examples for the use of genetically and chimeric liver humanized mice for studies in drug metabolism, disposition and toxicity is provided in Table 4. Within this field, genetically and liver humanized mice have been used for DDI studies to assess the role of individual proteins in drug metabolism, disposition or toxicity, for general toxicity testing, for studies to predict the human hazard to rodent nongenotoxic liver carcinogens, for studies related to human metabolites, for drug clearance predictions and to understand the relative contribution of liver and gut CYP3A4 to the metabolism of a compound. DDIs have been studied extensively in genetically and liver humanized mice. DDIs can be caused either through induction or inhibition of a drug metabolising enzyme (DME) or transporter by a (predator) drug, which is given in combination with a (victim) drug that is metabolised by the affected DME or transporter (Fig. 2). Induction usually works via the activation of a xenobiotic receptor by the predator drug, which then enhances the expression of DMEs and transporters. Inhibition results from the competitive or allosteric binding of the predator drug to these proteins. The interaction of a drug with xenobiotic receptors as well as with DMEs and transporters can vary significantly between species, such that extrapolation to humans based on preclinical in vivo DDI studies in wild-type animals is often difficult [4,9]. By contrast, the prediction of clinical DDIs solely from in vitro experiments and in silico modelling can be challenging as well [59]. Accordingly, genetically humanized mouse models for key proteins involved in drug metabolism and disposition and chimeric liver humanized mouse models provide a powerful bridge from in vitro to in vivo. Owing to the important role of CYP3A4 in drug metabolism, many studies in humanized mouse models have focused on DDIs mediated by this enzyme, either related to the induction of CYP3A4 via PXR (or CAR) [23,24,60–63] or its inhibition by competitive or time-dependent inhibitors [40,42,64–66]. Nevertheless, in vivo DDI studies involving other human xenobiotic receptors [67,68] and DMEs [44,45,69] have been described as

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TABLE 4

Selected examples for the use of genetically humanized and chimeric liver humanized mice for studies in drug metabolism, disposition and toxicity

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Application

Genetically humanized mice

Liver chimeric mice

DDI studies (inhibition) DDI studies (induction) Assessing role of individual proteins Toxicity testing NGC testing Human metabolite identification Nonspecific human protein binding by reactive metabolites Prediction of drug clearance Extrapolation of drug biofluid concentrations by PBPK modelling Studying CYP3A4 liver versus gut metabolism

[40–42,44,45,56,64,65] [21,23,24,62,63] [41,44,45,56,64,74,75,77–79,134,142] [68,71,84–86] [70,72,73,89,92,93] [74,85,99–101]

[66,69] [32,60,61,147–149] [150] [82,83,150,151] [90,91] [43,66,102–109] [110,111] [113,152] [114,115]

[112] [40–42]

Abbreviations: CYP, cytochrome P450; DDI, drug–drug interaction; NGC, nongenotoxic carcinogenicity; PBPK, physiologically based pharmacokinetics.

well. Of particular interest are studies where humanized mouse models enabled the quantitative prediction of clinically relevant DDIs [24,60]. Genetically humanized mouse models are particularly suited to assess the role of individual proteins in drug metabolism, disposition or toxicity. This is due to the fact that these mice are humanized for one or a few proteins only and that wild-type controls as well as corresponding knockout models, which neither express the mouse nor the human protein, are usually available. This enables the dissection of the role of the particular human protein. This approach has been applied to assess the contribution of various xenobiotic receptors [21,68,70–73], CYPs [41,44,45,74,75], phase 2 enzymes [76,77] and transporters [56,78–80] to the metabolism, disposition and toxicity of a wide range of different compounds. The lack of specific knockout controls makes the use of chimeric liver humanized mice less straightforward for this type of application, although the selective inactivation of a given protein by chemical inhibitors provides a potential opportunity to overcome this limitation. Unforeseen toxicity, including hepatotoxicity, is a major concern in drug development, because many compounds fail at late clinical stages or are either withdrawn after launch or acquire ‘black box’ warnings limiting their clinical use as a consequence of such issues [81]. Obviously, preclinical in vivo studies in wildtype animals often do not adequately predict such adverse events reliably. Differences in the pathways of drug metabolism and disposition, or differential susceptibilities of mouse and human cells to drug-induced toxicity, are potential factors that can contribute to the deficiency of traditional in vivo toxicological approaches to predict these failures. Although genetically and chimeric liver humanized mice can help to overcome species differences in drug metabolism and disposition, only the latter offer the opportunity to assess the susceptibility of human cells (i.e. hepatocytes) to the toxicity of a compound. Accordingly, chimeric liver humanized mice lend themselves to testing for human hepatotoxicity. The power of this approach was impressively demonstrated by the successful prediction of the human susceptibility to two hepatotoxic compounds that were not recognised as such by traditional preclinical toxicology studies [82,83]. However, as well the successes of the chimeric mice the utility of genetically humanized mouse models for xenobiotic receptors and CYPs in predicting human hepatotoxicity has also been demonstrated [68,71,84–86]. Clearly, if the human protein is expressed in 8

other organs than the liver, genetically humanized mouse models can also be used to assess drug toxicity in extrahepatic tissues, as recently demonstrated by the protective role of human MRP2 against cisplatin-mediated nephrotoxicity [80]. A special application of genetically and chimeric liver humanized mice in the field of toxicology is for human risk assessment of rodent nongenotoxic liver carcinogens. Many compounds that induce liver tumours in rats or mice do not show the same response in humans [87]. The relevance of drug-induced rodent liver tumour formation to humans is therefore questionable. Furthermore, many rodent nongenotoxic liver carcinogens induce their effects through interaction with the nuclear receptors PXR, CAR or PPARa and it was speculated that these receptors are responsible for the differential carcinogenic response between rodents and humans [88]. Genetically humanized mouse models for PXR/CAR [89] and PPARa [70,72,73] as well as chimeric liver humanized mouse models [90,91] demonstrated a more humanlike hyperplastic response to the rodent nongenotoxic liver carcinogens phenobarbital (CAR activator) and WY14643 (PPARa acti- Q11 vator), respectively. However, it should be noted that recent studies have questioned the simple hypothesis that the genetic humanization of PXR/CAR is sufficient to confer human-like liver tumour formation susceptibility in response to phenobarbital to these genetically humanized mice [92,93]. Drug metabolites can be associated with drug toxicity and therefore need to be considered for safety testing [94]. According to the regulatory guidance on metabolites in safety testing (MIST) emanating from the FDA and the International Conference on Harmonization (ICH), human metabolites can require separate safety assessment if they are not formed in sufficient concentrations in the plasma of the species used during the preclinical safety assessment of the candidate drug [95]. Genetically and chimeric liver humanized mouse models have been proposed as potential solutions, when the formation of a unique or disproportionate metabolite in humans prevents the use of wild-type animals for such studies [96,97]. Apart from the aspect of safety assessment, it is preferable to have a solid understanding of the major drug–metabolite profile in humans to be aware of potential concerns and, if necessary, mitigate such risks early on in the drug development process. Although in vitro approaches, for example exploiting human hepatocytes, are routinely used to identify human metabolites, these methods frequently fail to predict the accurate formation or abundance of a

