Putting Science to Work

The Heptox Platform

The Heptox Platform is a novel and validated mechanistic hepatotoxicity prediction system that integrates in vitro and in silico methods to predict toxicity in vivo.

Syngene’s Virtual Liver Model

At the heart of the prediction platform lies the Virtual Liver, a patented mathematical model of normal liver physiology. When coupled with targeted assays performed at Syngene’s state-of-art laboratory, it provides mechanistic insights into how a drug molecule impacts the liver. Specifically, it predicts three major types of drug-induced liver injury, which together account for more than 75% of the instances of toxicity.

    • Hepatocellular Injury
    • Cholestasis
    • Steatosis

Our model of normal liver physiology has been created using an extensive literature survey covering several hundred papers and 80 person-years of work. This model has pathways for Oxidative stress, Cholestasis, Steatosis, Energy depletion, and Cytoskeleton maintenance. It allows researchers to understand the evolution of DILI, answering questions such as:

      • What pathways are impacted?
      • How does this impact translate into biological changes over a short period, as well as longer periods of time?

This knowledge allows researchers to identify early markers that very specifically signal the impending approach of a specific form of toxicity. Our model is capable of predicting the impact of both small molecule and biological agents. The model is mechanistic and has not “been trained” on any one class of molecules. It is therefore not limited by chemical class – a lacuna of pure toxicogenomic approaches.

Advantages

  • Scientifically advanced hepatotoxicity prediction: 80 person-years of R&D have already been performed in developing the platform.  The platform  predicts the toxicity of several known drugs and toxins well.
  • Combines in silico and wet lab techniques: The challenge in hepatotoxicity prediction is the understanding of drug biotransformation and the impact of active metabolites on the liver. Syngene’s wet lab approach deals efficiently with these biotransformation issues.
  • Handles toxicogenomic data: Syngene’s platform allows pharmaceutical companies to input their toxicogenomic data and generate actionable hypotheses for further exploration along with suggested experiments to identify specific (potential) modes of toxicity.
  • Flexible business model: The existing models can be tailored to address issues of specific interest to an organization, e.g. the Steatosis module has been used to address key questions in cardiovascular disease for a pharma collaborator.
The Syngene Heptox™ Platform - Predicting Liver Toxicity

Syngene Heptox™ Platform - Predicting Liver Toxicity

  • Syngene Heptox platform is available for the pharma and biotech industries to test their lead compounds for liver toxicity, as well as for household chemical and personal care companies for safety assurance of their products.
  • The platform can be used as an aid for lead candidate selection, to identify toxic mechanisms and molecular initiating events.
  • The platform combines in vitro experiments with in silico predicts to provide a customer with actionable insights into the toxic effect of a compound on a rat and human liver.

How it works

After we receive the test compound, we perform a set of in vitro assays (see below for details) in our lab using either freshly isolated rat hepatocytes, or H4IIE or HepG2 cell lines depending on our customer’s preference. The compound’s impact on the liver is tested at multiple time points and concentrations using multiple biological replicates. The information gathered from these experiments is then input into our patented  in silico model representing normal healthy liver function. The model simulations predict whether the test compound has a toxic effect on the liver in vivo, the nature of the toxicity (necrosis, oxidative stress, cholestasis or steatosis,) and the key mechanisms behind it. The simulations provide insight into the underlying mechanisms and associated adaptive changes that lead to the toxic response. The customer receives a comprehensive report on the analysis after an interactive discussion session. This compound specific report is delivered within 4 weeks from when we receive the compound.

Our Virtual Liver model is also capable of predicting toxicity of compounds on the normal liver or patients with disease such as metabolic syndrome. Additional user generated experimental data can also be input into the model. Please contact us for more details, or take a look at the presentation  “Integrating Multiple Data Using Virtual Liver”  describing the possibility of integrating additional data (e.g. microarray, bile transporter data) in the model.

Types of compounds that can be tested

Since the model is mechanistic and has not been trained on a particular class of compound, any chemical class of compound can be tested.

The in vitro experiments

            -The Heptox&trade in vitro Assays-
          • Cellular ATP
          • Mitochondrial membrane potential
          • Complex I Activity
          • Complex II Activity
          • Mitochondrial transition pore opening assay
          • Glutathione Reductase Activity
          • Gamma glutamyl cysteine synthetase (gamma-GCS) Activity
          • Glutathione peroxidase activity
          • Cellular GSH
          • Cellular ROS
          • Cellular MDA
          • Cellular 4HNE
          • CPT1 Activity
          • Microsomal Triglyceride Transfer Protein (MTTP) Activity
          • Cellular TG
          • FAS Activity
          • Fatty acid transporter activity
          • FOF1 atpase activity
          • UDP Glucuronosyl Transferase (UGT) activity
          • Glutathione S-transferase (GST) assay
          • Cell viability (CCK assay)
          • BSEP transporter assay
          • NTCP transporter assay

The compound received from a customer is tested in 23 different assays. Our state-of-the-art laboratory uses the following cell types depending on a customer’s preference:

          • Freshly isolated rat hepatocytes that are phase I and phase II metabolically competent
          • H4IIE
          • HepG2 cell lines
          • Human hepatocytes

Primary cell-systems such as rat or human primary cells maintain their metabolic competence and are hence appropriate when the toxicity is believed to be generated due to a reactive metabolite.

We can generate primary rat hepatocytes in-house or work with commercially available human hepatocyte sources. Cell-lines are useful when the toxicity is believed to be associated with the parent compound alone.

The in silico model

In silico model

The results from the 19 assays are input into our patented Virtual Liver model (US PTO Patent 8,645,075 received in February 2014). The model contains over 200 coupled differential equations that model cellular energy, antioxidant, fatty acid, bile salt, and bilirubin homeostasis. The simulation based on the input from the in vitro assays affects several hundreds of intracellular molecules and biological pathways. The model has been validated with over 80 compounds and 6 pharma partners and all the simulated results so far have matched well with what has been reported in literature.

The Result

In silico model result

All the results from the experiments and the model are summarized in a comprehensive report returned to you within 4-6 weeks of receiving the compound. The report contains:

          • A detailed analysis of the compound for its liver toxicity potential across a range of exposures. We also summarize the information by creating a color-coded safety range; green, yellow, red indicating increasing toxicity potential.

Key toxicity mechanisms (or targets) impacted by the compound.

Please take a look at the sample report on  Troglitazone and  Tak 875 analysis

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