Cytotoxicity / Hepatotoxicity test & CYP450 Inhibition Profiling

Cytotoxicity / Hepatotoxicity test 

What about the toxicity of the test compound? Is the compound too toxic to be therapeutically useful?

The in vitro cytotoxicity test with primary hepatocytes is used to identify the cytotoxic potential of a test substance. The relative cell viability upon incubation with test article compared to the solvent control is determined (single point).

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Hepatocyte cells are incubated for 24 hours with known toxic and non-toxic compounds at a range of different concentrations. At the end of the incubation period the cells are loaded with the ATP-liteTM 1step ATP monitoring reagent and scanned using an automated plate reader with luminescence detection to determine the number of active/live cells.

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CYP450 Inhibition Profiling

Will the compound of interest lead to drug-drug interactions?  Does the compound of interest inhibit a key enzyme responsible for oxidative metabolism?

·         Cytochrome P450s (CYPs) are a superfamily of heme-containing enzymes that mediate the inactivation and metabolism of many drugs as well as endogenous substances.

·         Compounds that inhibit P450s may cause the toxic accumulation of other substrates.

·         CYP inhibition profiling examines the effects of a test compound on the metabolism of other known enzyme substrates of the five primary drug human metabolizing CYP: 1A2, 2B6, 2C9, 2D6, 3A4.

·         The levels of the CYP isoform marker substrate and metabolites are measured in the presence and absence of a test compound by LC/MS/MS.

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In an assay similar to the metabolic stability assay, liver microsomes are used to determine the CYP450 inhibition profile of test compounds by measuring the % metabolism of a known substrate. Microsomes (and NADPH regenerating system) are dispensed into a 96well plate containing a substrate and test compound (10 µM), and the reaction is allowed to proceed for 0.5 hours at 37°C with shaking. The reaction is quenched by the addition of MeOH, centrifuged and the amount of product is measured by LC/MS/MS.

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