Prediction of Drug Interactions with Methadone, Buprenorphine and Oxycodone from in vitro Inhibition of Metabolism: A Continuation

As submitted by the applicant: A near epidemic of opioid-related deaths arose over the past decade. Drug interactions that increase the concentration of active opioid are a potential mitigating factor to these mortalities. This danger is enhanced with time-dependent inhibitors (TDI), where cessation of inhibition requires rejuvenation of the inhibited enzyme. This potentially causal factor must be considered in the interpretation of forensic toxicology cases. Methadone, buprenorphine and oxycodone are three commonly used and abused opioids. While some potential for interaction may be intuited from cytochrome P450 (CYP)-specific metabolic pathways and the often-limited clinical studies with these opioids, the potential of many drug groups, in particular their potential for TDI remains unexplored. Having sensitive liquid chromatographic-tandem mass spectrometric (LC-MS/MS) assays to study the in vitro metabolism of methadone, buprenorphine and oxycodone, we proposed four years ago to test the hypothesis that in vitro inhibition of opioid metabolism can predict potential drug interactions. At first we hoped to simply screen a list of 100 potential inhibitors and characterize then as either reversible, and determine 50% inhibitory concentration (IC50) values, or irreversible, and determine the concentration required for half-maximal rate of inactivation at saturation (KI) and maximal rate of inactivation at saturation (kinact). We soon found we would also need to further optimize incubation conditions, substantiate positive controls, and increase replicates. These have been accomplished along with characterization of inhibition by the H2-receptor antagonists, proton pump inhibitors and azole antifungal agents. These studies also revealed previously unsuspected mechanisms of TDI for an H2-receptor antagonists and a number of proton pump inhibitors. To further test this hypothesis in a manner that permits exploration of mechanisms of inhibition, we now propose a continuation of these studies to: 1. Screen potential inhibitors in human liver microsomes (HLM) ± preincubation. 2. For inhibitory compounds in HLM, determine IC50 values for inhibition of specific CYPs. 3. For compounds that display TDI, dual-incubation experiments will be performed with indicated CYPs to determine TDI kinetics for KI and kinact. 4. Follow-up experiments will be conducted to clarify mechanism of TDI. The results of these findings will be disseminated through presentations at the annual meeting of the Society of Forensic Toxicologists and through publication in peer-reviewed journals. In this manner we propose to add to the knowledge base concerning the basic science of drug interaction potentials for three highly used opioids. The PI and his colleagues are aptly suited to carry out these studies. This project contains a research and/or development component, as defined in applicable law. ca/ncf