MQP-BIO-DSA-0262 Validation of Cytochrome P450 2C8 Inhibition Assay A Major Qualifying Project Report Submitted to the Faculty of the W ORCESTER POLYTECHNIC INSTITUTE In partial fulfillment of the requirements for the Degree of Bachelor of Science in Biology and Biotechnology By ______________________ Matthew Schulze April 24, 2008 APPROVED: _______________________ _______________________ Adrian Sheldon, Ph.D. David Adams, Ph.D. Associate Director (Invitro ADMET) Biology and Biotechnology Charles River Laboratories Preclinical Services W PI Project Advisor Major Advisor ABSTRACT One of the most important pharmaceutical properties of a drug candidate to be determined during any pre-clinical discovery is the effect of the specific test compound on the activity of Cytochrome P450, a key enzyme involved in drug metabolism. The purpose of this project was to validate a Cytochrome P450 2C8 (CYP2C8) enzyme inhibition assay in development at Charles River Labs (W orcester). The assay quantitates the in vitro inhibition of CYP2C8 by a test compound as measured by the amount of specific metabolite generated by CYP2C8-specific drug substrates Amodiaquine (AMOD) and Paclitaxel (PACL). The data presented here validate the assay. TABLE OF CONTENTS Abstract.......................................................................................................................................- 2 - Table of Figures ..........................................................................................................................- 4 - Acknowledgements.....................................................................................................................- 6 - Background.................................................................................................................................- 7 - Drug Metabolism & Biotransformation ..................................................................................- 7 - Cytochrome P450..................................................................................................................- 11 - History...............................................................................................................................- 12 - Biochemistry......................................................................................................................- 14 - Cytochrome P450 2C8 ..........................................................................................................- 19 - Structure of CYP2C8.........................................................................................................- 20 - Metabolism of CYP2C8 ....................................................................................................- 21 - Project Purpose .........................................................................................................................- 24 - Methods.....................................................................................................................................- 25 - Enzyme Titration...................................................................................................................- 26 - Substrate Titration.................................................................................................................- 27 - CYP2C8 Inhibition................................................................................................................- 27 - Results.......................................................................................................................................- 28 - Discussion.................................................................................................................................- 33 - Bibliography.............................................................................................................................- 35 - TABLE OF FIGURES Figure 1: Definitions of Specificity. (Monosson, 2008).............................................................- 1 - Figure 2: Visual Description of Oxidation and Reduction (Phase I) reactions. ("Mnemonic: Oxidation and Reduction.“, 13 Feb 2008) ..................................................................................- 1 - Figure 3: Model of the conserved tertiary structure of CYP monooxygenase. The heme is orange, substrate recognition site is red, and the heme coordinating I and L helices are green. (Urlacher & Eiben, 2006)............................................................................................................................- 1 - Figure 4: —Carbon monoxide-difference spectra for NADH-reduced (- - -) and Na2S2O4-reduced (–) rat liver microsomes.“ (Sato & Omura, 1978) ....................................................................- 1 - Figure 5: Drug discovery and development process. (—KinomeScan“, 13 Feb 2008)................- 1 - Figure 7: epoxidation of styrene to styrene oxide. (Urlacher & Eiben, 2006)............................- 1 - Figure 8: Hydroxylation of fatty acids to hydroxyl-fatty acids. (Urlacher & Eiben, 2006) .......- 1 - Figure 6: Hydroxylation of an aromatic compound naphthalene to 2-naphol/1-naphol. (Urlacher & Eiben, 2006)............................................................................................................................- 1 - Figure 9: The fold and active site for CYP. Black displays the substrate recognition sites (SRS). (Denisov et al., 2005)..................................................................................................................- 1 - Figure 10: Predicted secondary structure of Cytochrome P450 2C8 Dimer Enzyme. Light blue=turns; Pink=coils; Green=helices; Purple=strands; Magenta(center)=two palmitic acid molecules; Magenta(top and bottom)= hemes (Produced in MBT Protein W orkshop, Jan 2008)..- 1 - Figure 12: Metabolism of PACK to 6M-hydroxypaclitaxel. (Separations of Paclitaxel and Its Metabolite 6a-Hydroxypaclitaxel.).............................................................................................- 1 - Figure 11: Metabolism of AMOD to DEAQ. (Li et al., 2002) ...................................................- 1 - Figure 13: IC50 plots of amodiaquine inhibition in CYP2C8 by montelukast (•), candesartan cilexetil (), zafirlukast (), quercetin (), and gemfibrozil (). (W alsky et al., 2005) ........- 1 - Figure 15: IS Peak Area vs. Index of DEAQ using DESA-d3 as an IS......................................- 1 - Figure 14: IS Peak Area vs. Index of DESA-d3 using RSRP as an IS.......................................- 1 - Figure 16: Graphs of Average Area Ratio (Analyte Peak Area / IS Peak Area) vs. Enzyme Concentration for T=30 on the left and T=0 on the right. ..........................................................- 1 - Figure 17: Graph of Average Area Ratio (Analyte Peak / IS Peak) vs. substrate concentration (µM)............................................................................................................................................- 1 - Figure 18: Graph of Average Area Ratio (Analyte Peak / IS Peak) vs. substrate concentration (µM)............................................................................................................................................- 1 - Figure 20: IC50 of QRCT is 4.2 µM............................................................................................- 1 - Figure 19: IC50 of MONT is 0.3 µM...........................................................................................- 1 - ACKNOW LEDGEM ENTS First I would like to thank Adrian Sheldon for letting me use the Invitro ADMET lab at Charles River Laboratories. I would also like to thank Sarah Mitchell, Patty W alton, Susan Dearborn, and Ali Maarouf for showing me procedures that I was initially unfamiliar with, and getting me accustomed to the lab, for helping with my report writing, and most importantly for their guidance and direction. Thank you also to the Charles River Laboratories Preclinical Facilities in Shrewsbury and W orcester for providing the necessary methods, materials and laboratory space respectfully. Finally, I would like to thank Professor David Adams for his assistance in setting this project up, his help writing the report, and his continued support. BACKGROUND Drug Metabolism & Biotransformation A drug is defined as a substance taken for either medicinal or recreational use. After digestion, the substance circulates, enters the circulatory system, and begins to undergo metabolism or biotransformation. This process can be best described as —breaking down“ the drug whatever it may be, but it always involves the chemical modification or degradation of the drug. These changes can occur in any tissue, but most of them occur in hepatocytes. The liver produces numerous enzymes that alter drugs and toxins in order to clear them from the circulatory systems and excrete them in the bile. These enzymatic systems use a variety of reactions to metabolize drugs, such as oxidation, reduction, and hydrolysis. As these changes occur new metabolites are produced that may or may not remain active in vivo. However, the majority of these metabolites will enter the kidney where they can be processed and expelled in the urine, solid waste, or perspiration (—Liver“, 2005). Biotransformation is vital to the survival of an organism because it allows for necessary nutrients to be absorbed as they are transformed, metabolize drugs into metabolites that may be the therapeutic, and of course are a mechanism of defense against