Hepregen

Predictive Toxicology

Roughly 50 to 60% of drugs that progress through clinical trials ultimately fail in humans. Approximately 30% of that attrition is the result of liver injury. One pharmaceutical company estimated that clinical failures based on liver toxicity alone cost them more than $2 billion in the last decade, clearly demonstrating the need for more predictive pre-clinical models.

In Vivo Models
Various animal models exist to evaluate the toxicological effects of compounds. Rodent models are the gold standard, despite their low sensitivities in identifying clinical liabilities. Examples of hepatotoxicity resulting from metabolic pathway differences between humans and rodents are well documented. Furthermore, in vivo rodent studies put a significant strain on chemistry often requiring several grams of material to complete a study. Compound scale-up can take several months to complete, tie up significant chemistry resources and limit the number of molecules that can be assessed resulting lowering probability of identifying the optimum candidate molecule.

Conventional In Vitro Models
Isolated primary human hepatocytes in adherent or sandwich cultures are widely considered to be the most suitable model for in vitro testing. However, sandwich cultures are a declining model and loose functionality after only a few days. Their short-term functionality (3-5 days) does not allow for chronic dosing making it difficult to distinguish between drug-induced toxicity and the natural decline of hepatocyte health. Compounds must often be dosed at higher than efficacious concentrations to see toxicity in the short window of functionality. This can lead to results that are not clinically relevant and to problems of compound solubility. Even with highly sensitive high content imaging readouts, current sensitivities of sandwich cultures are estimated to be approximately 50 to 60%.

Limitations of short-term functional models:

• No chronic dosing
• Dosing at higher than efficacious concentrations
• Poor clinical relevance
• Low sensitivities

HepatoPac
HepatoPac’s long term viability and extended functionality make it a much more predictive model of clinical toxicity. Two different studies to detect the potential of drug induced liver injury using 35 and 41 clinically toxic compounds produced HepatoPac sensitivities of 69% and 81% respectively. Hepregen’s predictive toxicology assays use a variety of bulk assay endpoints to assess hepatotoxicity including:

• ATP Content
• Albumin secretion
• Urea synthesis
• ALT Release
• Cellular Glutathione Content

Hepregen scientists work closely with our customers to understand their needs and design an appropriate study plan that will address the scientific question at hand. Contact us for additional information, to schedule an appointment or learn more about HepatoPac.

Featured Resources

Poster: Assessment of a Micropatterned Hepatocyte Co-Culture System to Detect Compounds that Cause Drug Induced Liver Injury (DILI) in Humans
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Poster: Micropatterned Primary Hepatocyte Co-Cultures for Drug Metabolism and Toxicity Studies
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Mechanistic Toxicology

HepatoPac™ is an ideal tool for investigating pre-clinical and clinical mechanistic toxicity. Its phenotype remains stable over a several week period, allowing researchers to ask more complex questions of hepatotoxicity. HepatoPac can be used in a variety of applications to identify mechanistic toxicity resulting from:

• Reactive metabolites
• Enzyme inhibition
• Cholestasis
• GSH depletion
• Mitochondrial dysfunction
• Oxidative stress

Case Study: Glutathione Depletion
HepatoPac has been used to identify toxicity resulting from the depletion of glutathione (GSH), an antioxidant that prevents damage to important cellular components caused by reactive oxygen species. The addition of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, depletes cellular GSH levels as seen in the top graph on the right. N-acetyl-p-benzoquinone imine (NAPQI), the reactive metabolite of acetaminophen (APAP), becomes toxic when GSH is depleted. Co-administration of BSO and APAP potentiates hepatotoxicity caused by APAP as seen in the lower graph on the right.

The successful use of BSO to deplete GSH stores in HepatoPac provides a good model for studying toxicity of compounds that generate reactive metabolites.

Featured Resources

Poster: HepatoPac™ Allows Determination of Toxicity under Chronic Dosing and at Relevant Concentrations
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Poster: Global Gene Expression Changes Induced In Primary Human Hepatocytes by Thiazolidinediones Upon Repeat Dosing of HepatoPac™ Cultures
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HepatoPac-Kupffer Cell Model

The exact mechanisms of drug-induced liver injury (DILI) remain largely unknown. While, in most cases DILI is initiated by the bioactivation of drugs to chemically reactive metabolites, it is suspected that adaptive immune responses may contribute to idiosyncratic DILI. Activation of immune cells (e.g., Kupffer cells) can contribute to the progression of livery injury by producing pro-inflammatory mediators and secreting chemokines that further recruit inflammatory cells to the liver. The involvement of inflammation and its underlying mechanisms are becoming increasingly important in the early detection of drug-induced hepatotoxicity.

At present, there are few in vitro liver models that can initiate hepatic inflammatory responses for more than a few days. The HepatoPac ™ liver model has been supplemented with primary Kupffer cells, resident macrophages in the liver that line the walls of the sinusoids and have direct cell contact with hepatocytes. This enhanced platform has the unique ability to remain viable for more than 10 days while secreting inflammatory mediators in response to certain xenobiotics, which may lead to hepatotoxicity.

The Need for Better Models
Liver inflammation results from the early release of pro-inflammatory factors, reactive oxygen species and chemokines by Kupffer cells, which in turn recruit monocytes and neutrophils to further carry out the immune response in the liver. Inflammation stimulates the production of acute phase proteins and down-regulates enzymes involved in phase I and II metabolism, such as Cytochrome P450 in the liver. Inflammation-induced repression in P450 activity can lead to altered drug metabolism, changing the circulating plasma concentration of therapeutics in the body with the potential for increased and potentially fatal side-effects. Sandwich cultures of primary human hepatocytes, currently considered the most suitable for in vitro testing, are limited by a short culture life and low and inherently variable in their phenotypic stability. They lack both the temporal viability needed to carry out and characterize an inflammatory response as well as the cell types necessary to do so. There is a clear, unmet need for a model of primary human liver tissue that mimics both basal and inflamed conditions and is capable of predicting clinical outcomes.

HepatoPac Solution
HepatoPac co-cultures supplemented with Kupffer cells:

• Enable evaluation of inflammation-based toxicity
• Model both normal and inflamed physiological liver states
• Retain viability for more than 10 days in vitro
• May be used to monitor drug-drug interactions such as therapeutic protein-small molecule interactions

Featured Resources

Application Note: A Micropatterned Hepatocyte-Kupffer Cell Co-culture System to Study Inflammation in Drug Discovery
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Poster: A Micropatterned Culture with Human Hepatocytes and Kupffer Macrophages for Studying Inflammation-Drug Interactions
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