SOT 2018 Poster Session Wrap Up

We presented two posters at SOT this month in San Antonio that highlighted research performed by our Durham team. They worked with the Environmental Protection Agency (EPA) and Bristol-Myers Squibb (BMS).

The first poster, “Hepatobiliary Disposition of 15 Non-Therapeutic Chemicals in Sandwich-Culture Rat Hepatocytes using B-CLEAR® Technology”, describes results of a research collaboration between BioIVT and the Environmental Protection Agency (EPA) in which B-CLEAR® Technology was used to assess the hepatobiliary disposition of 15 non-therapeutic compounds in rat hepatocytes. The study was conducted as part of the EPA’s ToxCast™ Toxicity Forecaster.

High throughput in vitro screening provides surrogate toxicity data for thousands of chemicals occurring in commerce and the environment without traditional toxicity testing data. In vitro-in vivo extrapolation (IVIVE) via high throughput toxicokinetics (HTTK) allows screening data to be placed in a risk prioritization context. However, in vitro TK tools, such as hepatocyte suspension models underestimated toxicokinetic clearance (L/h/kg BW) of the selected compounds when compared to in vivo clearance. Possible reasons for underestimation include biliary excretion, which is not present in a hepatocyte suspension model.

The following compounds were selected for the study:

  • Diclosulam
  • Diniconazole
  • Ethametsulfuron methyl
  • Flumetsulam
  • Fulvestrant
  • Iodosulfuron-methyl-sodium
  • Mesotrione
  • Monobutyl phthalate
  • Oxytetracycline dihydrate
  • Penoxsulam
  • Perfluorooctanoic acid
  • Pyrithiobac-sodium
  • Quinclorac
  • Thidiazuron
  • Triflumizole

Results included the following observations:

  • Biliary efflux (BEI) results of < 20% for all compounds evaluated suggested biliary excretion of all compounds studied was low or slow. Although no biliary efflux is sometimes observed, a compound with high accumulation potential may still be extensively excreted into the bile a result of a slow excretion process.
  • The study indicated the importance of accounting for hepatic accumulation
  • The ratio of intracellular concentration (ICC) to nominal concentration was greater than 10X for diniconazole, ethametsulfuron-methyl, fulvestrant, and triflumizole.
  • Accumulation of three chemicals (Diclosulam, Quinclorac, and Monobutyl phthalate) was significantly over-predicted by the Armitage et al. (2014) partitioning model
  • The following compounds were found to accumulate in bile pockets: flumetsulam, iodosulfuron-methyl-sodium, mesotrione, and oxytetracycline dihydrate.

The second poster, “Prediction of Clinical Hepatotoxicity of Clinical Drug Candidates using the Novel C-DILI™ Assay: A Retrospective Case Study“, describes results of a research collaboration between BioIVT and Bristol-Myers Squibb (BMS) in which the C-DILI™ Assay was used to evaluate the risk of cholestatic hepatotoxicity of clinical drug candidates.

BMS provided compounds code-named, “A”, “B”, “C” and “D” to BioIVT but did not provide information on their chemical structures. BioIVT researchers evaluated the compounds using the C-DILI Assay and were able to predict the hepatotoxic potential that BMS had observed from in vivo studies. The study was implemented at BioIVT’s laboratory in Durham NC, and results were presented in the Late Breaker poster session at the Society of Toxicology 57th Annual Meeting and ToxExpo, March 11 – 15, 2018.

Each compound was evaluated at several concentrations up to doses at significant multiples of the human Cmaxconcentration. The assay used the QUALGRO™ Sensitization medium and a growth medium to determine bile acid-dependent (e.g. cholestatic) versus general hepatotoxicity. Hepatotoxicity categories were determined based upon the LDH leakage profile and intracellular ATP depletion. LDH leakage in only bile acid sensitization media suggests a cholestatic hepatotoxicity mechanism while marked LDH leakage in both media conditions suggests a general hepatotoxicity mechanism.

The C-DILI Assay predicted the following results:

  • Compound A: High cholestatic hepatotoxicity at 57X clinical Cmax.
  • Compound B: Low hepatotoxicity potential across the concentrations examined.
  • Compound C: High hepatotoxicity potential at 20X clinical Cmax.
  • Compound D: High hepatotoxicity potential at 18X clinical Cmax

The assay results effectively predicted in vivo observations including Compound D, a drug which BMS halted clinical trials resulting from increased ALT (>5.5XULN) and AST (≥3XULN) observed in healthy patients.

The study illustrates how the C-DILI Assay can be used to inform lead selection and optimization decisions.

About the C-DILI Assay

The C-DILI™ Assay is a novel in vitro method to evaluate a compound’s potential for cholestatic drug-induced liver injury (DILI). The C-DILI Assay has demonstrated high in vitro – in vivo correlation with compounds known to have a risk for cholestatic hepatotoxicity. It is an effective tool for assessing the risk of cholestatic DILI and providing data useful in lead selection and managing toxicity risk. At high concentrations, bile acids can cause liver toxicity by signaling apoptotic pathways as well as disrupting membranes. The liver tightly controls bile acid concentrations through multiple regulatory mechanisms including basolateral efflux, biliary efflux, synthesis, and metabolism. However, if control mechanisms are disrupted, bile acid concentrations can reach a “tipping point” resulting in hepatotoxicity. The C-DILI Assay combines all of these human-relevant processes in one simple threshold readout, providing a unique clinically-predictive assessment of cholestasis risk.

Study Report

The C-DILI Assay is specially constructed to use a simple LDH and ATP readout that is specific to compounds that inhibit bile acid transport and antagonize FXR and ATP readout. The readout provides a threshold value and is analogous to clinical tests for liver toxicity.

Process

TRANSPORTER CERTIFIED™ Hepatocytes are cultured in sandwich culture to re-establish physiologically-relevant uptake, metabolism, regulation and efflux function. The hepatocytes form a matrix with bile pockets and demonstrate transporter function, including BSEP, OSTs, and MRP3/4. After the culture has been established, BioIVT’s proprietary QUALGRO™ Sensitization Media is added to the wells, along with the test compound, and incubated for 24 hours. Cholestatic hepatotoxicity is evaluated by measuring lactate dehydrogenase (LDH) concentration and ATP content versus controls in a standard plate-reader assay. The response of the test article is compared to drugs with known clinical cholestatic effects. Comparison with negative controls and positive controls allow compounds to be bucketed for their clinical cholestatic hepatotoxicity potential. In addition, an incubation in standard QUALGRO Media can be performed in parallel to assess general hepatotoxicity potential of the test compound.

Applications

The C-DILI™ Assay integrates effects on BSEP, OSTs, MRP3/4, and FXR to delineate hepatotoxicity resulting from a build-up of intracellular bile acids and has been shown to be effective in predicting compounds that have clinical hepatocellular cholestatic toxicity.

Accelerate Decision-Making

Either as a stand-alone program or implemented with other studies the C-DILI Assay is designed to complement the R&D process. From early-stage screening through optimization and characterization the C-DILI Assay informs SAR, lead selection, and toxicity assessment programs. An added advantage is that the same system, and even the same cells, can be used for more in-depth studies later in characterization work. The data are all connected and consistent so there is no need to translate data from different model systems.

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