© Copyright 2017 Ryan P. Seguin Non-Dissociative Sequential Metabolism of Enoxacin to a Metabolic Intermediate Complex with Cytochrome P450 1A2 Ryan P. Seguin A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2017 Reading Committee: Kent L. Kunze, Chair Rheem A. Totah William M. Atkins Program Authorized to Offer Degree: Medicinal Chemistry University of Washington Abstract Non-Dissociative Sequential Metabolism of Enoxacin to a Metabolic Intermediate Complex with Cytochrome P450 1A2 Ryan P. Seguin Chair of the Supervisory Committee: Assoc. Professor Kent L. Kunze Medicinal Chemistry Cytochrome P450 enzymes constitute a superfamily of isoforms which accelerate the removal of foreign compounds from the body through oxidative biotransformation to generate polar metabolites that are more readily excreted from the body. Hence, susceptibility to oxidation by cytochrome P450 may largely determine the in vivo half-life, plasma level, and tissue exposure of small molecule drugs. As the P450 isoforms involved in drug metabolism contain large, flexible, and promiscuous active sites which can bind and metabolize multiple drugs, it is common for one drug to interfere with the metabolism of another. Inhibition of a specific P450 isoform may lead to drug-drug interactions (DDIs) through impairment of metabolic clearance of a coadministered drug and the build-up of this drug or its metabolites to toxic levels in the body. The use of in vitro data to predict, rationalize and, ultimately, prevent DDIs is a top priority in drug development. Accordingly, emphasis in this field of study has been placed on understanding which drug structures inhibit P450 and the mechanism by which inhibition occurs. This dissertation explores the mechanism of inhibition of cytochrome P450 1A2 (CYP1A2) by the fluoroquinolone antibacterial enoxacin. Enoxacin elicits clinically-significant DDIs with theophylline and caffeine due to potent CYP1A2 inhibition in vivo. However, enoxacin is characterized as a weak reversible inhibitor of CYP1A2 in vitro leading to severe underprediction of the DDIs with theophylline and caffeine. Herein, we have clarified the mechanism of inhibition as a time-dependent irreversible process where enoxacin is sequentially metabolized within the CYP1A2 active site to a mechanism-based inhibitor. Thus, CYP1A2 is inactivated by a metabolite of enoxacin through formation of a metabolic- intermediate (MI) complex. The mechanism of MI complex formation requires N-hydroxylation of the piperazine ring of enoxacin followed by -carbon hydroxylation and oxidative ring-opening to a nitroso metabolite that strongly ligates to the ferrous heme iron of CYP1A2. We elucidated the mechanism of CYP1A2 inactivation as sequential metabolism of the piperazine ring to an MI complex in recombinant CYP1A2 and confirmed that this mechanism is still viable in human liver microsomes. Circumstantial evidence was provided for MI complex formation with CYP1A2 in primary human hepatocytes. The DDIs with theophylline and caffeine were well-predicted by in vitro to in vivo predictions using apparent CYP1A2 inactivation parameters for the parent drug, enoxacin. Our results suggest that enoxacin is sequentially metabolized to an MI complex non-dissociatively within the CYP1A2 active site and that released metabolite intermediates do not significantly contribute to MI complex formation. The non-dissociative nature of the sequential metabolic inactivation process accounts for our success in predicting the DDIs with theophylline and caffeine using apparent inactivation parameters of the parent drug. Although enoxacin requires multiple CYP1A2-mediated oxidations to inactivate CYP1A2, enoxacin can be treated as a single-step inactivator (i.e. a true mechanism-based inhibitor) for DDI prediction purposes. In our view, non-dissociative sequential inactivation accounts not only for the success of our prediction, but also the potent inhibition of CYP1A2 in vivo. Our observation that N-hydroxy enoxacin, an intermediate hydroxylamine metabolite, is reduced back to enoxacin in human liver microsomes and human hepatocytes calls into question whether a dissociative sequential inactivation process would be viable in vivo. Futile cycling between enoxacin and the N-hydroxy enoxacin metabolite could theoretically prevent the sequential metabolic process from progressing beyond the hydroxylamine, thus highlighting the importance of non-dissociative sequential metabolism. In summary, we proposed enoxacin is a time-dependent inhibitor of CYP1A2 through non- dissociative sequential metabolism of the piperazine ring to an MI complex. This is the first report of sequential metabolism of a piperazine by a cytochrome P450 to an MI complex. It is