Sub-Lethal Hepatotoxicity
High-Content Toxicology

Adverse Drug Reactions Due to Hepatotoxicity

Adverse drug reactions (ADRs) and idiosyncratic drug reactions (IDRs), are a major cause of attrition in preclinical and clinical drug development¹. Post-marketing, they are the cause of black-box warnings, lawsuits, and withdrawal of approved drugs from the market.² Consequently, earlier and more cost-effective identification of potential mechanisms that cause adverse drug reactions have substantial potential for improving outcomes for both drug discovery and for patients.

ADRs can be caused by a variety of toxicities with a number of identified mechanisms. Major organ systems affected are liver, heart, and kidneys. The major toxic effect is hepatotoxicity, also known as drug-induced liver injury (DILI). Although biochemical mechanisms of drug-induced toxicities are complex, by combining these screening methods into a panel of assays, a risk assessment profile/decision-making guide can be obtained for each drug candidate. On the cellular level, it has been demonstrated that drug-induced liver injury may be caused by mitochondrial toxicity³ and formation of reactive metabolites.^{4, 5}

Contact us for help assessing the hepatotoxicity potential of your drug candidates.

Mechanistic Approach for Assessing the Hepatotoxicity Potential of Drug Candidates

In 2006 Pfizer developed a mechanistic approach that identifies minimum toxic concentration, organelle damage, and other markers of sub-lethal damage of early drug candidates.^{6, 7} This approach utilizes High Content Screening (HCS), aka High Content Analysis (HCA), to multiplex markers in mammalian cell lines, such as HepG2 cells, a well-characterized human hepatoma cell line.^{7, 8}

This new approach is a valuable addition to the existing cytotoxicity toolset. The advantages of High Content Screening include:

1. Detection of sub-lethal damage that can occur one or two logs lower in concentration than that identified with an IC50 curve of a classic cytoxicity assay.⁶
2. Detection of chronic damage to cellular organelles. Damage to liver frequently is not triggered by short-duration treatment.
3. Detection of cellular damage to cells of human origin. Liver metabolism differs significantly among species. In vivo toxicity studies predict only 50% to 60% of human hepatotoxicity.⁹

Apredica offers a multiplexed assay for sub-lethal hepatotoxicity, based on the literature^{5, 6, 7, 8} in this field and pioneered by Pfizer.^{6, 7}

Sub-Lethal Hepatotoxicity Readouts

• Gross mechanism of cell death (apoptosis or necrosis).
• Minimum toxic concentration.
• Mitochondrial potential changes as a leading indicator (or not).
• GI50 (cell growth inhibition).
• Ask us about oxidative stress, reactive oxygen species, or other cytotoxic mechanisms of interest to you.

Advantages of Apredica's Multiplexed Analysis

The markers for cell damage identified in the literature could be evaluated one at a time in different assays, but the power of multi-wavelength, automated High Content Screening allows cytotoxic markers to be analyzed simultaneously in individual cells, yielding several benefits over separate assays:

• Improved sensitivity and specificity in detection of human liver damage.⁷
• Better baseline comparison between markers.
• Lower cost per data point.

Hepatocyte Cell Models

Apredica's hepatotox assay is usually performed using the human liver carcinoma HepG2 cell line. Other cell lines are available, such as HepaRG, a human hepatocellular carcinoma-derived cell line with full metabolic activity, or rat hepatocytes for prediction of rat liver injury, or human hepatocytes. Please inquire to discuss the cell model appropriate for the stage of your project.

HepG2 is commonly used as a model of human hepatocytes. Although it is not as metabolically active as primary human hepatocytes, it is easily cultured and is very reproducible, allowing early identification of mechanisms of mammalian toxicity. Mechanistic liver toxicity assays based upon this model have demonstrated good concordance with human hepatotoxicity, as has been demonstrated in the literature.^{6, 7, 8}

Contact us for help assessing the hepatotoxicity potential of your drug candidates.

References

1. Uetrecht, J. (2008). Idiosyncratic drug reactions: past, present, and future. Chemical Research in Toxicology 21, 84-92.

2. Kaitin, KI. Obstacles and opportunities in new drug development. Clin Pharmacol Ther. 2008 Feb;83(2):210-2.

3. Dykens, JA & Will, Y. (2007). The significance of mitochondrial testing in drug development. Drug Discovery Today 12,777-785.

4. Williams, DP & Park, BK. (2003) Idiosyncratic toxicity: the role of toxicophores and bioactivation. Drug Discovery Today 8, 1044-1050.

5. Caldwell, GW & Yan, Z. (2006) Screening for reactive intermediates and toxicity assessment in drug discovery. Curr Opin Drug Discov Devel. 9:47-60.

6. Xu, JJ; Diaz, D; O'Brien, PJ. (2004) Applications of cytotoxicity assays and pre-lethal mechanistic assays for assessment of human hepatotoxicity potential. Chem Biol Interact. 150:115-28.

7. O'Brien, PJ; Irwin, W; Diaz, D; Howard-Cofield, H; Krejsa, CM; Slaughter, MR; Gao, B; Kaludercic, N; Angeline, N; Bernardi, P; Brain, P; Hougham, C. (2006). High concordance of drug-induced human hepatotoxicity with in vitro cytotoxicity measured in a novel cell-based model using high content screening. Archives of Toxicology 80,580-604.

8. Abraham VC, Towne DL, Waring JF, Warrior U, Burns DJ. Application of a high-content multiparameter cytotoxicity assay to prioritize compounds based on toxicity potential in humans. J Biomol Screening 13:527-537.

9. Olson, H; Betton, G; Robinson, D; Thomas, K; Monro, A; Kolaja, G; Lilly, P; Sanders, J; Sipes, G; Bracken, W; Dorato, M; Van Deun, K; Smith, Berger PB; Heller, A. (2000). Concordance of the toxicity of pharmaceuticals in humans and in animals. Regulatory Toxicology and Pharmacology 32, 56-67.