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human metabolite in the clinic [98]. There are numerous reports for the use of genetically [74,85,99–101] and chimeric liver [43,66,102– 109] humanized mice to identify selected human metabolites or to establish a human metabolite profile and these models are promising complements to traditional in vitro assays. Whether or not they will be suitable for the long term (greater than one month), toxicity studies required for drug registration have, however, yet to be established. Chimeric liver humanized mouse models have also been used to study the nonspecific binding of human proteins by reactive intermediates from idiosyncratic hepatotoxicants [110,111]. Therefore, these models might help to understand the mechanism of toxicity of such compounds or to evaluate hepatotoxic potential before first-in-human testing. Despite the potential utility of humanized mouse models to support various aspects of toxicity testing, their limitations in terms of costs, lack of historical background data or fragility of immunocompromised animals will most probably restrict the use of these models to case by case rather than generic applications for the foreseeable future. The accurate prediction of drug clearance in humans is of crucial importance in drug development, but has remained a challenging endeavour to date [112,113]. The approach of using humanized mouse models to improve human clearance predictions is relatively new and only a few studies have been published recently. One example described the successful use of a genetically humanized CYP3A4 mouse to predict the hepatic clearance of CYP3A4 substrates in humans [112]. In another study chimeric liver humanized mice were used to predict the hepatic clearance and half-life of selected drugs metabolised by CYP as well as non-CYP enzymes [113]. The authors from the latter study concluded that rank order of human clearance and half-life could be accurately determined and, although the prediction of absolute values might not be possible, the approach could be a useful adjunct to in vitro technologies to predict semi-quantitatively the pharmacokinetic characteristics of compounds in humans. The utility of chimeric liver humanized mice to evaluate human pharmacokinetics and toxicokinetics of drugs and their metabolites was further substantiated by two recent studies using such mice in combination with a simple physiologically based pharmacokinetic (PBPK) modelling approach [114,115]. A special application is the use of specific genetically humanized mouse models to assess the relative contribution of liver and gut CYP3A4 to the metabolism of a compound. This has become possible through the availability of humanized mice that express human CYP3A4 selectively in the liver or in the gut, or together in both organs [41]. By using these mouse models it was demonstrated that intestinal CYP3A4 has a predominant role in the first-pass metabolism of docetaxel, triazolam and lopinavir after oral administration of these compounds [40–42].

Guidance for the selection of genetically and chimeric liver humanized mouse models for studies in drug metabolism and toxicity There are no universal rules for the selection of genetically and/or chimeric liver humanized mouse models for studies in drug metabolism and toxicity, but such decisions need to be made on a case-by-case basis, taking various factors into account. Nevertheless, it is possible to provide some guidance for the selection of the sort of approach that can be used. As a general rule of thumb it can

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be argued that the advantages of chimeric liver over genetically humanized mice increase the more the liver supersedes the relevance of other organs, and the more enzymes or pathways involved in drug metabolism, disposition and toxicity there are that need to be considered. By contrast, genetically humanized mouse models can have benefits where extrahepatic organs are involved (either with or without contributions from the liver) and where drug metabolism, disposition and toxicity are predominantly mediated by a single or a few proteins. For example, genetically humanized mice are gaining increasing traction for studies related to drug transporters. Owing to the fact that these proteins are often involved in extrahepatic tissue distribution, for example in the intestine, kidney or blood–brain barrier, chimeric liver humanized mice have little value for such studies, except for studies targeted on hepatic uptake or efflux. From the applications perspective, there is a smooth transition from preferred uses of genetically humanized mice, via applications practicable in both types of models, to studies favourably conducted in chimeric liver humanized mice. The first category is clearly represented by studies aimed to assess the relative contribution of liver and gut CYP3A4 to the metabolism of a compound, which is only feasible in specific genetically humanized CYP3A4 models. Furthermore, genetically humanized mice are usually preferred for the assessment of the role of individual proteins in drug metabolism, disposition or toxicity, owing to the fact that the availability of knockout and wild-type controls enables the dissection of the function of a particular human protein. As explained above, transporter studies are usually a domain of genetically humanized mice as well. By contrast, chimeric liver humanized mice are the first choice for the identification of unknown human metabolites or to establish a human metabolite profile. Essentially, chimeric liver humanized mice can be used to study the liver metabolism of all types of small molecules and without any knowledge of the enzymes involved in their turnover. From the safety assessment perspective genetically humanized mice might come into play if the enzyme accounting for the formation of the respective metabolite has been identified and a corresponding transgenic model is available. Another obvious preferred field of activity for chimeric liver humanized mice is in hepatotoxicity testing. Intuitively, the possibility to study the susceptibility of human hepatocytes to the toxicity of a compound appears to be of greater human relevance than assessing this effect on mouse hepatocytes from a genetically humanized mouse model. Recent studies in chimeric liver humanized mice with known human hepatotoxins have confirmed the value of this approach [82,83]. Furthermore, the availability of dual liver–immune-system humanized mice [36,37] might provide an additional benefit for compounds that exert their hepatotoxic effect via an interaction of the liver and immune system or where changes in the expression of drug-metabolising enzymes or transporters by infections alters drug metabolism and disposition [116]. Compared with that, the use of genetically humanized mouse models in hepatotoxicity testing is more limited, but might add value in understanding the involved mechanism of action. Furthermore, genetically humanized mice offer the opportunity to study drug toxicity in extrahepatic tissues. Model selection is more challenging for applications that are practicable and potentially valuable in both types of models and

