Elsevier

Toxicology

Volume 246, Issue 1, 3 April 2008, Pages 9-17
Toxicology

Review
Chimeric mice with humanized liver

https://doi.org/10.1016/j.tox.2007.11.012Get rights and content

Abstract

Recently, chimeric mice with humanized liver were established by transplanting human hepatocytes into an urokinase-type plasminogen activator+/+/severe combined immunodeficient transgenic mouse line. The replacement with human hepatocytes is more than 80–90% and is higher than any other chimeric mouse reported previously. In drug development, the liver is one of the most important organs because it is mainly involved in the pharmacokinetics of drugs and is frequently damaged by many drugs due to the accumulation of drugs and/or metabolites. The pharmacokinetics could affect the efficacy and toxicity of a drug, and thus prediction of the human pharmacokinetics is important for developing new drugs without adverse reactions and toxicity. Extrapolation from experimental animals or in vitro studies to the human in vivo pharmacokinetics is still difficult. To date, human hepatocytes and liver microsomes are recognized as better tools and are frequently used to estimate the human pharmacokinetics. We thought that chimeric mice with humanized liver could become a new tool for estimating the human toxicity and pharmacokinetics. At first, metabolism, which plays an essential role in pharmacokinetics, was investigated in the chimeric mice. In the liver of the chimeric mice, human drug metabolizing enzymes were found to be expressed and to reflect the capacities and genetic polymorphism of the donor. In an in vivo study on metabolism, human specific metabolites could be detected in the serum of the chimeric mice indicating that the chimeric mice could be used as an in vivo model to address human metabolism. These results suggested that the chimeric mice could overcome the species differences in drug metabolism and be used to evaluate drug toxicity due to genetic polymorphism. The reasons for drug interaction are often enzyme induction and inhibition. By the treatment with a typical inducer of cytochrome P450 (P450), which is the central drug-metabolizing enzyme, P450s expressed in the liver of the chimeric mice were found to possess induction potencies. After the treatment with a specific inhibitor of human P450, the area under the curve of the P450 metabolite was significantly decreased in the chimeric mice but not in the control mice. Therefore, it was indicated that the chimeric mice could be useful for assessing drug interactions in vivo. Moreover, drug excretion was determined to be humanized because cefmetazole was mainly excreted in urine both in the chimeric mice and humans but in the feces in control uPA−/−/SCID mice. Drug transporters expressed in the liver of the chimeric mice were also humanized.

In this review, studies of the chimeric mice with humanized liver, particularly on metabolism and excretion, are summarized and the possibility of using the chimeric mice is proposed for the advanced prediction of human pharmacokinetics and toxicity.

Introduction

There is large interindividual variability in the efficacy and toxicity of drugs, which may be mainly caused by differences in the drug pharmacokinetics. The pharmacokinetics can be determined by ADME (absorption, distribution, metabolism, and excretion), especially drug metabolism. Drug metabolism consists of the phase I reactions (oxidation, reduction, and hydrolysis) and phase II reaction (conjugation), which occur predominantly in the liver. Therefore, the liver is the essential organ to determine the drug pharmacokinetics. One of the phase I enzymes, cytochrome P450 (P450, CYP), plays a central role in drug metabolism. P450 can metabolize various compounds including xenobiotic and endogenous compounds (Nelson et al., 1996). In addition, bioactivation leading to toxicity can sometimes be initiated by some P450s. Recently, the phase II reaction has been well studied since a parent drug and/or phase I metabolites are frequently excreted after conjugation. Furthermore, several drug-metabolizing enzymes have been shown to be polymorphic (Ingelman-Sundberg, 2002, Miners et al., 2002) indicating that genetic polymorphism can affect differences in the drug pharmacokinetics.

The mechanism of drug interactions can be often explained by the induction and inhibition of drug metabolizing enzymes. Serious drug interactions involving P450 have been reported (Dresser et al., 2000, Niemi et al., 2003). The QT prolongation caused by the inhibition of CYP3A4 by a coadministered drug resulted in the withdrawal of terfenadine and cisapride from the market. The prediction of adverse drug reactions is essential in drug development.

