Humanized liver models

Although the assessments of the overall capacity of these models to replicate human hepatotoxicity are stiU in progress, these emerging examples suggest that these models could be very valuable, particularly in cases of potential cholestatic liver injury due to transporter inhibition, an application where preclinical studies have shown limited predictivity of DILI (Morgan et al., 2013). 

Johnson, M. D., Cool, M. H., Salvatore, R, Asico, L. D., Jose, P. A., Taylor, S. I., and Westphal, H. (1996). Early neonatal death in mice homozygous for a null aUele of the insulin receptor gene. Nat Genet 12,106-109. Akinleye, A., Furqan, M., Mukhi, N., Ravella, P, and Liu, D. (2013). MEK and the inhibitors from bench to bedside. J Hematol Oncol 6, 27. 

Blasco, R. B., Francoz, S., Santamaria, D., Canamero, M., Dubus, P, Charron, J., Baccarini, M., and Barbacid, M. (2011). c-Raf, but not B-Raf, is essential for development of K-Ras oncogene-driven non-small cell lung carcinoma. Cancer Cell 19, 652-663. 

Bogaards, J. J., Bertrand, M., Jackson, R, Oudshoom, M. J., Weaver, R. J., van Bladeren, P. J., and Walther, B. (2000). Determining the best animal model for human cytochrome P450 activities a comparison of mouse, rat, rabbit, dog, micropig, monkey and man. Xenobiotica 30, 1131-1152. 

Bronson, S. K. and Smithies, O. (1994). Altering mice by homologous recombination using embryonic stem cells. J Biol Chem 269, 27155-27158. 

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Humanized liver models Degree and consistency of human hepatocyte colonization Animal health and histopathology Severely immunocompromised Unable to recapitulate extrahepatic metabolism, potentially leading to a hybrid metabolic profile and disposition Compensatory effects on extrahepatic tissues Detection of human-predominant toxicities (i.e., fialuridine, bosentan) Modeling human induction liabilities (i.e., rifampicin) Prediction of human PK where in vitro models are limited... 

Ohtsuki, S., Kawakami, H., Inoue, T., Nakamura, K., Tateno, C., Katsukura, Y, Obuchi, W, Uchida, Y, Kamiie, J., Horie, T., and Terasaki, T. (2014). Validation of uPA/SCID mouse with humanized liver as a human liver model protein quantification of transporters, cytochromes P450, and UDP-glucuronosyltransferases by LC-MS/MS. Drug Metab Dispos 42, 1039-1043. 

In the case of dmg interactions involving metabolic inhibition, little increase in the substrate concentration is expected when the inhibition constant (K ) determined in in vitro studies using human liver samples is larger than the inhibitor concentration in vivo. Various approaches have been adopted using mathematical models in attempts to quantitatively predict in vivo dmg interactions from in vitro data [5]. 

IkB kinase-p is a key regulatory enzyme in the NF-kB pathway, and inhibition of this enzyme has the potential for yielding treatments for inflammatory and autoimmune diseases. Morwick et al. [53] report on the optimization of a pM IKKp inhibitor with low aqueous solubility, moderate human liver microsome stability, and inhibition of several CYPs (3A4, 2C9, 1A2) with pM potencies. Modulation of the thiophene core (other thiophene isomer, pyrimidine and oxazole) produces compounds of similar potency to the hit. Fusing the 5-phenyl moiety to the thiophene to form a thieno[2,3-b]pyridine core increases aqueous solubility of the series as well as reduces the CYP liability. While the optimized compound still shows pM IKK(S potency, the aqueous solubility, HLM stability and CYP profiles are much improved. A pharmacophore model was generated that enabled scaffold hopping to yield this new chemotype (Scheme 7). 

Researchers focused on the metabolically competent human hepatoma cell line HepG2 as a model of human liver. HepG2 cells are a well-known hepatoma cell line that retains many of the morphological characteristics of liver parenchymal cells. This model is often used as a useful tool for HRA/ERA-oriented chemical risk assessment due to the expression of antioxidant and xenobiotic metabolizing enzymes (in particular phase I and phase II enzymes responsible for the bioactivation/detoxification of various xenobiotics) that can be induced or inhibited by dietary and non-dietary agents [28-30]. 

Strobl GR, von Kruedener S, Stockigt J, Guengerich FP, Wolff T. 1993. Development of a pharmacophore for inhibition of human liver cytochrome P-450 2D6 molecular modeling and inhibition studies. J Med Chem 36 1136-1145. 

Since a high yield isolation procedure of rat hepatocytes was described in 1969 [6], hepatocytes have become the model of choice for drug transport studies in the liver in vitro [7]. With this procedure, isolated hepatocytes from many species have been prepared, including hepatocytes from rat, mouse, chicken, dog, fish, hamster, pig, cow, sheep and monkey liver (for an extensive review see reference [8]). Before 1976, only relatively small numbers of human hepatocytes could be isolated, due to the use of non-perfusion techniques [9]. Bojar et al. [10] were the first to use a perfusion technique on human livers which greatly enhanced the yield of hepatocytes. In principle the procedure that is now commonly used, is based on the one described by Seglen [6] for rat hepatocytes. Either a biopsy wedge with intact capsula on... 

Sakiyama, Y., Yuki, H., Moriya, T., Hattori, K., Suzuki, M., Shimada, K., Honma, T. Predicting human liver microsomal stability with machine learning techniques. J. Mol. Graph. Model. 2008,... 

Mancy, A., Broto, P., Dijols, S., Dansette, P. M., and Mansuy, D. (1995) The substrate binding site of human liver cytochrome P450 2C9 an approach using designed tienilic acid derivatives and molecular modeling. Biochemistry 34, 10,365-10,375. 

Wolff, T., Distlerath, L. M., Worthington, M. T., et al. (1985) Substrate specificity of human liver cytochrome P-450 debrisoquine 4-hydroxylase probed using immunochemical inhibition and chemical modeling. Cancer Res. 45, 2116-2122. 

Comparative Toxicokinetics. Metabolic pathways and mechanisms of hepatotoxicity of carbon tetrachloride have been the subject of many studies in intact animals and in vitro, and are therefore better understood than for many other chemicals. However, there are apparently no data on metabolism of carbon tetrachloride in humans. It would be valuable to conduct in vitro experiments with human liver samples and hepatocytes to determine whether metabolic pathways and toxic metabolites are similar to those found in animals. It would also be beneficial to identify an animal model in which MFO systems develop in uteroas they do in the human fetus. 

Quercetin has been found to inhibit P-gp-mediated efflux of ritonavir in Caco-2 cells (47), to reduce the oxidation of acetaminophen in rat liver microsomes and HepG2 cells (48), and to inhibit the metabolism of midazolam and quinidine in human liver microsomes (49). It did not have an effect on CYP3A4-mediated metabolism and P-gp-mediated transport of saquinavir (41). Rutin was demonstrated to moderately increase the uptake of idar-ubicin in an isolated perfused rat lung model, and also the outflow recovery of the major metabolite idarubicinol, possibly by affecting P-gp (45). Nobe-litin and tangeretin were shown to inhibit OATP-B-mediated uptake of estrone-3-sulfate into human embryonic kidney cells (23). 

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