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where decisions need to be made on a case-by-case basis. These decisions need to take the knowledge about the characteristics of the test compound into account. In the case of DDI studies this includes information about the pathways of drug metabolism, disposition and clearance known to be involved, and the expected clinical co-medications. If, for example, the major pathway of test compound clearance is via hepatic metabolism, in vitro studies indicate the involvement of multiple DMEs and the expected clinical co-medication involves either inhibitors or inducers of these DMEs, chimeric liver humanized mice might be the preferred model. If, under otherwise identical conditions, a single enzyme such as CYP3A4 dominates the metabolism of the test compound, genetically and liver humanized mice might be equally suited. If, in the latter case, the test compound and the inhibitor or inducer are given via oral administration and gut metabolism significantly contributes to first-pass metabolism of the test compound, a genetically humanized mouse might be selected. The intention of these examples is to sensitise the reader to the necessity to consider various aspects carefully to enable informed decisions to be made about the model of choice and to maximise the predictability of the study. In the case of human risk assessment of rodent nongenotoxic carcinogens, the predictability of PXR/CAR genetically humanized mice is currently not clear [89,92,93], whereas all available data indicate a good accordance in the responses of humans and PPARa-humanized mice to PPARa agonists [70,72,73]. The limited results from chimeric liver humanized mice treated with CAR and PPARa activators look promising [90,91], but long-term studies will be required to assess whether the tumorigenic risk in humans is accurately predicted. The authors of this article doubt the general predictability of human drug clearance by currently available humanized mouse models with high accuracy, and rather take the position that this application is restricted to compounds that fulfil certain criteria. The prediction of human clearance of compounds that are primarily metabolised by CYP3A4 appears to be possible with reasonable accuracy in genetically humanized CYP3A4 mice [112] and this concept might be extendable to substrates of other CYPs and their respective genetically humanized mouse models. However, accurate predictions for compounds with complex pathways of clearance appear unachievable in genetically humanized mice. Data from chimeric liver humanized mouse models might contribute to clearance predictions when contextualised with corresponding in vitro data [113]; however, this approach appears inappropriate if nonhepatic, for example renal, clearance is a major factor. Furthermore, the residual mouse hepatocytes in chimeric liver humanized mice can significantly obscure the contribution of the human cells to drug clearance, specifically for compounds that are more rapidly metabolised by murine DMEs. The endeavour to predict human drug clearance by utilising genetically or chimeric liver humanized mouse models and the selection of the appropriate model therefore requires a profound understanding of the characteristics of a compound and is only feasible on a case-by-case basis.

Concluding remarks: trends and perspectives Genetically and chimeric liver humanized mouse models are emerging as promising tools for improved prediction of the 10

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pharmacokinetic, DDI and safety characteristics of compounds in humans. From the previous sections it should have become evident that these models are just at the beginning to fulfil their potential for these applications and more work will be required for their wider acceptance. However, some of the trends and perspectives on the path to achieving this goal are clear. Although the various potential applications of genetic and chimeric liver humanized mice in drug, metabolism, disposition and toxicity are reasonably well defined now (Table 4), it appears that more data will be required to demonstrate the robustness and accuracy in predicting clinical outcomes for each of these uses. For example, initial studies with selected compounds indicate the utility of genetically and chimeric liver humanized mice in quantitatively predicting clinical DDIs [24,60], but the robustness of such predictions over a wide range of different compounds with distinct characteristics is currently unclear. In a similar manner, the successful prediction of human hepatotoxicity with selected compounds in chimeric liver humanized mice is promising [82,83], but how well will these mice predict the human susceptibility for compounds with different mode of actions? Further, how reliably will genetic and chimeric liver humanized mice predict the human hazard to a rodent nongenotoxic carcinogen? Our view is that it will not only be necessary to determine the promise of these tools but also to define their limitations clearly, so that studies can be designed with a maximum likelihood of success. It can be expected that the increasing use of genetically and chimeric liver humanized mice by academia and industry and the growing number of publications will help to establish a solid database. In our opinion humanized mice will not be stand-alone tools to predict human outcomes in normal cases but that results obtained from such studies will need to be contextualised with in silico predictions, in vitro data and, if available, clinical results. Accordingly, these in vivo models will be a complement to established technologies, rather than a replacement. We anticipate an increasing combined use of these different approaches to select the most promising drug candidates, to increase the confidence before firstin-human tests, to determine accurate starting doses for clinical studies or to explain unexpected clinical findings. Where meaningful, one important aspect and probable future trend will be the increasing inclusion of data from humanized mouse models for quantitative predictions of human outcomes. The integration of modules for humanized mouse models into commercially available PBPK modelling simulators might be a possible trend in the future. In the case of genetically humanized mouse models for proteins involved in drug metabolism and disposition, we do see three major trends for the future: (i) a continuation of the generation of more-complex, multiple-humanized mice, which will allow the study of the interaction of compounds with various pathways of drug metabolism and disposition in a single mouse model; (ii) the availability of an increasing number of transporter humanized mouse models, which will satisfy the growing demand to consider the role of transporters in drug pharmacokinetics, DDI and toxicity; and (iii) the use for efficacy testing, either by combination with genetically engineered disease models or chemically induced disease phenotypes, in order to assess drug efficacy on a human metabolic background.

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For chimeric liver humanized mice we anticipate the following major developments: (i) an increasing robustness in the generation of mice highly reconstituted with human hepatocytes (>90%), although it is not foreseeable that full reconstitution will be achieved in the near future; (ii) an elimination of mouse-specific pathways of drug metabolism and disposition in the liver (and maybe in other organs) by corresponding genetic alterations, as in the recently described chimeric liver humanized model carrying a deletion of the mouse Cyp3a gene cluster [117]; (iii) a robust availability of liver humanized mouse models with additional cellular humanizations, similar to the recently described dual liver–immune-system reconstitution [36]; and (iv) a higher degree of reconstitution with hepatocytes derived from human iPSCs. It also appears conceivable to aggregate genetically and chimeric liver humanized mouse models to combine the benefits of both approaches, although this endeavour will be technically challenging and laborious. Furthermore, it is possible to predict that genetically and chimeric liver humanized rats will become available in the near to medium-term future. The application of gene editing technologies has enabled the efficient generation of knockout rats [118,119] and it is only a question of time before researchers combine rats that are deficient for proteins involved in drug metabolism and disposition with corresponding genetic humanizations. Nevertheless, it should be noted that establishing a comparable panel of genetically humanized rat models to that which currently exists in the mouse field remains a very long way off. First attempts to generate chimeric liver humanised rats have been made as well [120–122]. Further optimisation will be required to achieve a robust and efficient repopulation of rat liver with human hepatocytes, but there is reason to believe that this can be accomplished within the next few years.

