It is a common practice in drug discovery organizations
to screen new chemical entities in order to predict future drug-drug
interactions. For this purpose, there are two main assay strategies, one based
on recombinant cytochrome P450 (rCYP) enzymes and fluorescent detection, and
other on human liver microsomes (HLM) and liquid chromatography coupled to
mass spectrometry. Many authors have reported a poor correlation between both
technologies, giving rise to concerns about the usefulness of fluorometric
methods for predicting drug-drug interactions. In this study, we investigated
the role that compound aqueous kinetic solubility may play in this lack of correlation. We found that drug discovery compounds with unacceptable kinetic
solubility, measured by a turbidimetric solutibility assay, tended to yield
higher IC50 values in in vitro models based on human liver microsomes,
whereas compounds with kinetic solubility values higher than 50 μM
showed very similar IC50 values in both in vitro models. Our results show that the turbidimetric solubility
assay is a useful tool to identify those discovery compounds that may require
further investigation in order to avoid overlooking future drug-drug
Cite this paper
Pérez, J. , Díaz, C. , Salado, I. , Pérez, D. , Peláez, F. , Genilloud, O. and Vicente, F. (2013) Evaluation of the effect of compound aqueous solubility in cytochrome P450 inhibition assays. Advances in Bioscience and Biotechnology, 4, 628-639. doi: 10.4236/abb.2013.45083.
 Zlokarnik, G., Grootenhuis, P.D. and Watson, J.B. (2005) High throughput P450 inhibition screens in early drug discovery. Drug Discovery Today, 10, 1443-1450.
 Bell, L., et al. (2008) Evaluation of fluorescenceand mass spectrometry-based CYP inhibition assays for use in drug discovery. Journal of Biomolecular Screening, 13, 343-353. doi:10.1177/1087057108317480
 McMasters, D.R., et al. (2007) Inhibition of recombinant cytochrome P450 isoforms 2D6 and 2C9 by diverse drug-like molecules. Journal of Medicinal Chemistry, 50, 3205-3213. doi:10.1021/jm0700060
 Rogge, M.C. and Taft, D.R. (2010) Preclinical drug development. 2nd Edition, D.A.T.P. Sciences, 187, Informa Healthcare USA, Inc., New York.
 Krippendorff, B.F., et al. (2007) Optimizing classification of drug-drug interaction potential for CYP450 isoenzyme inhibition assays in early drug discovery. Journal of Biomolecular Screening, 12, 92-99.
 Crespi, C.L., Miller, V.P. and Penman, B.W. (1997) Microtiter plate assays for inhibition of human, drug-metabolizing cytochromes P450. Analytical Biochemistry, 248, 188-190. doi:10.1006/abio.1997.2145
 Di, L., et al. (2007) Comparison of cytochrome P450 inhibition assays for drug discovery using human liver microsomes with LC-MS, rhCYP450 isozymes with fluorescence, and double cocktail with LC-MS. International Journal of Pharmaceutics, 335, 1-11.
 Cohen, L.H., et al. (2003) In vitro drug interactions of cytochrome p450: An evaluation of fluorogenic to conventional substrates. Drug Metabolism and Disposition, 31, 1005-1015. doi:10.1124/dmd.31.8.1005
 Di, L. and Kerns, E.H. (2003) Profiling drug-like properties in discovery research. Current Opinion in Chemical Biology, 7, 402-408. doi:10.1016/S1367-5931(03)00055-3
 Gibbs, M.A., et al. (1999) Inhibition of cytochrome P450 3A (CYP3A) in human intestinal and liver microsomes: Comparison of Ki values and impact of CYP3A5 expression. Drug Metabolism and Disposition, 27, 180187.
 Lipinski, C.A., et al. (2001) Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced Drug Delivery Reviews, 46, 3-26.
 Bergstrom, C.A., et al. (2002) Experimental and computational screening models for prediction of aqueous drug solubility. Pharmaceutical Research, 19, 182-188.
 Lipinski, C.A. (2000) Drug-like properties and the causes of poor solubility and poor permeability. Journal of Pharmacological and Toxicological Methods, 44, 235249. doi:10.1016/S1056-8719(00)00107-6
 Stresser, D.M., et al. (2000) Substrate-dependent modulation of CYP3A4 catalytic activity: Analysis of 27 test compounds with four fluorometric substrates. Drug Metabolism and Disposition, 28, 1440-1448.
