The interaction between biological systems and an administered drug are marked by a complex series of events and effects. In general, the removal of a drug from the body is accelerated by enzyme-catalyzed steps which transform the drug molecule into a series of metabolites which are more readily eliminated from the body. These biotransformation reaction occur primarily in the liver and are principally mediated by cytochrome P450 enzymes. It is now evident that the participation of the cytochrome P450 enzymes in biotransformation reactions is a two-edged sword leading to both desirable and undesirable consequences. The cytochrome P40 enzymes exhibit broad and overlapping substrate specificity's and product selectivities. In addition their distribution and levels of expression in different tissues exhibits high inter individual variability which may be dramatically altered by exposure to drugs and environmental contaminants. As a consequence, it has often been difficult to associate a particular biochemical endpoint such as toxicity or drug clearance with a particular P450 isozyme or sub-family of isozymes. Knowledge of the role played by each isozyme is a prerequisite to understanding the participation of these enzymes in toxicity and drug metabolism.

 One of the primary goals of our research is to develop simple in vitro models and methods which may be used to understand and predict the impact of human cytochrome P450 dependent oxidation reactions on drug clearance and toxicity. One method which we are developing is the use of isozyme-specific inhibitors which may be used to determine which P450 enzymes are responsible for the metabolism of a given drug. Idealy an arsenal of isozymes specific inhibitors will be developed which will allow for the rapid identification of those isozymes which are responsible for the metabolism of a new drug or environmental contaminant. The approach we are using to address this problem is to design isoform selective "suicide substrates" for each P450 enzyme by incorporating certain types of reaction chemical substructures into known isoform selective substrates. A suicide substrate is a compound which irreversibly inactivates an enzyme when a reactive species is unmasked during and attempted oxidation step leading to covalent attachment of the substrate to the enzyme active site. Since the active site of the enzyme is covalently modified by these types of inhibitors a second goal of this research is to identify amino acids which occupy the active site of each enzyme.

 A second major goal in our laboratory is to explore the molecular basis of metabolic basis of metabolic drug-drug interactions and to develop a framework which may be used to predict the likelihood of an interaction at an early stage in drug development. The change in the rate metabolism of one drug caused by the presence of a second competing drug in vitro can provide qualitative information about drug-drug interactions. In order to apply this knowledge to the prediction of a drug interaction in vitro, a number of parameters such as inhibitory potency, enzyme specificity and concentration of the inhibitory drug in the liver need to be determined or estimated. The scope of this research includes metabolically-based drug-drug interactions and to establish kinetic and pharmacokinetic models which relate inhibitor concentrations in plasma to the magnitude of inhibitory effect on drug clearance.

Recent Publications

• J. K. Racha, A. E. Rettie, and K. L. Kunze, "Mechanism-Based Inactivation of Human Cytochrome P450 1A2 by Furafylline: Detection of a 1:1 Adduct to Protein and Evidence for the Formation of a Novel Imidazomethide Intermediate," Biochemistry, 37, 7407-7419 (1998).

 
• J. K. Racha and K. L. Kunze, "Stereochemical Evidence for Decomposition of Reactive Intermediates by Active Site Water in the Metabolism of 8-Alkyl Xanthines by P450 1A2," J. Am. Chem. Soc., 120, 5337-5338 (1998).

 
• K. A. Regal, W. N. Howald, R. M. Peter, C. A. Gartner, K. L. Kunze and S. D. Nelson, "Subanomolar Quantification of Caffeine's In Vitro Metabolites by Stable Isotope Dilution Gas Chromatography-Mass Spectrometry," J. Chromatogr. B. Biomed. Sci. Appl., 708, 75-85 (1998).

 
• J. M. Fisher, S. A. Wrighton, P. B. Watkins, P. Schmiedlin-Ren, J. C. Calamia, D. D. Shen, K. L. Kunze and K. E. Thummel, "First-Pass Midazolam Metabolism Catalyzed by 1alpha,25-dihydroxy Vitamin D3-Modified Caco-2 Cell Monolayers," J. Pharmacol. Exp. Ther., 289, 1134-1142 (1999).

 
• J. M. Fisher, S. A. Wrighton, J. C. Calamia, D. D. Shen, K. L. Kunze and K. E. Thummel, "Midazolam Metabolism Modified Caco-2 Monolayers: Effects of Extracellular Protein Binding," J. Pharmacol. Exp. Ther., 289, 1143-1150 (1999).

 
• M. A. Gibbs, K. E. Thummel, D. D. Shen and K. L. Kunze, "Inhibition of CYP3A in Human Intestinal and Liver Microsomes: Comparison of Ki Values and Impact of CYP3A5 Expression," Drug Metab. Dispos., 27, 180-187 (1999).

 
• M. A. Gibbs, K. L. Kunze, W. N. Howald and K. E. Thummel, "Effect of Inhibitor Depletion on Inhibitory Potency:  Tight Binding Inhibition of CYP3A by Clotrimazole," Drug Metab. Dispos., 27, 596-599 (1999).

 
• M. A. Gibbs, M. T. Baillie, D. D. Shen, K. L. Kunze and K. E. Thummel, "Persistent Inhibition of CYP3A4 by Ketoconazole in Modified Caco-2 Cells," Pharm. Res., 17, 299-305 (2000).

 
• K. E. Thummel, K. L. Kunze and D. D. Shen, "Metabolically-Based Drug-Drug Interactions: Principles and Mechanisms," in Metabolic Drug Interactions (R. Levy, K. E. Thummel, W. F. Trager, P. D. Hansten and M. Eichelbaum, eds.), Williams and Wilkins, Philadelphia, PA, 3-20 (2000).

 
• C. Yao, K. L. Kunze, E. D. Kharasch, Y. Wang, W. F. Trager, I. Ragueneau and R. H. Levy, "Fluvoxamine-Theophylline Interaction: Gap Between In Vitro and In Vivo Inhibition Constants Towards CYP1A2," Clin. Pharm., in press.

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This page last updated: June 17, 2002