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Genetically and chimeric liver humanized mice are neither officially accepted by regulatory authorities nor is their use for any particular application explicitly recommended. Accordingly, the current use of these in vivo models is more targeted on decision making rather than on regulatory purposes. It is conceivable that the use of these tools for specific applications could become part of regulatory guidelines in the future, but this requires a much more extensive validation of these tools than that available to date and it will certainly be many years from now. Nevertheless, at various conferences and in peer-reviewed publications [94] representatives from the FDA have taken an open-minded attitude towards the use of humanized mouse models to support regulatory submissions, if scientifically reasonable. In such cases sponsors are encouraged to involve the regulators in the early phases of the project. It is therefore not unlikely that data from studies in genetically humanized mouse models for genes involved in drug metabolism and disposition or chimeric liver humanized mice will be included occasionally as supporting information in regulatory submissions. We wish to conclude this review by advising investigators not to use genetically and chimeric liver humanized mouse models imprudently to predict clinical outcomes. Essentially, these models are still mice and studies need to be well thought out and carefully executed. If so, genetically and chimeric liver humanized mice do have significant potential to increase the predictability of clinical drug pharmacokinetics, DDIs and toxicity and to contribute to improving the success rates in drug development.

Conflicts of interest The authors declare no financial conflicts of interest. The authors alone are responsible for the content and writing of this paper.

References

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1 Scheer, N. et al. (2013) Generation and utility of genetically humanized mouse models. Drug Discov. Today 18, 1200–1211 2 Shultz, L.D. et al. (2007) Humanized mice in translational biomedical research. Nat. Rev. Immunol. 7, 118–130 3 Lin, J.H. (1995) Species similarities and differences in pharmacokinetics. Drug Metab. Dispos. 23, 1008–1021 4 Martignoni, M. et al. (2006) Species differences between mouse, rat, dog, monkey and human CYP-mediated drug metabolism, inhibition and induction. Expert Opin. Drug Metab. Toxicol. 2, 875–894 5 Owen, A. and Curley, P. (2014) Species similarities and differences in pharmacokinetics and distribution of antiretroviral drugs. In Humanized Mice for HIV Research (for HIV Researchpp. 339–360, Springer Science and Business Media 6 Stanley, L.A. et al. (2006) PXR and CAR: nuclear receptors which play a pivotal role in drug disposition and chemical toxicity. Drug Metab. Rev. 38, 515–597 7 Nishimuta, H. et al. (2013) Species differences in hepatic and intestinal metabolic activities for 43 human cytochrome P450 substrates between humans and rats or dogs. Xenobiotica 43, 948–955 8 Furukawa, T. et al. (2014) Species differences in intestinal glucuronidation activities between humans, rats, dogs and monkeys. Xenobiotica 44, 205–216 9 Chu, X. et al. (2013) Species differences in drug transporters and implications for translating preclinical findings to humans. Expert Opin. Drug Metab. Toxicol. 9, 237–252 10 Scheer, N. and Wolf, C.R. (2013) Xenobiotic receptor humanized mice and their utility. Drug Metab. Rev. 45, 110–121 11 Scheer, N. and Wolf, C.R. (2014) Genetically humanized mouse models of drug metabolizing enzymes and transporters and their applications. Xenobiotica 44, 96–108 12 Bissig, K.D. and Grompe, M. (2013) Response to ‘‘Can ‘humanized’ mice improve drug development in the 21st century?’’. Trends Pharmacol Sci. 34, 425

13 Peltz, G. (2013) Can ‘humanized’ mice improve drug development in the 21st century? Trends Pharmacol. Sci. 34, 255–260 14 Peltz, G. (2013) Reply to bissig and grompe. Trends Pharmacol. Sci. 34, 426 15 Strom, S.C. et al. (2010) Chimeric mice with humanized liver: tools for the study of drug metabolism, excretion, and toxicity. Methods Mol. Biol. 640, 491–509 16 Cheung, C. and Gonzalez, F.J. (2008) Humanized mouse lines and their application for prediction of human drug metabolism and toxicological risk assessment. J. Pharmacol. Exp. Ther. 327, 288–299 17 Shen, H.W. et al. (2011) Humanized transgenic mouse models for drug metabolism and pharmacokinetic research. Curr. Drug Metab. 12, 997–1006 18 Devoy, A. et al. (2012) Genomically humanized mice: technologies and promises. Nat. Rev. Genet. 13, 14–20 19 Hu, Y. et al. (2014) Development and characterization of a novel mouse line humanized for the intestinal peptide transporter PEPT1. Mol. Pharm. 11, 3737– 3746 20 Giacomini, K.M. et al. (2010) Membrane transporters in drug development. Nat. Rev. Drug Discov. 9, 215–236 21 Scheer, N. et al. (2008) A novel panel of mouse models to evaluate the role of human pregnane X receptor and constitutive androstane receptor in drug response. J. Clin. Invest. 118, 3228–3239 22 Felmlee, M.A. et al. (2008) Cytochrome P450 expression and regulation in CYP3A4/CYP2D6 double transgenic humanized mice. Drug Metab. Dispos. 36, 435– 441 23 Ma, X. et al. (2008) A double transgenic mouse model expressing human pregnane X receptor and cytochrome P450 3A4. Drug Metab. Dispos. 36, 2506–2512 24 Hasegawa, M. et al. (2011) Quantitative prediction of human pregnane X receptor and cytochrome P450 3A4 mediated drug-drug interaction in a novel multiple humanized mouse line. Mol. Pharmacol. 80, 518–528

