Mechanistic Toxicology

HepatoPac®, an in vitro micropatterened co-culture of primary hepatocytes and supportive stromal cells, is an ideal tool for investigating pre-clinical and clinical mechanistic toxicity. HepatoPac co-cultures remain highly functional for four weeks and are available for multiple animal species, allowing researchers to ask more complex questions of hepatotoxicity. HepatoPac is highly predictive of in vivo human toxicity and can be used in a variety of applications to identify acute and chronic mechanistic toxicity

HepatoPac hepatocyte island utilized in High Content Imaging (HCI) to determine method of toxicity

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  • Mitochondrial dysfunction - MTT, ATP
  • Reactive metabolites - GSH, CYP activity, metabolite formation
  • Cholestasis - Transporter activity, bile secretion
  • Steatosis - Lipid and phospholipid accumulation
  • Apoptosis - Caspases
  • Immune-mediated toxicity - Cytokine secretion, ATP, ALT, etc.
  • Oxidative Stress

Access the highly predictive power of HepatoPac using our Human HepatoPac Tox Kit or through Hepregen Contract Toxicity Services. HepatoPac co-cultures are available for a variety of animal species including rat, monkey and dog enabling you to choose a pre-clinical animal model best suited to predict human toxicity.

THE NEED:
Established in vitro models for detecting hepatotoxicity, such as mono- and sandwich hepatocyte cultures are viable for only 3-5 days, limiting their sensitivity, use in detection of chronic toxicity and in turn their value as a predictive tool. There is a need for an improved in vitro model of primary human liver tissue that is predictive of clinical outcomes and can be used with existing industrial automation for high-throughput screening to significantly reduce the number of late stage drug failures.

THE SOLUTION:
HepatoPac's ability to closely mimic the human liver's functionality as well as its extended in vitro viability make it well suited for use in both acute and chronic mechanistic toxicology studies. Hepatocytes in HepatoPac co-cultures retain their in vivo-like morphology, express liver genes, metabolize compounds using active Phase I/II drug metabolism enzymes, secrete diverse liver-specific products, and display functional bile canaliculi for up to 4 weeks in vitro. Additionally, HepatoPac co-cultures are available for a variety of species including human, monkey, rat and dog enabling researchers to identify the most relevant animal model for future pre-clinical studies.

HepatoPac Demonstrates Extended Physiologically Relevant Metabolic Activity
HepatoPac maintains high level viability and functionality over long culture times and exhibits the complete metabolic machinery required to replicate complex hepatic activity.

HepatoPac shows consistent CYP3A4 activity over time
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CYP3A4 activity (as assessed by Testosterone 6-beta-hydroxylation) was determined for human HepatoPac co-cultures derived from three different donors. CYP3A4 activity was maintained for over three weeks for all three human HepatoPac co-cultures.
HepatoPac shows reproducible ATP activity over time
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The long-term viability assessments (as assessed by ATP levels) were conducted for three human HepatoPac co-cultures derived from different donors. ATP content was stable for all three co-cultures over 28 days of culture.

 

Case Study: Reactive Metabolite Toxicity
Many types of drugs can be bioactivated leading to formation of reactive metabolites. This metabolic activation is often the initial event in many chemically-induced toxicities. In a study of compounds withdrawn from the US market due to hepatotoxicity, there was evidence of reactive metabolite formation in 5 out of 6 drugs that were withdrawn. Acetaminophen (APAP) is a commonly used antipyretic and analgesic compound that has hepatotoxic potential. It is primarily metabolized by glucuronidation and sulfation. However, its hepatotoxicity is mediated by its toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI), which is generated by liver cytochrome P450s (CYP2E1 and CYP1A2) and is detoxified by conjugation with hepatic glutathione (GSH). During an overdose with APAP, cellular GSH is depleted, and cellular functions are impaired by binding of NAPQI to macromolecules, which can lead to cell death. Using APAP as a model toxicant and mimicking cellular glutathione (GSH) depletion by addition of BSO to cultures, we have shown the utility of human HepatoPac for detecting reactive metabolite toxicities. Addition of 200 µ M L-buthionine (S,R)-sulfoximine (BSO), an inhibitor of GSH synthesis, to human HepatoPac co-cultures reduces GSH content by about 80% and potentiated acetaminophen-induced ATP depletion in these cultures.

Treatment of HepatoPac with APAP shows GSH depletion
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The addition of BSO at 200uM depletes GSH levels to below 20% of control.  In the absence of BSO, APAP reduces cellular GSH levels at 10 x Cmax.

HepatoPac treated with APAP shows GSH depletion
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Co-administration of APAP and BSO potentiates hepatotoxicity caused by APAP as measured by ATP.

Technical Resources

Type Title
Application Note HepatoPac®: A Bioengineered Micro-liver Platform for Predictive and Mechanistic Toxicology Studies
Poster Evaluation of the Toxicity Profiles of Selected Bioactivated Compounds in Primary Rat Hepatocytes Cultured in Micropatterned Cocultures
Application Note A Micropatterned Hepatocyte-Kupffer Cell Co-culture System to Study Inflammation in Drug Discovery
Poster HepatoPac® Allows Determination of Toxicity under Chronic Dosing and at Relevant Concentrations
Poster Global Gene Expression Changes Induced In Primary Human Hepatocytes by Thiazolidinediones Upon Repeat Dosing of HepatoPac® Cultures