The author(s) shown below used Federal funds provided by the U.S. Department of Justice and prepared the following final report: Document Title: Prediction of Drug Interactions with Methadone, Buprenorphine and Oxycodone from In Vitro Inhibition of Metabolism Author(s): David E. Moody, Ph.D. Document No.: 250127 Date Received: July 2016 Award Number: 2011-DN-BX-K532 This report has not been published by the U.S. Department of Justice. To provide better customer service, NCJRS has made this federally funded grant report available electronically. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. February 2016 Research and Development in Forensic Toxicology Prediction of drug interactions with methadone, buprenorphine and oxycodone from in vitro inhibition of metabolism Final Technical Report Submitted electronically to: U.S. Department of Justice Office of Justice Programs National Institute of Justice Prepared by: David E. Moody. Ph.D. Director of the Center for Human Toxicology, and Research Professor of Pharmacology and Toxicology [email protected] / 801-581-5117 NIJ Award Number: 2011-DN-BX-K532 Reporting Period: January 1, 2012 to December 31, 2015 Recipient Organization: University of Utah 75 South 2000 East, Room 222 Salt Lake City, UT 84112-8930 Organizational DUNS: 0090953650000 EIN: 876000525 This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Table of Contents Cover Page …………………………………………………………………………………… 1 Table of Contents ……………………………………………………………………..……… 2 Abstract ……………………………………………………………………………….……… 6 Executive Summary ………………………………………………………………..………… 7 ES 1.0 Introduction ………………………………………………………………………. 7 ES 2.0 Major Goals / Aims …………………………………………………………….… 7 ES 3.0 Accomplishments Under Goals …………………………………………………… 8 ES 3.1 Background …………………………………………………………… 8 ES 3.2 Accomplishments under Aim 1.A - Optimization of Incubation Conditions: HLM …………………………………………………….. 10 ES 3.3 Accomplishments under Aims 2 and 3: HLM Screens, IC50 Determinations and Extrapolations to In Vivo Potency ……………… 10 ES 3.4 Accomplishments under Aims 1B and 4 - Studies on TDI Kinetic Determinations and Follow Up experiments …………………...……. 11 ES 4.0 Conclusions …………………………………………………………………..….. 11 ES 4.1 Summary of Accomplishments …………………………………..….. 11 ES 4.2 Implications for Policy and Practice …………………………………. 12 ES 4.2.1 What is the impact of the project on the criminal justice system? …………………………………………………… 12 ES 4.2.2 How has it contributed to crime laboratories? ………...…. 12 ES 4.3 Implications for Further Research ……………………………………. 12 Technical Report ……………………………………………………………….……………. 13 1.0 Introduction ………………………………………………………………………….. 13 2.0 Major Goals / Aims …………………………………………………………..…….... 15 3.0 Accomplishments Under Goals ……………………………………………………… 16 3.1 Background …………………………………………………………………….......... 16 3.2 Accomplishments under Aim 1.A - Optimization of Incubation Conditions: HLM.... 19 3.2.1 Initial Studies with Oxycodone and Methadone ……………………………… 19 3.2.2 (Re)Optimization of Assays with 20-µM substrate ……………………………….. 20 3.2.3 Background Formation With Use of 20 µM Substrates …………………………. 21 3.2.4 Negative Controls ± Solvent ……………………………………………………………..….. 22 3.2.5 Positive Controls for TDI …………………………………………………………………..… 23 3.3 Accomplishments under Aims 2 and 3: HLM Screens, IC50 Determinations and Extrapolations to In Vivo Potency ………………………………………………..... 23 3.3.1 Methadone and Oxycodone Inhibition by H2-Receptor Antagonists and Proton Pump Inhibitors with Substrate at 2.0-µM ……………………....…. 23 2 This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. 3.3.2 Buprenorphine, Methadone and Oxycodone Inhibition by Azole Antifungal Agents with Substrate at 20-µM ……………………………………….…. 27 3.4 Accomplishments under Aims 1B and 4 - Studies on TDI Kinetic Determinations and Follow Up experiments ………………………………………………………… 33 3.4.1 TDI Positive Controls ………………………………………………………… 33 3.4.2 Determining TDI kinetics for Cimetidine and Different CYP-mediated Pathways ………………………………………………………………….…. 36 3.4.3 TDI and PPI ………………………………………………………………..… 38 3.4.4 Establishing MBI …………………………………………………………….. 41 4.0 Conclusions ……………………………………………………………………..…… 42 4.1 Summary of Accomplishments …………………………………………….… 42 4.2 Implications for Policy and Practice ……………………………………….… 42 4.2.1 What is the impact of the project on the criminal justice system? …... 