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25 Nelson, D.R. et al. (2004) Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants. Pharmacogenetics 14, 1–18 26 Guengerich, F.P. (2008) Cytochrome p450 and chemical toxicology. Chem. Res. Toxicol. 21, 70–83 27 Foster, J.R. et al. (2014) Chimeric rodents with humanized liver: bridging the preclinical/clinical trial gap in ADME/toxicity studies. Xenobiotica 44, 109–122 28 Grompe, M. and Strom, S. (2013) Mice with human livers. Gastroenterology 145, 1209–1214 29 Rhim, J.A. et al. (1994) Replacement of diseased mouse liver by hepatic cell transplantation. Science 263, 1149–1152 30 Rhim, J.A. et al. (1995) Complete reconstitution of mouse liver with xenogeneic hepatocytes. Proc. Natl. Acad. Sci. U. S. A. 92, 4942–4946 31 Sandgren, E.P. et al. (1991) Complete hepatic regeneration after somatic deletion of an albumin-plasminogen activator transgene. Cell 66, 245–256 32 Azuma, H. et al. (2007) Robust expansion of human hepatocytes in Fah / /Rag2 / /Il2rg / mice. Nat. Biotechnol. 25, 903–910 33 Hasegawa, M. et al. (2011) The reconstituted ‘humanized liver’ in TK-NOG mice is mature and functional. Biochem. Biophys. Res. Commun. 405, 405–410 34 Washburn, M.L. et al. (2011) A humanized mouse model to study hepatitis C virus infection, immune response, and liver disease. Gastroenterology 140, 1334–1344 35 Zhang, R.R. et al. (2015) Human hepatic stem cells transplanted into a fulminant hepatic failure Alb-TRECK/SCID mouse model exhibit liver reconstitution and drug metabolism capabilities. Stem Cell Res. Ther. 6, 49 36 Wilson, E.M. et al. (2014) Extensive double humanization of both liver and hematopoiesis in FRGN mice. Stem Cell Res. 13, 404–412 37 Strick-Marchand, H. et al. (2015) A novel mouse model for stable engraftment of a human immune system and human hepatocytes. PLoS One 10, e0119820 38 Adams, D.H. et al. (2010) Mechanisms of immune-mediated liver injury. Toxicol. Sci. 115, 307–321 39 Krishna, D.R. and Klotz, U. (1994) Extrahepatic metabolism of drugs in humans. Clin. Pharmacokinet. 26, 144–160 40 ter Heine, R. et al. (2011) An integrated pharmacokinetic model for the influence of CYP3A4 expression on the in vivo disposition of lopinavir and its modulation by ritonavir. J. Pharm. Sci. 100, 2508–2515 41 van Herwaarden, A.E. et al. (2007) Knockout of cytochrome P450 3A yields new mouse models for understanding xenobiotic metabolism. J. Clin. Invest. 117, 3583– 3592 42 van Waterschoot, R.A. et al. (2009) Inhibition and stimulation of intestinal and hepatic CYP3A activity: studies in humanized CYP3A4 transgenic mice using triazolam. Drug Metab. Dispos. 37, 2305–2313 43 Bateman, T.J. et al. (2014) Application of chimeric mice with humanized liver for study of human-specific drug metabolism. Drug Metab. Dispos. 42, 1055–1065 44 Scheer, N. et al. (2012) Modeling human cytochrome P450 2D6 metabolism and drug-drug interaction by a novel panel of knockout and humanized mouse lines. Mol. Pharmacol. 81, 63–72 45 Scheer, N. et al. (2012) Generation and characterization of novel cytochrome P450 Cyp2c gene cluster knockout and CYP2C9 humanized mouse lines. Mol. Pharmacol. 82, 1022–1029 46 Nagamoto, Y. et al. (2015) Efficient engraftmentof human iPS cell-derived hepatocyte-like cells in uPA/SCID mice by overexpression of FNK, a Bcl-xmutant gene. Cell Transplant. 24, 1127–1138 47 Takebe, T. et al. (2014) Generation of a vascularized and functional human liver from an iPSC-derived organ bud transplant. Nat. Protoc. 9, 396–409 48 Zhu, S. et al. (2014) Mouse liver repopulation with hepatocytes generated from human fibroblasts. Nature 508, 93–97 49 Foquet, L. et al. (2014) Vaccine-induced monoclonal antibodies targeting circumsporozoite protein prevent Plasmodium falciparum infection. J. Clin. Invest. 124, 140–144 50 Vaughan, A.M. et al. (2012) Complete Plasmodium falciparum liver-stage development in liver-chimeric mice. J. Clin. Invest. 122, 3618–3628 51 Bissig, K.D. et al. (2010) Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment. J. Clin. Invest. 120, 924–930 52 Kosaka, K. et al. (2013) A novel TK-NOG based humanized mouse model for the study of HBV and HCV infections. Biochem. Biophys. Res. Commun. 441, 230–235 53 Meuleman, P. and Leroux-Roels, G. (2008) The human liver-uPA-SCID mouse: a model for the evaluation of antiviral compounds against HBV and HCV. Antiviral Res. 80, 231–238 54 Dorner, M. et al. (2013) Completion of the entire hepatitis C virus life cycle in genetically humanized mice. Nature 501, 237–241 55 Fujiwara, S. et al. (2012) A novel animal model for in vivo study of liver cancer metastasis. World J. Gastroenterol. 18, 3875–3882

