483. TOXICITY AND METABOLISM OF STRUCTURALLY SIMILAR FENTANYL ANALOGS WITH THE INTEGRATED APPROACH USING <i>IN VITRO</i> EXPERIMENTS AND <i>IN SILICO</i> PREDICTIONS

Abstract Background Opioid addiction and overdosing toxicity caused by fentanyl analogs (FAs) are a serious problem worldwide. Various structurally modified FAs, including positional isomers, have been designed and synthesized to evade legal restrictions. Considering these circumstances, it is deemed important to establish the methods 1) quickly predicting the risk of FAs by understanding their metabolic fate and possible toxicity and 2) identifying the metabolites to prove their intake. Aims &amp; Objectives This study tried to clarify the metabolism and toxicity of structurally similar FAs and to find the possible marker compounds to prove their intake. Method As FAs, we used 2-Methyl-N-phenyl-N-[1-(2-phenylethyl)-4-piperidinyl] propanamide (IBF) and its halogenated derivatives, N-(2-fluorophenyl)-2-methyl-N-[1-(2-phenylethyl)piperidin-4-yl] propanamide (2F-IBF) and N-(4-chlorophenyl)-2-methyl-N-[1-(2-phenylethyl)piperidin-4-yl] propanamide (4Cl-IBF). In vitro metabolism in human liver microsomes was done using human liver microsomes (HLMs). IBFs were incubated with HLMs for up to 4 hours. Samples were collected at the designated time points and were analyzed using LCMS-IT-TOF and LC-MS/MS. The in silico metabolism was predicted by GLORYx and the in silico neurotoxicity and blood-brain barrier (BBB) permeability was predicted by ProTox 3.0. Results The in vitro metabolic half-lives of IBF, 2F-IBF, and 4Cl-IBF were 210, 99, and 145 min, respectively. In all IBFs, N-dealkylated, monohydroxylated, and N-oxidized metabolites were identified as major metabolites. The obtained in vitro metabolites of IBFs were mostly similar to those predicted by in silico prediction by GLORYx. The in silico toxicity prediction revealed that both parent IBFs and their metabolites might show neurotoxicity and that parent IBFs relatively highly penetrate brain through blood-brain barrier (BBB) rather than their metabolites. Discussion &amp; Conclusions The relatively longer in vitro metabolic half-lives of IBFs, compared to other FAs, suggest that parent IBFs might show more significant effects than other FAs. Alternatively, parent IBFs, especially IBF, would be available to confirm their intake rather than other FAs. The results of in vitro metabolism demonstrated that all IBFs share the same metabolic pathway. Moreover, three major metabolites of IBFs would be markers to differentially prove the intake of each IBF since they possess the unique structure of each IBF. In silico predictions by GLORYx suggest the usefulness of the software in predicting the metabolic behavior of FAs. In silico toxicity predictions by ProTox 3.0 suggest the relatively potent neurotoxicity of parent IBFs rather than their metabolites since parent IBFs might possess potent neurotoxicity with relatively higher BBB permeability. In conclusion, these results suggest that the expansion of this approach to other structurally similar FAs including newly synthesized FAs might contribute to the quick introduction of the pharmacokinetic and toxicological properties to the public, subsequently preventing the abuse of FAs together with quick legal restriction. This work was supported by JSPS KAKENHI Grant Number JP24K13548.