suggested that MI complex formation may result from sequential metabolism of other alicyclic amine-containing substrates and that alicyclic amines may be a new structural alert for MI complex formation. Table of Contents List of Figures ........................................................................................................................... v List of Tables .......................................................................................................................... viii Acknowledgements ................................................................................................................. ix Chapter 1: Introduction ............................................................................................................ 1 1.1 Dissertation Purpose and Organization ........................................................................ 1 1.2 Drug-Drug Interactions ................................................................................................. 3 1.3 Prediction of Drug-Drug Interactions from in vitro Cytochrome P450 Inhibition Data .... 4 1.4 Mechanism-Based (Time-Dependent) Inhibition .......................................................... 7 1.4.1 The Cytochrome P450 Catalytic Cycle .................................................................. 8 1.4.2 Mechanism-Based Inactivation of Cytochrome P450 ...........................................10 1.4.3 Metabolic-Intermediate Complex Formation with Cytochrome P450 ....................11 1.5 Discovery, Development, and Use of Fluoroquinolone Antibacterials ..........................12 1.6 The Fluoroquinolone Enoxacin ...................................................................................14 1.7 Interaction of Fluoroquinolones with Cytochrome P450 1A2 .......................................15 Chapter 2: The Mechanism of CYP1A2 Inactivation by Enoxacin ....................................... 22 2.1 Introduction .................................................................................................................22 2.2 Experimental ...............................................................................................................24 2.2.1 Materials. .............................................................................................................24 2.2.2 1H NMR Spectroscopy. ........................................................................................24 2.2.3 Organic Synthesis. ...............................................................................................25 2.2.4 HPLC-MS/MS (MRM) Method for Quantification of Acetaminophen (APAP). .......29 2.2.5 HPLC-MS/MS (MRM) Method for Quantification of Enoxacin and Metabolites. ....30 2.2.6 Identification of Metabolites by High-Resolution Mass Spectrometry. ..................31 2.2.7 Oxidation of N-OH-ENX by Potassium Ferricyanide. ............................................32 2.2.8 Time-Dependent Inhibition of CYP1A2 Activity (Phenacetin o-deethylase). .........32 2.2.9 Reversibility of CYP1A2 Inhibition to Dialysis and Ferricyanide Treatments. ........33 2.2.10 Ferricyanide Reversal of the MI Complex: LC-MS Analysis of the Released Ligand. 34 2.2.11 Spectral Scanning for MI Complex Formation and CO Binding to Residual P450. 35 2.2.12 Spectral Ferricyanide Reversal in CYP1A2 Bactosomes......................................37 2.2.13 Substrate Depletion and CYP1A2 Activity Remaining (Partition Ratio).................38 2.2.14 Metabolite Formation Time Courses. ...................................................................38 i 2.2.15 Data analysis. ......................................................................................................39 2.2.16 Static Prediction of CYP1A2 MBI on Active Enzyme and AUCi/AUC. ...................40 2.3 Results ........................................................................................................................42 2.3.1 Synthesis of N-OH-ENX and 18O-labeled N-OH-ENX. ..........................................42 2.3.2 Isolation of an Aqueous Solution of the Cyclic Nitrone of Enoxacin. .....................42 2.3.3 CYP1A2 MI Complex Formation from ENX and N-OH-ENX, but not DES-ENX. ..43 2.3.4 Confirmation of the MI Complex by Spectral Ferricyanide Reversal. ....................45 2.3.5 Evaluation of ENX, N-OH-ENX, and DES-ENX as Time-Dependent Inhibitors of CYP1A2. ............................................................................................................................48 2.3.6 Reversibility to Dialysis and Ferricyanide: Assessing the Contribution of MI Complex Formation to CYP1A2 Activity Loss. ....................................................................51 2.3.7 High-Resolution