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56 Salphati, L. et al. (2014) Evaluation of organic anion transporting polypeptide 1B1 and 1B3 humanized mice as a translational model to study the pharmacokinetics of statins. Drug Metab. Dispos. 42, 1301–1313 57 van Waterschoot, R.A. et al. (2008) Midazolam metabolism in cytochrome P450 3A knockout mice can be attributed to up-regulated CYP2C enzymes. Mol. Pharmacol. 73, 1029–1036 58 van Waterschoot, R.A. et al. (2009) Intestinal cytochrome P450 3A plays an important role in the regulation of detoxifying systems in the liver. FASEB J. 23, 224–231 59 Wienkers, L.C. and Heath, T.G. (2005) Predicting in vivo drug interactions from in vitro drug discovery data. Nat. Rev. Drug Discov. 4, 825–833 60 Hasegawa, M. et al. (2012) Investigation of drug-drug interactions caused by human pregnane X receptor-mediated induction of CYP3A4 and CYP2C subfamilies in chimeric mice with a humanized liver. Drug Metab. Dispos. 40, 474– 480 61 Katoh, M. et al. (2005) In vivo induction of human cytochrome P450 3A4 by rifabutin in chimeric mice with humanized liver. Xenobiotica 35, 863–875 62 Kim, S. et al. (2008) Quantitative relationship between rifampicin exposure and induction of Cyp3a11 in SXR humanized mice: extrapolation to human CYP3A4 induction potential. Drug Metab. Lett. 2, 169–175 63 Ma, X. et al. (2007) The PREgnane X receptor gene-humanized mouse: a model for investigating drug-drug interactions mediated by cytochromes P450 3A. Drug Metab. Dispos. 35, 194–200 64 Granvil, C.P. et al. (2003) Expression of the human CYP3A4 gene in the small intestine of transgenic mice: in vitro metabolism and pharmacokinetics of midazolam. Drug Metab. Dispos. 31, 548–558 65 Kazuki, Y. et al. (2013) Trans-chromosomic mice containing a human CYP3A cluster for prediction of xenobiotic metabolism in humans. Hum. Mol. Genet. 22, 578–592 66 Nishimura, T. et al. (2013) Using chimeric mice with humanized livers to predict human drug metabolism and a drug-drug interaction. J. Pharmacol. Exp. Ther. 344, 388–396 67 Flaveny, C.A. et al. (2009) Ligand selectivity and gene regulation by the human aryl hydrocarbon receptor in transgenic mice. Mol. Pharmacol. 75, 1412–1420 68 Moriguchi, T. et al. (2003) Distinct response to dioxin in an arylhydrocarbon receptor (AHR)-humanized mouse. Proc. Natl. Acad. Sci. U. S. A. 100, 5652–5657 69 Katoh, M. et al. (2007) In vivo drug metabolism model for human cytochrome P450 enzyme using chimeric mice with humanized liver. J. Pharm. Sci. 96, 428–437 70 Cheung, C. et al. (2004) Diminished hepatocellular proliferation in mice humanized for the nuclear receptor peroxisome proliferator-activated receptor alpha. Cancer Res. 64, 3849–3854 71 Li, F. et al. (2013) Human PXR modulates hepatotoxicity associated with rifampicin and isoniazid co-therapy. Nat. Med. 19, 418–420 72 Morimura, K. et al. (2006) Differential susceptibility of mice humanized for peroxisome proliferator-activated receptor alpha to Wy-14,643-induced liver tumorigenesis. Carcinogenesis 27, 1074–1080 73 Yang, Q. et al. (2008) The PPAR alpha-humanized mouse: a model to investigate species differences in liver toxicity mediated by PPAR alpha. Toxicol. Sci. 101, 132– 139 74 Derkenne, S. et al. (2005) Theophylline pharmacokinetics: comparison of Cyp1a1( / ) and Cyp1a2( / ) knockout mice, humanized hCYP1A1_1A2 knockin mice lacking either the mouse Cyp1a1 or Cyp1a2 gene, and Cyp1(+/+) wild-type mice. Pharmacogenet. Genomics 15, 503–511 75 Dragin, N. et al. (2007) Generation of ‘humanized’ hCYP1A1_1A2_Cyp1a1/1a2( / ) mouse line. Biochem. Biophys. Res. Commun. 359, 635–642 76 Cai, H. et al. (2010) A humanized UGT1 mouse model expressing the UGT1A1*28 allele for assessing drug clearance by UGT1A1-dependent glucuronidation. Drug Metab. Dispos. 38, 879–886 77 Sugamori, K.S. et al. (2011) Liver-selective expression of human arylamine Nacetyltransferase NAT2 in transgenic mice. Drug Metab. Dispos. 39, 882–890 78 Higgins, J.W. et al. (2014) Utility of Oatp1a/1b-knockout and OATP1B1/3humanized mice in the study of OATP-mediated pharmacokinetics and tissue distribution: case studies with pravastatin, atorvastatin, simvastatin, and carboxydichlorofluorescein. Drug Metab. Dispos. 42, 182–192 79 van de Steeg, E. et al. (2013) Influence of human OATP1B1, OATP1B3, and OATP1A2 on the pharmacokinetics of methotrexate and paclitaxel in humanized transgenic mice. Clin. Cancer Res. 19, 821–832 80 Wen, X. et al. (2014) Transgenic expression of the human MRP2 transporter reduces cisplatin accumulation and nephrotoxicity in Mrp2-null mice. Am. J. Pathol. 184, 1299–1308 81 Kola, I. and Landis, J. (2004) Can the pharmaceutical industry reduce attrition rates? Nat. Rev. Drug Discov. 3, 711–715