There are many reports regarding drug interactions and toxicity of various drugs, although the drugs were approved only after their safety was thought to have been confirmed in preclinical and clinical studies. In the preclinical stage, the pharmacokinetics of a drug candidate are investigated using human-derived sources or experimental animals. The results from experimental animals and in vitro studies sometimes wrongly predict the human pharmacokinetics and toxicity. Many researchers have made much effort to overcome such difficulties including those caused by species differences. Nowadays, human liver microsomes and human hepatocytes in primary culture are recognized as better tools and are frequently used during drug development. Human liver microsomes can be stored for a few years without the loss of enzyme activities but cannot be used to evaluate the induction potencies. Human hepatocytes express all the drug metabolizing enzymes, but a novel technique for culture is needed to avoid decreasing the enzyme activities. Such in vitro models have various advantages and limitations, as have been described previously (Gomez-Lechon et al., 2003, Rodrigues and Rushmore, 2002).

To develop an artificial human liver is one of the best approaches for predicting human pharmacokinetics and safety. An urokinase-type plasminogen activator (uPA)+/+/severe combined immunodeficient transgenic mouse line, in which the liver could be replaced by 80–90% with human hepatocytes, was established in Japan (Tateno et al., 2004). In this review, basic researches concerning drug metabolism and drug interactions are summarized for the application of chimeric mice in drug development and toxicology.

Section snippets

Generation of chimeric mice with humanized liver

In 2001, chimeric mice with partially humanized liver were described by Dandri et al. (2001) and Mercer et al. (2001). In the former report, the livers in uPA/recombinant activation gene-2 mice could be repopulated with approximately 15% human hepatocytes (Dandri et al., 2001). In the latter, a portion the liver in uPA+/+/SCID mice was replaced with human hepatocytes (Mercer et al., 2001). Since they were investigating on hepatitis virus, their chimeric mice with low replacement might be

Cytochrome P450

The most important drug metabolizing enzyme involving phase I reactions is P450. One of the major isoforms, CYP3A4, has been reported to be responsible for the metabolism of more than 50% of clinical drugs (Pelkonen et al., 1998). In chimeric mice, human CYP3A4 mRNA and human CYP3A4 protein could be detected in an hAlb concentration-dependent manner by real-time reverse-transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively, which detected human CYP3A4 but not

Induction of P450 in chimeric mice with humanized liver

Enzyme induction of a drug-metabolizing enzyme is a long-term consequence of chemical exposure and leads to an elevation of the enzyme activity (Lin and Lu, 2001). Drug interactions caused by the induction of P450, especially CYP3A4, are sometimes a serious problem because the induction may result in changes in the efficacy and toxicity of a drug (Pascussi et al., 2003, Niemi et al., 2003). Many CYP3A4 inducers are used as drugs in clinical practice and they can induce CYP3A4 at clinically used

Inhibition of P450 in chimeric mice with humanized liver

Enzyme inhibition is an acute decrease of metabolism by a co-administered drug or a time-dependent decrease in the amount of an enzyme by several factors (Pelkonen et al., 1998). Drug interactions are often caused by the inhibition of P450 activities (Dresser et al., 2000). The prediction of human pharmacokinetic parameters from in vitro technologies has progressed (Houston and Galetin, 2003) but quantitative extrapolations from in vitro to human in vivo and from experimental animals to humans

In vivo excretion in chimeric mice with humanized liver

A drug is mostly eliminated by biliary and urinary excretion. To elucidate the excretion of a drug as well as the metabolism is essential for understanding the pharmacokinetics and toxicity. Species differences in the excretory pathway may make the extrapolation from experimental animals to humans difficult. In the case of an antibiotic agent, cefmetazole was mainly excreted in urine in humans (Ko et al., 1989) but in feces in rats and mice in an unchanged form (Murakawa et al., 1980, Okumura

Conclusions

The chimeric mice have been shown to exhibit a humanized profile of drug metabolism, induction and inhibition of drug metabolizing enzymes, and excretion in in vivo studies. This chimeric mouse line should be a promising model for evaluating the in vivo pharmacokinetics in humans. In recent drug development, adverse pharmacokinetic and bioavailability have reduced as a cause of attrition although toxicology has increased (Kola and Landis, 2004). Drug-induced hepatotoxicity is one of the major

Acknowledgments

This work was supported by a Research on Advanced Medical Technology, Health, and Labor Sciences Research Grant from the Ministry of Health, Labor, and Welfare of Japan. We thank Mr. Brent Bell for reviewing the manuscript.

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