Hyunyoung Lee, Hyun-Ji Kim, So-Young Park, Kwang-Hyeon Liu. DTT 2023;2:88-94. https://doi.org/10.58502/DTT.23.0010

Background/Objectives: Although donepezil, a reversible acetylcholinesterase inhibitor, has been in use since 1996, its metabolic characteristics remain poorly characterized. Therefore, this study aims to investigate the in vivo metabolism of donepezil using liquid chromatography–high-resolution mass spectrometry (LC-HRMS) based on a molecular networking (MN) approach integrated with a non-targeted metabolomics approach. Methods: After the oral administration of donepezil (30 mg/kg) in rats, urine, feces, and liver samples were collected for LC-HRMS analysis. Chromatographic and spectrometric data were processed through MN and multivariate data analysis to identify the in vivo metabolites of donepezil. Results: A total of 50 metabolites were characterized, including 23 newly identified metabolites. Donepezil was biotransformed by O-demethylation, N-debenzylation, and hydroxylation, and these metabolites are further conjugated with glucuronic acid and sulfurous acid. N-Desbenzyldonepezil (M4), didesmethyldonepezil (M5), and N-desbenzyldonepezil (M4) were identified as the most abundant metabolites in urine, feces, and liver samples, respectively. Conclusions: The metabolic characteristics of donepezil in rats were comparable to those in humans, indicating that a rat is a reliable model for studying donepezil metabolism. This study indicates that a MN approach combined with a metabolomics approach is a reliable tool to identify unknown metabolites of drugs and drug candidates.

Zhuoning Liang, Do-Hyeon Gwon, Bae-Gon Kang, Kwang-Hyeon Liu, So-Young Park. DTT 2025;4:108-14. https://doi.org/10.58502/DTT.25.0005

Background: Ritonavir (RTV), an HIV-1 (human immunodeficiency virus-1) protease inhibitor, has low aqueous solubility and poor oral bioavailability, requiring large amounts of excipients that result in bulky dosage forms and poor patient compliance. We aimed to develop an RTV-loaded polymeric micelle formulation with high drug loading capacity that improves aqueous solubility, reduces the excipient burden and associated excipient-related adverse effects, and remains stable after lyophilization without a cryoprotectant. Methods: RTV-loaded micelles were prepared from the amphiphilic block copolymer poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-b-PCL) by thin-film hydration. Particle size and the polydispersity index (PDI) were measured by dynamic light scattering and encapsulation efficiency and drug content by HPLC, before and after lyophilization. The drug-to-excipient ratio was compared with the commercial product (Norvir^®), and pharmacokinetics were evaluated in male Sprague-Dawley rats after single oral administration. Results: Micelles showed a mean particle size of 25.9 ± 0.09 nm, a PDI of 0.224 ± 0.008, and an encapsulation efficiency of 98.9 ± 1.54%, with particle size and drug content retained after lyophilization and reconstitution without any cryoprotectant. Drug content reached 27.3% (w/w) versus 12.5% for the commercial product, a 2.6-fold reduction in excipients per unit dose. In rats, the micelle formulation showed pharmacokinetic parameters comparable to those of the Norvir^® suspension (C_max, 2243.9 ± 1226.4 vs. 2106.1 ± 835.9 ng/mL; AUC_last, 6695.3 ± 2162.6 vs. 8178.2 ± 2892.1 h·ng/mL; T_max, 0.9 ± 0.6 vs. 1.5 ± 0.5 h), with no significant differences. Conclusions: The PEG-b-PCL micelle achieved high RTV loading, substantially reduced excipients, and provided oral exposure comparable to the commercial product, supporting its potential as an alternative oral delivery system.

Perfluorooctanesulfonate (PFOS) causes neurodevelopmental toxicity in zebrafish embryos; however, the temporally dynamic biomolecular mechanisms across exposure windows remain unclear. This study explored the biomolecular mechanisms underlying PFOS-induced neurodevelopmental toxicity using a methyl tert-butyl ether (MTBE)-based extraction method combined with a multi-omics approach to identify early biomarkers by profiling metabolomic and lipidomic perturbations of zebrafish larvae exposed to 5 μM PFOS in single biological samples collected at 72, 96, and 120 h post-fertilization (hpf). We first showed that MTBE-based extraction enhanced analytical resolution—yielding a greater number of dysregulated metabolites—compared with methanol-based extraction. The multi-omics analysis revealed that PFOS causes neurodevelopmental toxicity mainly by disrupting energy metabolism. Based on the metabolite and lipid profiles across all developmental stages, we propose biomolecular fingerprints of early PFOS-induced neurodevelopmental toxicity, including l-DOPA, isocitric acid, l-citrulline, sulfates, acetyl-l-carnitine, arachidonic acid, eicosenoic acid, and eicosadienoic acid. Interestingly, the levels of these biomarkers commonly increased at 72 hpf, suggesting that this is a relevant time point for biomarker detection. Our findings demonstrate that time-resolved multi-omics analysis is useful for identifying decisive early biomarkers, which may further refine our mechanistic understanding of PFOS-induced neurodevelopmental toxicity.