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82 Xu, D. et al. (2014) Fialuridine induces acute liver failure in chimeric TK-NOG mice: a model for detecting hepatic drug toxicity prior to human testing. PLoS Med 11, e1001628 83 Xu, D. et al. (2015) Chimeric TK-NOG mice: a predictive model for cholestatic human liver toxicity. J. Pharmacol. Exp. Ther. 352, 274–280 84 Cheng, J. et al. (2009) Rifampicin-activated human pregnane X receptor and CYP3A4 induction enhance acetaminophen-induced toxicity. Drug Metab. Dispos. 37, 1611–1621 85 Cheung, C. et al. (2011) Rapid induction of colon carcinogenesis in CYP1Ahumanized mice by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and dextran sodium sulfate. Carcinogenesis 32, 233–239 86 Cheung, C. et al. (2005) The Cyp2e1-humanized transgenic mouse: role of Cyp2e1 in acetaminophen hepatotoxicity. Drug Metab. Dispos. 33, 449–457 87 Lake, B.G. (2009) Species differences in the hepatic effects of inducers of CYP2B and CYP4A subfamily forms: relationship to rodent liver tumour formation. Xenobiotica 39, 582–596 88 Boverhof, D.R. et al. (2011) Transgenic animal models in toxicology: historical perspectives and future outlook. Toxicol. Sci. 121, 207–233 89 Ross, J. et al. (2010) Human constitutive androstane receptor (CAR) and pregnane X receptor (PXR) support the hypertrophic but not the hyperplastic response to the murine non-genotoxic hepatocarcinogens phenobarbital and chlordane in vivo. Toxicol. Sci. 116, 452–466 90 Tateno, C. et al. (2015) Chimeric mice with hepatocyte-humanized liver as an appropriate model to study human peroxisome proliferator-activated receptoralpha. Toxicol. Pathol. 43, 233–248 91 Yamada, T. et al. (2014) Human hepatocytes support the hypertrophic but not the hyperplastic response to the murine nongenotoxic hepatocarcinogen sodium phenobarbital in an in vivo study using a chimeric mouse with humanized liver. Toxicol. Sci. 142, 137–157 92 Braeuning, A. et al. (2014) Phenobarbital-mediated tumor promotion in transgenic mice with humanized CAR and PXR. Toxicol. Sci. 140, 259–270 93 Luisier, R. et al. (2014) Phenobarbital induces cell cycle transcriptional responses in mouse liver humanized for constitutive androstane and pregnane x receptors. Toxicol. Sci. 139, 501–511 94 Robison, T.W. and Jacobs, A. (2009) Metabolites in safety testing. Bioanalysis 1, 1193–1200 95 Frederick, C.B. and Obach, R.S. (2010) Metabolites in safety testing: ‘‘MIST’’ for the clinical pharmacologist. Clin. Pharmacol. Ther. 87, 345–350 96 Kamimura, H. et al. (2010) Assessment of chimeric mice with humanized liver as a tool for predicting circulating human metabolites. Drug Metab. Pharmacokinet. 25, 223–235 97 Powley, M.W. et al. (2009) Safety assessment of drug metabolites: implications of regulatory guidance and potential application of genetically engineered mouse models that express human P450s. Chem. Res. Toxicol. 22, 257–262 98 Anderson, S. et al. (2009) Predicting circulating human metabolites: how good are we? Chem. Res. Toxicol. 22, 243–256 99 Chen, C. et al. (2006) Urinary metabolite profiling reveals CYP1A2-mediated metabolism of NSC686288 (aminoflavone). J. Pharmacol. Exp. Ther. 318, 1330– 1342 100 Li, F. et al. (2012) Metabolomics reveals the metabolic map of procainamide in humans and mice. Biochem. Pharmacol. 83, 1435–1444 101 Shen, H.W. et al. (2010) Effects of monoamine oxidase inhibitor and cytochrome P450 2D6 status on 5-methoxy-N,N-dimethyltryptamine metabolism and pharmacokinetics. Biochem. Pharmacol. 80, 122–128 102 Barnes, A.J. et al. (2014) Endogenous and xenobiotic metabolite profiling of liver extracts from SCID and chimeric humanized mice following repeated oral administration of troglitazone. Xenobiotica 44, 174–185 103 Igawa, Y. et al. (2014) In vitro and in vivo metabolism of a novel chymase inhibitor, SUN13834, and the predictability of human metabolism using mice with humanized liver. Xenobiotica 44, 154–163 104 Kamimura, H. et al. (2015) Formation of the accumulative human metabolite and human-specific glutathione conjugate of diclofenac in TK-NOG chimeric mice with humanized livers. Drug Metab. Dispos. 43, 309–316 105 Samuelsson, K. et al. (2014) Troglitazone metabolism and transporter effects in chimeric mice: a comparison between chimeric humanized and chimeric murinized FRG mice. Xenobiotica 44, 186–195 106 Samuelsson, K. et al. (2012) Pharmacokinetics and metabolism of midazolam in chimeric mice with humanised livers. Xenobiotica 42, 1128–1137 107 Sanoh, S. et al. (2012) Predictability of metabolism of ibuprofen and naproxen using chimeric mice with human hepatocytes. Drug Metab. Dispos. 40, 2267–2272 108 Sanoh, S. et al. (2012) Prediction of human metabolism of FK3453 by aldehyde oxidase using chimeric mice transplanted with human or rat hepatocytes. Drug Metab. Dispos. 40, 76–82

