Hepatocyte Stability Assay (mouse, rat, dog, monkey, human)

• Metabolic stability, as defined as the percentage of parent compound lost over time, is assessed in the presence of cryopreserved hepatocytes.

• The liver is the most important site of drug metabolism in the body. Approximately 60% of marketed compounds are cleared by hepatic CYP-mediated metabolism.1 Hepatocytes contain the full complement of hepatic drug metabolizing enzymes (both phase I and phase II) maintained within the intact cell and are routinely used to determine the in vitro intrinsic clearance of a compound.1-3

Readout: % remaining at time point, t1/2 determination, and intrinsic clearance (Clint) estimation

Controls: Phenacetin, Diclofenac, Dextromethorphan, Omeprazole, Midazolam, 7-EC

Phenacetin, Diclofenac, Omeprazole, Dextromethorphan, Midazolam and 7-ethoxycoumarin (7-EC) are all recommended in vitro enzyme specific probe substrates for P450s.4,5 Phenacetin is the preferred probe for screening CYP1A2-based drug interaction.4-7 Diclofenac is a substrate for CYP2C9.4,5,8-10 Omeprazole (OMP), is metabolized by CYP2C19 and CYP3A4.11 Dextromethorphan is an in vitro substrate for studying CYP2D6 inhibition.12 Midazolam is a recommended probe for CYP3A4.4,5,13 Known species differences exist in 7-ethoxycoumarin (7-EC) metabolism.14

Assay Description:

Working solutions of each compound are prepared from 10 mM stock solution in DMSO and diluted to a final concentration of 2 μM in incubation medium.

Mixtures of control/test compound working solutions and pre-incubated hepatocyte suspension (2 million viable cells/mL in incubation medium) are incubated in CO2 atmosphere for 2 hours.  Aliquots are removed at each time point (0, 15, 30,  60, 90, and 120 mins).  For each aliquot, the reaction is stopped by adding internal standard / quenching solution (terfenadine and tolbutamide in methanol/ACN) and vortexing.  The samples are centrifuged and the supernatant is removed for LC-MS/MS analysis.

The MS detection is performed by using a SCIEX API 4000 Q trap instrument. Each compound is analyzed by reversed phase HPLC using a Kinetex 2.6μ C18 100Å column (3.0 mm X 30 mm, Phenomenex). Mobile phase – Solvent A: water with 0.1% formic acid,  solvent B: ACN with 0.1% formic acid. The amount of parent compound is determined on the basis of the peak area ratio (compound area to IS area) for each time point.

Data Analysis:

The slope of the the Ln(% remaining) vs time point line is used to calculate t1/2 according to the following formula:

Half life (t1/2) = – ln(2) / Slope

Slope = Terminal elimination rate constant (-K)

The estimation of Clint (in µL/min/million cells) is calculated using the following equation:

Clint = ln(2) * 1000 / T1/2/ 1 (million cells/mL)

Abbreviations:

ACN                         Acetonitrile

DMSO                     Dimethylsulfoxide

7-EC                        7-ethoxycoumarin

HPLC                       High-performance liquid chromatography

LC                             Liquid chromatography

MS                           Mass spectrometry

Literature:

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11. Andersson, T.; Miners, J. O.; Veronese, M. E.; Birkett, D. J.; Br. J. Clin. Pharmacol. 37, 597, (1994).
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13. Galetin, A.; Ito, K.; Hallifax, D. and Houston, J. B.; J. Pharmacol. Exp. Therap. 314, 180, (2005).
14. Li, A. P., et al. “Cryopreserved human hepatocytes: characterization of drug-metabolizing enzyme activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability, and drug-drug interaction potential” Chem. Biol. Interact. 121, 17, (1999).