Introduction

By: Robert Gates, Biofiles Volume 3 Article 7

        Robert Gates

Robert Gates
Product Manager
Email: robert.gates@sial.com

Over the past three decades non-cellular and cellular systems have supplanted whole animal studies for the preliminary screening of drug candidates and well as for initial drug metabolism and toxicology studies. In 1991 inadequate metabolic and pharmacokinetic parameters were a major reason for the failure of new drug candidates submitted for FDA approval. In vitro testing methods allowed xenobiotics to be tested in human cells or with human enzymes with more clearly defined study parameters and end points. Toxic or metabolically inert compounds could be identified earlier in the drug development process, and these methods coupled with animal studies and Phase I clinical trials provided more complete information on drug metabolism, availability, and pharmacokinetics in humans. The failure rate for deficiencies in these metabolic parameters decreased to 10% by 2000. (1)

Orally delivered drugs are primarily metabolized in the liver. Phase I reactions are carried out by a series of cytochrome P450-containing enzymes (CYP) that carry out primarily oxidation, reduction and hydrolysis reactions. The CYP isoforms most involved in drug metabolism are the CYP1A family, CYP2A6, CYP2B6, the CYP2C family, CYP2D6, CYP2E1, and the CYP3A family. CYP3A4 is the most abundant isoform in human liver and is involved in the metabolism of approximately half of all currently marketed drugs. (1,2) Phase II reactions are conjugation reactions with glucuronide, glutathione, and sulfonates. Conjugation tends to make the molecules more water soluble. Phase III is excretion by the kidneys (hydrophilic compounds and conjugates) or via the bile into the intestine (lipophilic molecules). (1,3)

Model System for Drug Metabolism Studies

Several cellular models have been used to study drug metabolism in vitro. Human hepatocyte primary cultures are the closest model to the in vivo situation. The hepatocyte membrane retains transporters for the uptake and excretion of xenobiotics and intact intracellular Phase I and Phase II metabolic pathways. (3,4) This model has been of limited use due to the low availability and high variability of human liver samples; although primarily hepatocytes can be cryopreserved. Cultured hepatocytes are also phenotypically unstable; by 48 hrs enzyme activity has declined by 60–80%, and mRNA declines to 15% of original levels by 4 hrs. (3)

Hepatoma cell lines (HepG2 and Mz-Hep-1) have the advantage of being highly available. They are easy to culture, have an unlimited life span and stable phenotype. Unfortunately these cells express drug metabolizing enzymes only at very low levels or not at all. Hepatomas transfected with a single CYP are used to screen for enzyme compounds that inhibit CYP activity. These cells can also be used to study hepatotoxicity in vitro. (3,4)

Recently methods have been reported for producing hepatocyte-like cells from embryonic stem cells. Embryonic stem cells are pluripotent cells that can be maintained in a undifferentiated state. In nonadherent culture they aggregate to form embryoid bodies (EBs) that differentiate into primordial ectoderm, mesoderm and endoderm cells that can further differentiate into progenitors of organ-specific cell types. Methods for culturing mouse embryonic stem cells (mESC) are well established, and methods obtaining mouse hepatocyte-like cells have been described. (5-7) In their study, Tsutsui, et al., (7) showed that mESC-derived hepatocyte-like cells expressed several p450 isoforms and that the products of testosterone hydroxylation by these hepatocytes were identical to those produced by cultured fetal hepatocytes. However, several hydroxylation products characteristic of metabolism by adult hepatocytes were not produced by mESC-derived hepatocytes.

Standardized protocols for culturing human embryonic stem cells (hESCs) are not well established (5), but methods for the production of hepatocyte-like cells from hESCs have recently been described. (2,6,8-11) Both hepatoblasts and bile duct (cholangiocyte) progenitors are separated from the EBs derived from hESCs. The cholangiocyte progenitors are distinguished from hepatic progenitors by having high levels of cytokeratins 7 and 17. Unlike adult hepatic stem cells, hepatoblasts from hESC express α-fetoprotein (AFP) and do not express the adult stem cell markers NCAM and claudin 3. (2,6) In addition to AFP, hepatocyte-like cells expressed the markers Foxa2, α-1-antitrypsin, albumin, HNF4a, and GATA4. Hepatocyte functions were also observed, such as albumin secretion, glycogen accumulation, urea production, and the accumulation of dyes and low density lipoproteins. (2,6,8,9,11) By 21 days in culture, Phase I enzymes such as Cyp 1A2, Cyp 3A4, and Cyp 7A1 and phase II conjugating enzymes such as glutathione transferase were expressed or could be induced by xenobiotic challenge. (2,6,8,11)