REVIEWS

109 Tanoue, C. et al. (2013) Prediction of human metabolism of the sedative-hypnotic zaleplon using chimeric mice transplanted with human hepatocytes. Xenobiotica 43, 956–962 110 Yamazaki, H. et al. (2015) Zone analysis by two-dimensional electrophoresis with accelerator mass spectrometry of in vivo protein bindings of idiosyncratic hepatotoxicants troglitazone and flutamide bioactivated in chimeric mice with humanized liver. Toxicol. Res. 4, 106–111 111 Yamazaki, H. et al. (2010) Approach for in vivo protein binding of 5-n-butylpyrazolo[1,5-a]pyrimidine bioactivated in chimeric mice with humanized liver by two-dimensional electrophoresis with accelerator mass spectrometry. Chem. Res. Toxicol. 23, 152–158 112 Mitsui, T. et al. (2014) A useful model capable of predicting the clearance of cytochrome 3A4 (CYP3A4) substrates in humans: validity of CYP3A4 transgenic mice lacking their own Cyp3a enzymes. Drug Metab. Dispos. 42, 1540–1547 113 Sanoh, S. et al. (2012) Prediction of in vivo hepatic clearance and half-life of drug candidates in human using chimeric mice with humanized liver. Drug Metab. Dispos. 40, 322–328 114 Adachi, K. et al. (2015) Human biofluid concentrations of mono(2ethylhexyl)phthalate extrapolated from pharmacokinetics in chimeric mice with humanized liver administered with di(2-ethylhexyl)phthalate and physiologically based pharmacokinetic modeling. Environ. Toxicol. Pharmacol. 39, 1067–1073 115 Miyaguchi, T. et al. (2015) Human urine and plasma concentrations of bisphenol A extrapolated from pharmacokinetics established in in vivo experiments with chimeric mice with humanized liver and semi-physiological pharmacokinetic modeling. Regul. Toxicol. Pharmacol. 72, 71–76 116 Morgan, E.T. et al. (2008) Regulation of drug-metabolizing enzymes and transporters in infection, inflammation, and cancer. Drug Metab. Dispos. 36, 205–216 117 Kato, K. et al. (2015) Development of murine Cyp3a knockout chimeric mice with humanized liver. Drug Metab. Dispos. 43, 1208–1217 118 Geurts, A.M. et al. (2009) Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325, 433 119 Li, D. et al. (2013) Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat. Biotechnol. 31, 681–683 120 Igarashi, Y. et al. (2008) Engraftment of human hepatocytes in the livers of rats bearing bone marrow reconstructed with immunodeficient mouse bone marrow cells. Xenotransplantation 15, 235–245 121 Tachibana, A. et al. (2013) Repopulation of the immunosuppressed retrorsinetreated infant rat liver with human hepatocytes. Xenotransplantation 20, 227–238 122 Wu, C.H. et al. (2001) Human hepatocytes transplanted into genetically immunocompetent rats are susceptible to infection by hepatitis B virus in situ. J. Viral Hepat. 8, 111–119 123 Zhang, J. et al. (2002) Modulation of acetaminophen-induced hepatotoxicity by the xenobiotic receptor CAR. Science 298, 422–424 124 Xie, W. et al. (2000) Reciprocal activation of xenobiotic response genes by nuclear receptors SXR/PXR and CAR. Genes Dev. 14, 3014–3023 125 Zhou, J. et al. (2006) A novel pregnane X receptor-mediated and sterol regulatory element-binding protein-independent lipogenic pathway. J. Biol. Chem. 281, 15013–15020 126 Lichti-Kaiser, K. and Staudinger, J.L. (2008) The traditional Chinese herbal remedy tian xian activates pregnane X receptor and induces CYP3A gene expression in hepatocytes. Drug Metab. Dispos. 36, 1538–1545 127 Scheer, N. et al. (2010) In vivo responses of the human and murine pregnane X receptor to dexamethasone in mice. Drug Metab. Dispos. 38, 1046–1053 128 Igarashi, K. et al. (2012) Development of humanized steroid and xenobiotic receptor mouse by homologous knock-in of the human steroid and xenobiotic receptor ligand binding domain sequence. J. Toxicol. Sci. 37, 373–380 129 Cheung, C. et al. (2005) Differential metabolism of 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine (PhIP) in mice humanized for CYP1A1 and CYP1A2. Chem. Res. Toxicol. 18, 1471–1478 130 Jiang, Z. et al. (2005) Toward the evaluation of function in genetic variability: characterizing human SNP frequencies and establishing BAC-transgenic mice carrying the human CYP1A1_CYP1A2 locus. Hum. Mutat. 25, 196–206 131 Zhang, Q.Y. et al. (2005) Generation and characterization of a transgenic mouse model with hepatic expression of human CYP2A6. Biochem. Biophys. Res. Commun. 338, 318–324 132 Wei, Y. et al. (2012) Generation and characterization of a CYP2A13/2B6/2F1transgenic mouse model. Drug Metab. Dispos. 40, 1144–1150 133 Lofgren, S. et al. (2008) Generation of mice transgenic for human CYP2C18 and CYP2C19: characterization of the sexually dimorphic gene and enzyme expression. Drug Metab. Dispos. 36, 955–962 134 Corchero, J. et al. (2001) The CYP2D6 humanized mouse: effect of the human CYP2D6 transgene and HNF4alpha on the disposition of debrisoquine in the mouse. Mol. Pharmacol. 60, 1260–1267

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135 Morgan, K. et al. (2002) Production of a cytochrome P450 2E1 transgenic mouse and initial evaluation of alcoholic liver damage. Hepatology 36, 122–134 136 Yu, A.M. et al. (2005) Potential role for human cytochrome P450 3A4 in estradiol homeostasis. Endocrinology 146, 2911–2919 137 van Herwaarden, A.E. et al. (2005) Midazolam and cyclosporin a metabolism in transgenic mice with liver-specific expression of human CYP3A4. Drug Metab. Dispos. 33, 892–895 138 Cheung, C. et al. (2006) Growth hormone determines sexual dimorphism of hepatic cytochrome P450 3A4 expression in transgenic mice. J. Pharmacol. Exp. Ther. 316, 1328–1334 139 Li, Y. et al. (1996) Establishment of transgenic mice carrying human fetus-specific CYP3A7. Arch. Biochem. Biophys. 329, 235–240 140 Leff, M.A. et al. (1999) Prostate-specific human N-acetyltransferase 2 (NAT2) expression in the mouse. J. Pharmacol. Exp. Ther. 290, 182–187 141 Chen, S. et al. (2005) Tissue-specific, inducible, and hormonal control of the human UDP-glucuronosyltransferase-1 (UGT1) locus. J. Biol. Chem. 280, 37547– 37557 142 Fujiwara, R. et al. (2010) Developmental hyperbilirubinemia and CNS toxicity in mice humanized with the UDP glucuronosyltransferase 1 (UGT1) locus. Proc. Natl. Acad. Sci. U. S. A. 107, 5024–5029 143 Yueh, M.F. et al. (2011) Inhibition of human UGT2B7 gene expression in transgenic mice by the constitutive androstane receptor. Mol. Pharmacol. 79, 1053–1060

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144 Scheer, N. et al. (2012) Generation and characterization of a novel multidrug resistance protein 2 humanized mouse line. Drug Metab. Dispos. 40, 2212–2218 145 van de Steeg, E. et al. (2012) Complete OATP1B1 and OATP1B3 deficiency causes human Rotor syndrome by interrupting conjugated bilirubin reuptake into the liver. J. Clin. Invest. 122, 519–528 146 Tateno, C. et al. (2004) Near completely humanized liver in mice shows humantype metabolic responses to drugs. Am. J. Pathol. 165, 901–912 147 Kakuni, M. et al. (2014) Chimeric mice with humanized livers: a unique tool for in vivo and in vitro enzyme induction studies. Int. J. Mol. Sci. 15, 58–74 148 Katoh, M. et al. (2005) In vivo induction of human cytochrome P450 enzymes expressed in chimeric mice with humanized liver. Drug Metab. Dispos. 33, 754–763 149 Yamazaki, H. et al. (2013) In vivo drug interactions of the teratogen thalidomide with midazolam: heterotropic cooperativity of human cytochrome P450 in humanized TK-NOG mice. Chem. Res. Toxicol. 26, 486–489 150 Foster, J.R. et al. (2012) Differential effect of troglitazone on the human bile acid transporters, MRP2 and BSEP, in the PXB hepatic chimeric mouse. Toxicol. Pathol. 40, 1106–1116 151 Kakuni, M. et al. (2012) Chimeric mice with a humanized liver as an animal model of troglitazone-induced liver injury. Toxicol. Lett. 214, 9–18 152 Sanoh, S. and Ohta, S. (2014) Chimeric mice transplanted with human hepatocytes as a model for prediction of human drug metabolism and pharmacokinetics. Biopharm. Drug Dispos. 35, 71–86

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