- 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
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.
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)
HPLC High-performance liquid chromatography
LC Liquid chromatography
MS Mass spectrometry
- McGinnity, D. F.; Soars, M. G.; Urbanowicz, R. A. and Riley, R. J.; Drug Metab. Disp. 32, 1247, (2004).
- LeCluyse, E. L. and Alexandre, E.; Methods Mol. Biol. 640, 5, (2010).
- Coe, K. J.; Koudriakova, T.; “Metabolic Stability Assessed by Liver Microsomes and Hepatocytes:” Methods in Pharmacology & Toxicology 151, (2008).
- Tucker, G. T.; Houston, J. B.and Huang, S.M.; Pharm. Res. 18, 1071, (2001).
- 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).
- Butler, M. A.; Iwasaka, M.; Guengerich, F. P.; Kadlubar, F. F.; Biochemistry 86, 7696, (1989).
- Sesardic, D.; Boobis, A. R.; Edwards, R. J.; Davies, D. S.; Br. J. Clin. Pharmacol. 26, 363, (1988).
- Shen, S.; Marchick, M. R.; Davis, M.R.; Doss, G. A.; Pohl, L.R.; Chem. Res. Toxicol. 12, 214, (1999).
- Tang, W.; Stearns, R. A.; Wang, R. W.; Chiu, S. H.; Baillie, T. A.; Chem. Res. Toxicol. 12, 192, (1999).
- Yasar, U.; Eliasson, E.; Forslund-Bergengren, C.; Tybring, G.; Gadd, M.; Sjöqvist, F.; Dahl, M. L.; Eur. J. Clin. Pharmacol. 57, 729, (2001).
- Andersson, T.; Miners, J. O.; Veronese, M. E.; Birkett, D. J.; Br. J. Clin. Pharmacol. 37, 597, (1994).
- Huang, S. M.; Temple, R.; Throckmorton, D. C.; Lesko, L. J.; Clin. Pharmacol. Ther. 81, 298, (2007).
- Galetin, A.; Ito, K.; Hallifax, D. and Houston, J. B.; J. Pharmacol. Exp. Therap. 314, 180, (2005).
- 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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