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Model Systems for Metabolomics and Drug Toxicity Studies

hESCs and hESC-derived progenitor cells may also be useful in determining the effect of xenobiotics on cellular metabolism. The metabolome comprises all the small molecule substrates, intermediates and products of cellular metabolism. Approximately 7500 compounds currently make up the human metabolome including about 2500 metabolites, 3500 food components, and 1200 drug products. (12) These metabolites can be separated chromatographically from tissue, urine, or serum samples and identified by nuclear magnetic resonance (NMR) or mass spectroscopy (MS). The Consortium on Metabonomic Toxicology has proposed predicting the liver or kidney toxicity of novel compounds by multivariate analysis of the urine metabolome, assuming that compounds with similar adverse effects would produce similar abnormalities in the metabolic profile of urine. (13)

Xenobiotics are sometimes teratogenic and knowing the effects of these compounds on cellular metabolism of ESCs or various organ progenitor cells may indicate the pathways involved in drug action and/or toxicity in utero. (14) Human and mouse ESC-derived neurons or cardiomyocytes have also been used to screen for drug toxicities. (5,15-17)

Changes in the metabolome of hESCs or differentiated cells derived from hESC may provide markers of potential drug toxicity. Recently, Cezar, et al. (14) studied the effect of valproate, an anticonvulsant compound with higher teratogenic risk, on the secreted metabolome of three hESC cell lines. Compounds were separated chromatographically and identified by electrospray ionization time-of-flight MS. At moderate valproate concentrations the tryptophan breakdown product, kynurenine, and several glutamate pathway metabolites were elevated. At higher concentrations, the GABA pathway metabolites were also elevated. This study showed that metabolomics methodology could be applied to ESCs in culture.

In the future hESCs and cells derived from hESCs may provide suitable model systems for studying drug metabolism and toxicity in vitro.


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References

  1. Baranczewski, P., et al. Introduction to in vitro estimation of metabolic stability and drug interactions of new chemical entities in drug discovery and development. Pharmacol. Rep. 58, 453-472 (2006).
  2. Agarwal, S., et al., Efficient differentiation of functional hepatocytes from human embryonic stem cells., Stem Cells, 16, 1117-1127 (2008).
  3. Castell, J.V., et al., Hepatocyte cell lines: their use, scope, and limitations in drug metabolism studies. Expert Opin. Drug Metab. Toxicol., 2, 183-212 (2006).
  4. Donato, M.T., et al., Cell lines: a tool for in vitro drug metabolism studies. Curr. Drug Metab., 9, 1-11 (2008).
  5. Pouton, C.W., and Haynes, J.M., Embryonic stem cells as a source of models for drug discovery. Nat. Rev. Drug Disc. 6, 605-616 (2007).
  6. Shiraki, N., et al., Differentiation of mouse and human embryonic stem cells into hepatic lineages. Genes Cells, e-pub, May 20, 2008.
  7. Tsutsui, M., et al., Characterization of cytochrome p450 expression in murine embryonic stem cell-derived hepatic tissue system. Drug Metab. Disp. 34, 696-701 (2006).
  8. Baharvand, H., et al., Differentiation of human embryonic stem cells into functional hepatocyte-like cells in a serum-free adherent culture condition. Differentiation, 76, 465-477 (2008).
  9. Chiao, E., et al., Isolation and transcriptional profiling of purified hepatic cells derived from human embryonic stem cells. Stem Cells, e-pub, June 5, 2008.
  10. Lavon, N., and Benvenisty, N., Directed differentiation of human embryonic stem cells into hepatic cells, in Human Embryonic Stem Cells: the practical handbook. (Sullivan, S., et al., eds.) pp 186-194. Wiley: Chichester, 2007.
  11. Soderdahl, E.M, et al., Expression of drug metabolizing enzymes in hepatocyte-like cells derived from human embryonic stem cells. Biochem. Pharmacol., 74, 496-503 (2007).
  12. Fiehn, O., Combining genomics, metabolome analysis, and biochemical modeling to understand metabolic networks. Comp. Funct. Genomics, 2, 155-168 (2001).
  13. Ebbels, T.M.D., et al., Prediction and classification of drug toxicity using probabilistic modeling of temporal metabolic data: the Consortium on Metabonomic Toxicology screening approach. J. Proteome Res. 6, 4407-4422 (2007).
  14. Cezar, G.G., et al., Identification of small molecules from human embryonic stem cells using metabolomics. Stem Cells Dev. 16, 869-882 (2007).
  15. Cezar, G.G., Can human embryonic stem cells contribute to the discovery of safer and more effective drugs? Curr. Opin. Chem. Biol. 11, 405-409 (2007).
  16. Chaudhary, K.W., et al., Embryonic stem cells in predictive cardiotoxicity: laser capture microscopy enables assay development. Tox. Sci., 90, 149-158 (2006).
  17. Davila, J.C., et al., Use and application of stem cells in toxicology. Tox. Sci., 79, 214- 223 (2004).

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