조혜진 (영남대)

김주현 (영남대학교)

조혜진 (영남대)

김민규 (영남대학교)

김주현 (영남대학교)

Chlorogenic acid (CGA) and its isomer cryptochlorogenic acid (CCGA) are widely found in natural products and known for their pharmacological effects, including antioxidant, anticancer, antiviral, anticoagulant, and antithrombotic activities. Both compounds are metabolized to caffeic acid (CA) by carboxylesterase (CES). In early drug development, rodents are commonly used for pharmacokinetic studies. However, they possess more CES isozymes and show significantly higher CES activity than humans. Among rodents, mice exhibit higher CES activity than rats. These metabolic differences can influence the PK profiles of CGA and CCGA, requiring careful interpretation when extrapolating mouse data to humans. This study aimed to determine optimal ex vivo conditions for inhibiting plasma CES activity in rodents to prevent errors in CGA plasma concentration measurements. Bis(4-nitrophenyl) phosphate (BNPP), a known CES inhibitor, was used in this study. Irinotecan, an ester-containing drug, was selected to assess CES inhibition by BNPP. A sensitive LC-MS/MS method was developed to assess the inhibitory effect of BNPP. Additionally, an accurate LC-HRMS method for CGAs and CA quantification in mouse plasma was established and validated. In interspecies comparisons, mice exhibited markedly higher CES activity for CGA, CCGA and irinotecan than rats and humans. The optimal BNPP concentration for CES inhibition in rodent plasma was evaluated using irinotecan as a model compound. The established BNPP condition is currently being applied to rodent plasma containing CGAs to evaluate its broader applicability, with further in vivo pharmacokinetic studies planned to validate its effectiveness. These results highlight significant differences in plasma CES activity between mice and humans. The variation in BNPP concentration required for CES inhibition across different compounds suggests the need for compound-specific optimization. Plasma metabolic enzyme activity differences across species were managed to enable accurate clinical pharmacokinetic interpretation, highlighting the importance of post-sampling handling.