• 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:

1. McGinnity, D. F.; Soars, M. G.; Urbanowicz, R. A. and Riley, R. J.; Drug Metab. Disp. 32, 1247, (2004).

2. LeCluyse, E. L. and Alexandre, E.; Methods Mol. Biol. 640, 5, (2010).

3. Coe, K. J.; Koudriakova, T.; “Metabolic Stability Assessed by Liver Microsomes and Hepatocytes:” Methods in Pharmacology & Toxicology 151, (2008).

4. Tucker, G. T.; Houston, J. B.and Huang, S.M.; Pharm. Res. 18, 1071, (2001).

5. Bjornsson, T. D.; Callaghan, J. T.; Einolf, H. J.; et al.; “The conduct of in vitro and in vivo drug-drug interaction studies: A Pharmaceutical Research and Manufacturers of America (PhRMA) perspective”; Drug Metab. Dispos. 31, 815, (2003).

6. Butler, M. A.; Iwasaka, M.; Guengerich, F. P.; Kadlubar, F. F.; Biochemistry 86, 7696, (1989).

7. Sesardic, D.; Boobis, A. R.; Edwards, R. J.; Davies, D. S.; Br. J. Clin. Pharmacol. 26, 363, (1988).

8. Shen, S.; Marchick, M. R.; Davis, M.R.; Doss, G. A.; Pohl, L.R.; Chem. Res. Toxicol. 12, 214, (1999).

9. Tang, W.; Stearns, R. A.; Wang, R. W.; Chiu, S. H.; Baillie, T. A.; Chem. Res. Toxicol. 12, 192, (1999).

10. Yasar, U.; Eliasson, E.; Forslund-Bergengren, C.; Tybring, G.; Gadd, M.; Sjöqvist, F.; Dahl, M. L.; Eur. J. Clin. Pharmacol. 57, 729, (2001).

11. Andersson, T.; Miners, J. O.; Veronese, M. E.; Birkett, D. J.; Br. J. Clin. Pharmacol. 37, 597, (1994).

12. Huang, S. M.; Temple, R.; Throckmorton, D. C.; Lesko, L. J.; Clin. Pharmacol. Ther. 81, 298, (2007).

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).

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