LC–MS/MS-Based Quantitative Investigation of the Exposure Characteristics of Psilocin, the Active Metabolite of Psilocybin, in Rats

Background: Psilocybin has shown promising antidepressant effects; however, pharmacokinetic data on psilocybin and the associated active metabolite, psilocin, remain limited. Thus, this study aimed to develop and validate a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for quantifying psilocin and to investigate the associated pharmacokinetics, tissue distribution, excretory characteristics in rats, and transmembrane transport properties. Methods: An LC–MS/MS method for determining psilocin in rat plasma was developed and validated. Male Sprague-Dawley (SD) rats received intravenous (i.v.) psilocybin (1 mg/kg) or oral psilocybin at low, medium, and high doses (1, 3, and 10 mg/kg, respectively). Plasma psilocin concentrations were measured serially. The medium-dose group (3 mg/kg psilocybin) included both male and female rats to evaluate sex-related differences. In male rats administered 3 mg/kg psilocybin orally, tissue samples were collected at various time points to quantify psilocin concentrations across numerous tissues. Following oral administration of 10 mg/kg psilocybin to male rats, excretory samples were collected over time to characterize the excretion profile of psilocin. Additionally, transmembrane transport was assessed using Caco-2 cells and LLC-PK1 cells overexpressing human MDR1 (hMDR1). Results: The bioanalytical method employed met all validation criteria. Following intravenous administration, psilocin demonstrated a clearance of 4.4 ± 0.4 L/h/kg and an volume of distribution at steady state of 1.5 ± 0.1 L/kg. Oral administration of psilocybin resulted in nonlinear pharmacokinetics; dose-normalized psilocin area under the concentration–time curve (AUC) increased by 63% (medium dose) and 102% (high dose) compared to the low-dose group. No statistically significant differences in pharmacokinetic parameters were observed between male and female subjects. After oral administration of the medium dose, psilocin was extensively distributed to the brain and major tissues, with tissue-to-plasma partition coefficient (Kp) values exceeding 1 in all cases (excluding fat). Psilocin exhibited moderate permeability in vitro (Papp = 12.39 ± 1.31 × 10-6 cm/s). In P-glycoprotein (P-gp)-expressing monolayer cell models, the efflux ratio was 1.26, which is below the threshold of 2, indicating that psilocin is not a P-gp substrate. Conclusions: Psilocybin is rapidly metabolized to psilocin in vivo. The pharmacokinetic profile of psilocin is characterized by rapid clearance, extensive tissue distribution, nonlinear kinetics, and low cumulative recovery. The favorable membrane permeability and P-gp-mediated efflux resistance of psilocin likely contribute to efficient penetration of the blood–brain barrier and broad tissue distribution.