42 4.2.2 How has it contributed to crime laboratories? ……………………..... 43 4.3 Implications for Further Research …………………………………………… 43 5.0 References ………………………………………………………………………….... 43 6.0 Training and Professional Development Opportunities ……………………….…….. 48 7.0 Dissemination of Results ……………………..……………………..……………..… 49 8.0 Glossary of Abbreviations …………………………………………………………… 50 List of Tables Table 1. Drugs to test for inhibition potential ……………………..…………………..….. 17 Table 2. Optimized Incubation Conditions for Buprenorphine, Methadone and Oxycodone Metabolism Assays ……………………..……………………...…… 21 Table 3. Approximate IC50 Determinations for H2-Receptor Antagonists and Proton Pump Inhibitors Incubated with HLM ± Pre-Incubation ………………………... 24 Table 4. IC50s and Estimates of Ki and In Vivo Inhibitory Potential at Average and 10X Average Plasma Concentrations of Inhibitor ……………………..……………… 25 Table 5. Examples of the Impact of Plasma and Microsome Drug Binding on Calculation of Inhibitory Potential: Oxycodone Metabolism by CYP3A4 …………………... 26 Table 6. Approximate IC50 Determinations for Azole Antifungals Incubated with HLM ± Pre-Incubation ……………………..……………………..…………………….. 28 Table 7. In Vitro Reversible Inhibition of CYP-Mediated Metabolism of Methadone, Buprenorphine and Oxycodone by Azole Antifungal Agents …………………… 30 Table 8. Extrapolation of IC50 Determinations to Ki and Inhibitory Potential at Average and 10 x Average Plasma Concentration of Azole Inhibitors ……………..…….. 32 3 This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Table 9. IC50 Determination for Proton Pump Inhibitors (PPIs) and Their Metabolites on Buprenorphine Metabolism: Effect of Pre-incubation with Inhibitor …….….. 40 List of Figures Figure 1. Metabolic routes under study with primary cytochrome P450 (CYP450) involved in metabolism indicated ……………………..……………………...… 18 Figure 2. Hypothetical effect of reversible versus irreversible inhibitors on the plasma concentration of a target drug during a pharmacokinetic drug interaction ……... 19 Figure 3. Initial optimization of oxycodone metabolism in HLM ………………………… 20 Figure 4. Buprenorphine utilization and norbuprenorphine formation in HLM, CYP3A4 and CYP2C8 incubated with 20-µM buprenorphine ……………………………. 21 Figure 5. Amount of metabolite detected in HLM that were incubated for 0-time with no inhibitors added (i.e., background samples) ……………………………………. 22 Figure 6. Effect of addition of 1% DMSO and pre-incubation on the activity of the negative controls to which no inhibitor is added ………………………………... 22 Figure 7. Effect of pre-incubation on inhibition by the positive controls 5 µM troleandromycin (Trol), 5 µM gemfibrozil glucuronide (Gem), 0.5 µM paroxetine (Parox), and 10 µM ThioTEPA …………………………………...… 23 Figure 8. Plot of upper concentration of inhibitor used to not exceed 1% DMSO in incubation versus log of the octanol-water partition coefficient (X Log P3) ….... 28 Figure 9. Inhibition of the CYP450 mediated metabolism using the highest achievable concentration for non-CYP3A4 pathways, and highest concentration used in IC50 determinations for CYP3A4 pathways ……………………………………... 29 Figure 10. Ranking of IC50 determinations for CYP3A4 mediated pathways …………….... 31 . Figure 11. Determination of kinetics for MBI of CYP3A4 metabolism of buprenorphine by troleandomycin (TAO) …………………………………………………….… 34 Figure 12. Determination of kinetics for MBI by TAO of CYP3A4 metabolism of oxycodone to noroxycodone, racemic methadone to R-EDDP, and racemic methadone to S-EDDP ……………………..……………………..…………..… 35 Figure 13. Determination of kinetics for MBI by gemfibrozil glucuronide of CYP2C8 metabolism of buprenorphine …………………………………………….…….. 36 4 This document is a research report submitted to the U.S. Department of Justice. This report has not been published by the Department. Opinions or points of view expressed are those of the author(s) and do not necessarily reflect the official position or policies of the U.S. Department of Justice. Figure 14. Inhibition of buprenorphine (20-µM) metabolism in HLM by the H2-receptor antagonists ± 15 minute pre-incubation ……………………..………………….. 36 Figure 15. Inhibition of 20-µM oxycodone and methadone metabolism ± 15 minute pre-incubation with cimetidine ………………………………………………….. 37 Figure 16. Kobs versus inhibitor concentration nonlinear regression plots for TDI by cimetidine ……………………..……………………..………………………. 37 Figure