 Wang, R.W., et al. (2000) Human cytochrome P-450 3A4: In vitro drug-drug interaction patterns are substrate-dependent. Drug Metabolism and Disposition, 28, 360-366.
 Korzekwa, K.R., et al. (1998) Evaluation of atypical cytochrome P450 kinetics with two-substrate models: Evidence that multiple substrates can simultaneously bind to cytochrome P450 active sites. Biochemistry, 37, 41374147. doi:10.1021/bi9715627
 Kenworthy, K.E., et al. (1999) CYP3A4 drug interactions: Correlation of 10 in vitro probe substrates. British Journal of Clinical Pharmacology, 48, 716-727.
 Galetin, A., et al. (2005) CYP3A4 substrate selection and substitution in the prediction of potential drug-drug interactions. Journal of Pharmacology and Experimental Therapeutics, 314, 180-190. doi:10.1124/jpet.104.082826
 Lin, J.H. (2000) Sense and nonsense in the prediction of drug-drug interactions. Current Drug Metabolism, 1, 305331. doi:10.2174/1389200003338947
 Walsky, R.L. and Obach, R.S. (2004) Validated assays for human cyto-chrome P450 activities. Drug Metabolism and Disposition, 32, 647-660. doi:10.1124/dmd.32.6.647
 Margolis, J.M. and Obach, R.S. (2003) Impact of nonspecific binding to microsomes and phospholipid on the inhibition of cytochrome P4502D6: implications for relating in vitro inhibition data to in vivo drug interactions. Drug Metabolism and Disposition, 31, 606-611.
 Chauret, N., et al. (1999) Description of a 96-well plate assay to measure cytochrome P4503A inhibition in human liver microsomes using a selective fluorescent probe. Analytical Biochemistry, 276, 215-226.
 Pérez, J., Sánchez, M. and Peláez, F. (2004) Suitability of DMSO as a solvent for the compounds to be tested in cytochrome 3A4, 2C9 and 2D6 P450 isoforms inhibition assays. Annual Meeting of Society of Bimolecular Screening, Madrid.
 Di Marco, A., et al. (2005) Development and validation of a high-throughput radiometric CYP3A4/5 inhibition assay using tritiated testosterone. Drug Metabolism and Disposition, 33, 349-358. doi:10.1124/dmd.104.002873
 Fowler, S.M., et al. (2002) CYP3A4 active site volume modification by mutagenesis of leucine 211. Drug Metabolism and Disposition, 30, 452-456.
 Kroemer, H.K., et al. (1993) Identification of P450 enzymes involved in metabolism of verapamil in humans. Naunyn Schmiedebergs Archives of Pharmacology, 348, 332-337. doi:10.1007/BF00169164
 Busse, D., et al. (1995) Cytochromes of the P450 2C sub-family are the major enzymes involved in the O-demethylation of verapamil in humans. Naunyn Schmiedebergs Archives of Pharmacology, 353, 116-121.
 Venkatakrishnan, K., von Moltke, L.L. and Greenblatt, D.J. (1999) CYP2C9 is a principal low-affinity phenacetin O-deethylase: Fluvoxamine is not a specific CYP1A2 inhibitor. Drug Metabolism and Disposition, 27, 15191522.
 Tracy, T.S., et al. (1999) Cytochrome P450 isoforms involved in metabolism of the enantiomers of verapamil and norverapamil. British Journal of Clinical Pharmacology, 47, 545-552.
 Wang, Y.H., Jones, D.R. and Hall, S.D. (2004) Prediction of cytochrome P450 3A inhibition by verapamil enantiomers and their metabolites. Drug Metabolism and Disposition, 32, 259-266. doi:10.1124/dmd.32.2.259
 Racha, J.K., et al. (2003) Substrate dependent inhibition profiles of fourteen drugs on CYP3A4 activity measured by a high throughput LCMS/MS method with four probe drugs, midazolam, testosterone, nifedipine and terfenadine. Drug Metabolism and Pharmacokinetics, 18, 128-138.
 Rodrigues, A.D. (1999) Integrated cytochrome P450 reaction phenotyping: Attempting to bridge the gap between cDNA-expressed cytochromes P450 and native human liver microsomes. Biochemical Pharmacology, 57, 465-480.