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TECH NOTE

A Novel Cell-Based Model for Toxicity Assessment Using Cellartis Enhanced hiPS-HEP Cells

Overview

Late-stage withdrawal of candidate drugs due to unforeseen hepatotoxicity is costly, both in terms of time and money. The current screening methods for toxicity and safety assessment do not adequately identify hepatotoxic compounds. The HepG2 hepatoma cell line is frequently used for toxicity testing, however the lack of functional drug metabolizing enzyme expression in this cell line limits its usefulness to assessing only parental drug toxicity. Toxicity testing using primary human hepatocytes is the gold standard today, but given the limitations of this model (e.g., high inter-donor variation, limited access to material from the same donor, and notable decrease in drug metabolic function in culture), there is a need for improved human cell-based models for safety and toxicity assessment of candidate drugs.

Cellartis Enhanced hiPS-HEP cells provide an unlimited source of human hepatocytes. These cells are derived from induced pluripotent stem (iPS) cells and express functional drug-metabolizing enzymes in a similar range to primary human hepatocytes. Here, we demonstrate the sensitivity and specificity of Cellartis Enhanced hiPS-HEP cells to well-known toxic drugs.

Results

Cellartis Enhanced hiPS-HEP cells were exposed to the nontoxic/toxic analog pairs pioglitazone/troglitazone and levofloxacin/trovafloxacin. Cell viability was assessed using the WST-1 cell proliferation assay (Figure 1). Cellartis Enhanced hiPS-HEP cells are sensitive to the toxic compounds troglitazone and trovafloxacin; no toxicity was detected for the nontoxic structural analogs pioglitazone and levofloxacin.

Cellartis Enhanced hiPS-HEP cells can distinguish between toxic and nontoxic analogs

Figure 1. Cellartis Enhanced hiPS-HEP cells can distinguish between toxic and nontoxic analogs. Troglitazone and trovafloxacin are known toxic compounds that have nontoxic analogs, pioglitazone and levofloxacin, respectively. Dose-response curves were plotted after exposure to these compounds (n=3; mean ± SEM). The hiPS-HEP cells display the expected sensitivity and specificity to these drugs.

The toxicity of compounds known to induce clinical drug induced liver injury (DILI) (benzbromarone, amiodarone, tolcapone, flutamide, and imipramine) was tested using Cellartis Enhanced hiPS-HEP cells. Previous data using primary human hepatocytes, the gold standard for hepatotoxicity testing, identified in vitro toxicity for only two of these compounds (benzbromarone and amiodarone, Xu et al., 2008). In contrast, toxicity was detected for all of these compounds when tested using Cellartis Enhanced hiPS-HEP cells (Figure 2 and Table I).

Toxicity to known DILI-causing compounds is identified using in vitro toxicity testing with Cellartis Enhanced hiPS-HEP cells

Figure 2. Toxicity to known DILI-causing compounds is identified using in vitro toxicity testing with Cellartis Enhanced hiPS-HEP cells. hiPS-HEP cells were exposed to each compound for 24 hr, cell viability was assessed by WST-1 assay, and dose-response curves were plotted (n=3; mean ± SEM). Cellartis Enhanced hiPS-HEP cells were sensitive to all of the tested compounds, while primary human hepatocytes were unable to detect toxic effects of imipramine and flutamide (see Table I, below).

Conclusions

As we have reported, Cellartis Enhanced hiPS-HEP cells stably express drug metabolizing enzymes in the same range as primary human hepatocytes. hiPS-HEP cells could distinguish between toxic and nontoxic structural analogs, troglitazone from pioglitazone and trovafloxacin from levofloxacin. When exposed to compounds known to cause clinical DILI, Cellartis Enhanced hiPS-HEP cells respond with the expected sensitivity and specificity. This is in contrast to primary human hepatocytes, the long-considered gold standard in toxicity testing, which were insensitive to two of the test compounds, imipramine and flutamide (Xu et al., 2008). Given that the data from Cellartis Enhanced hiPS-HEP cells correlates with clinical response, these cells provide a new cell-based model for in vitro hepatotoxicity testing.

Methods

Cell culture

Cryopreserved Cellartis Enhanced hiPS-HEP cells were thawed and plated in 96-well plates according to the recommended protocol. Cells were maintained for eleven days in culture, with a medium change every other day before the start of the toxicity assay.

Toxicity assessment

Cellartis Enhanced hiPS-HEP cells were exposed to hepatotoxic and non-hepatotoxic compounds (Table I); six concentrations were used, with a maximum of 500 µM. The cells were incubated with the compounds for 24 hr prior to cell viability analysis using the WST-1 assay. Dose-response curves and the TC50 (toxic concentration that kills 50% of the cells) were calculated and compared to published data (Xu et al., 2008).

Table I. Toxicity Data for Various Compounds

Compounds

Status Label (FDA)

Severity (FDA)

Clinical DILI (Xu et al., 2008)

PHH
(Xu et al., 2008)

hiPS-HEP

TC50 (µM)

Troglitazone

WD

NA

Positive

Positive

Positive

73

Pioglitazone

-

-

Negative

Negative

Negative

-

Trovafloxacin

WD

NA

Positive

Positive

Positive

118

Levofloxacin

-

-

Negative

Negative

Negative

-

Benzbromarone

WD

NA

Positive

Positive

Positive

90

Amiodarone

BW

8

Positive

Positive

Positive

48

Tolcapone

BW

8

Positive

NT

Positive

113

Flutamide

BW

8

Positive

Negative

Positive

-

Imipramine

AR

3

Positive

Negative

Positive

154

* AR: adverse reaction, BW: black box warning, DILI: drug induced liver injury, NA: not applicable, NT: not tested, PHH: primary human hepatocytes, TC50: toxic concentration that kills 50% of the cells, WD: withdrawn

Reference

Xu, J.J. et al. (2008) Cellular imaging predictions of clinical drug-induced liver injury. Toxicol Sci. 105:97–105.

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