© Copyright 2018 Faith M. Stevison In Vitro to In Vivo Translation of Complex Drug-Drug Interactions Involving Retinoic Acid Faith M. Stevison A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2018 Reading Committee: Nina Isoherranen, Chair Kenneth E. Thummel Yvonne S. Lin Program Authorized to Offer Degree: Pharmaceutics University of Washington Abstract In Vitro to In Vivo Translation of Complex Drug-Drug Interactions Involving Retinoic Acid Faith M. Stevison Chair of the Supervisory Committee: Professor and Vice Chair, Nina Isoherranen Pharmaceutics all-trans-retinoic acid (atRA), the active metabolite of vitamin A, is a ligand for several nuclear receptors and acts as a critical regulator of many physiological processes. The cytochrome P450 26 (CYP26) enzymes are responsible for atRA clearance and are potential drug targets to increase concentrations of endogenous atRA in a tissue-specific manner. The first project of this thesis aimed to establish the relationship between CYP26 inhibition, by the potent CYP26A1 and B1 inhibitor talarozole, and altered atRA concentrations in tissues, and to quantify the increase in endogenous atRA concentrations necessary to alter atRA signaling in target organs. The second part of this thesis focused on evaluating exogenous atRA and retinoid dosing on gene regulation. Several in vitro and preclinical studies have suggested that atRA down-regulates CYP2D6 expression and activity. Both atRA and its stereoisomer 13-cis retinoic acid (13cisRA) are used clinically, and steady state exposures of atRA are similar after dosing with atRA or 13cisRA. The second aim of this thesis was to determine whether 13-cis retinoic acid (13cisRA) or its active metabolites, atRA and 4-oxo-13cisRA, cause a drug-drug interaction (DDI) with CYP2D6, decreasing the clearance of the CYP2D6 probe dextromethorphan. Further, the effects of 13cisRA and its metabolites (i.e., atRA and 4-oxo-13cisRA) on CYP2D6 and CYP3A4 in human hepatocytes were determined and used to predict whether the in vivo results could be correctly predicted from in vitro. Following a single 2.5-mg/kg dose of talarozole to mice, atRA concentrations increased up to 5.7-, 2.7-, and 2.5-fold in serum, liver, and testis, respectively, resulting in induction of Cyp26a1 in the liver and testis and Rarβ and Pgc1β in liver. The increase in atRA concentrations was well-predicted from talarozole pharmacokinetics and in vitro data of CYP26 inhibition. After multiple doses of talarozole, a significant increase in atRA concentrations was observed in serum but not in liver or testis. This lack of increase in tissue atRA concentrations correlated with increase in CYP26A1 expression in liver and testis. The increased atRA concentrations in serum without a change in liver suggest that CYP26B1 in extrahepatic sites plays a key role in regulating systemic atRA exposure. The putative DDI between 13cisRA and dextromethorphan was studied in clinical study in eight healthy volunteers. The geometric mean ratio (GMR; 90% CI) of dextromethorphan area under the curve from time zero to infinity (AUC0-∞) prior to and after 13cisRA treatment was 0.822 (0.677 – 0.998) indicating that dextromethorphan clearance and CYP2D6 activity was increased following 13cisRA treatment. The dextrorphan-to-dextromethorphan AUC0-∞ ratio and dextrorphan formation clearance (Clf) were also increased consistent with CYP2D6 induction. In addition, the Clf of 3-methoxymorphinan, a CYP3A4-specific metabolite of dextromethorphan, and the Clf of 6β-hydroxycortisol, an endogenous marker of CYP3A4 activity, were also increased suggesting that CYP3A4 expression was also induced by 13cisRA. Quantitative DDI predictions were made with data for CYP2D6 mRNA down-regulation in human hepatocytes assuming that 13cisRA and its metabolites competitively bind to the same receptor to elicit CYP2D6 down-regulation. An ~50% decrease in CYP2D6 activity and 2-fold increase in dextromethorphan AUC were predicted to be observed in vivo after dosing with 13cisRA. These data demonstrate a clear disconnect between in vitro and in vivo CYP2D6 down-regulation. In contrast to data with CYP2D6, data from hepatocytes support the induction of CYP3A4 observed in the clinical study. This is the first study to fully characterize the concentration-response effect of retinoids on CYP2D6 activity and highlights the difficulty in translating in vitro observations of CYP down-regulation to the clinic. TABLE OF CONTENTS List of Figures ................................................................................................................................ iv List of Tables ................................................................................................................................. vi Chapter 1. Introduction ................................................................................................................... 1 1.1 Retinoic acid isomers as important endogenous signaling molecules and therapeutic agents ......................................................................................................................................... 2 1.2 Synthesis, elimination, and pharmacokinetics of atRA and 13cisRA ............................ 5 1.3 CYP down-regulation and drug-drug interactions with retinoids ................................. 11 1.4 Hypothesis and aims ..................................................................................................... 14 Chapter 2. Inhibition of the all trans-retinoic acid (atRA) hydroxylases CYP26A1 and CYP26B1 results in dynamic, tissue-specific changes in endogenous atRA signaling ................................. 19 2.1 Introduction ................................................................................................................... 20 2.2 Materials and methods .................................................................................................. 22 2.2.1 Chemicals and reagents............................................................................................. 22 2.2.2 Animal care and talarozole treatments ...................................................................... 22 2.2.3 Determination of talarozole pharmacokinetic parameters in the mouse ................... 23 2.2.4 Talarozole protein binding ........................................................................................ 25 2.2.5 Prediction of CYP26 inhibition following single or multiple doses of talarozole .... 25 2.2.6 Quantitation of atRA in mouse serum and tissues .................................................... 26 2.2.7 Quantification of mRNA and protein changes in mouse liver and testis .................. 27 2.2.8 Immunohistochemistry of Stra8 in mouse testis ....................................................... 29 i 2.2.9 Statistical analysis ..................................................................................................... 29 2.3 Results ........................................................................................................................... 30 2.3.1 Effect of single dose talarozole on CYP26 activity and endogenous atRA concentrations ....................................................................................................................... 30 2.3.2 Effects of multiple doses of talarozole on CYP26 activity and endogenous atRA concentrations ....................................................................................................................... 32 2.3.3 Effect of CYP26 inhibition on atRA signaling in mouse liver and testis ................. 33 2.4 Discussion ..................................................................................................................... 34 Chapter 3. Investigation of clinical drug-drug interactions involving 13-cis retinoic acid and cytochrome P450s ......................................................................................................................... 46 3.1 Introduction ................................................................................................................... 47 3.2 Materials and methods .................................................................................................. 49 3.2.1 Chemicals and reagents............................................................................................. 49 3.2.2 Clinical study protocol .............................................................................................. 49 3.2.3 Quantitation of study drugs and metabolites in serum.............................................. 50 3.2.4 Pharmacokinetic analysis .......................................................................................... 53 3.2.5 Animal care and retinoid treatments ......................................................................... 54 3.2.6 Quantification of mRNA in mouse liver ................................................................... 54 3.2.7 Statistical analysis ..................................................................................................... 55 3.3 Results ........................................................................................................................... 56 3.3.1 Clinical study ............................................................................................................ 56 3.3.2 Changes in mRNA in mouse liver ...........................................................................