In vitro and in vivo metabolism of psilocybin’s active metabolite psilocin

Abstract

<i>In vivo</i>, psilocybin is rapidly dephosphorylated to psilocin which induces psychedelic effects by interacting with the 5-HT_2A receptor. Psilocin primarily undergoes glucuronidation or conversion to 4-hydroxyindole-3-acetic acid (4-HIAA). Herein, we investigated psilocybin's metabolic pathways <i>in vitro</i> and <i>in vivo</i>, conducting a thorough analysis of the enzymes involved. Metabolism studies were performed using human liver microsomes (HLM), cytochrome P450 (CYP) enzymes, monoamine oxidase (MAO), and UDP-glucuronosyltransferase (UGT). <i>In vivo</i>, metabolism was examined using male C57BL/6J mice and human plasma samples. Approximately 29% of psilocin was metabolized by HLM, while recombinant CYP2D6 and CYP3A4 enzymes metabolized nearly 100% and 40% of psilocin, respectively. Notably, 4-HIAA and 4-hydroxytryptophol (4-HTP) were detected with HLM but not with recombinant CYPs. MAO-A transformed psilocin into minimal amounts of 4-HIAA and 4-HTP. 4-HTP was only present <i>in vitro</i>. Neither 4-HIAA nor 4-HTP showed relevant interactions at assessed 5-HT receptors. In contrast to <i>in vivo</i> data, UGT1A10 did not extensively metabolize psilocin <i>in vitro</i>. Furthermore, two putative metabolites were observed. <i>N</i>-methyl-4-hydroxytryptamine (norpsilocin) was identified <i>in vitro</i> (CYP2D6) and in mice, while an oxidized metabolite was detected <i>in vitro</i> (CYP2D6) and in humans. However, the CYP2D6 genotype did not influence psilocin plasma concentrations in the investigated study population. In conclusion, MAO-A, CYP2D6, and CYP3A4 are involved in psilocin's metabolism. The discovery of putative norpsilocin in mice and oxidized psilocin in humans further unravels psilocin's metabolism. Despite limitations in replicating phase II metabolism <i>in vitro</i>, these findings hold significance for studying drug-drug interactions and advancing research on psilocybin as a therapeutic agent.