Evaluation of stabilizing additives to protect activities of cytochrome P450 enzymes for in vitro drug testing and pharmacogenetic studies: Focus on CYP2D6

• Published in: Biochimica et biophysica acta. General subjects Vol. 1869; no. 4; p. 130770
• Main Authors: Yamoune, Sabrina, Koch, Henner, Delev, Daniel, Weber, Yvonne, Stingl, Julia Carolin
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2025
• Subjects: Biocatalysis engineering; brain; Brain - enzymology; Brain - metabolism; Brain drug metabolism; CYP in vitro stability; CYP2D6; cysteine; cytochrome P-450; Cytochrome P-450 CYP2D6 - chemistry; Cytochrome P-450 CYP2D6 - metabolism; Cytochrome P-450 Enzyme System - metabolism; dithiothreitol; Dithiothreitol - chemistry; Dithiothreitol - pharmacology; drug development; drugs; Enzyme Stability - drug effects; enzymes; Humans; liver; liver microsomes; Local drug metabolism; Microsomes, Liver - enzymology; Microsomes, Liver - metabolism; organoids; Pharmacogenomic Testing - methods; pharmacokinetics; phosphorylcholine; safety assessment; Brain drug metabolism; DTT; DMSO; SOD; EtOH; Biocatalysis engineering; EDTA; MS/MS; AUC; BSA; CYP in vitro stability; PC; CYP2D6; MeOH; PMSF; Local drug metabolism; ROS; CYP; DLPC; KPB; Cys; GSH; HPLC
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2025.130770

In vitro and ex vivo studies on drug metabolism and stability are vital for drug development and pre-clinical safety assessment. Traditional in vitro models, such as liver enzyme (S9) fractions and microsomes, often fail to account for individual variability. Personalized models, including 3D cell models and organoids, offer promising alternatives but may not fully replicate physiological processes, especially for Cytochrome P450 (CYP) families involved in extrahepatic metabolism. A major challenge in these studies is the low stability and expression of CYP enzymes. This study aimed to stabilize native CYP activity in vitro by developing an optimized buffer formulation. Initial experiments using recombinant CYP supersomes and liver microsomes identified 45 μM cysteine, 4 mM dithiothreitol (DTT), and 300 μM phosphocholine (PC) as the most effective stabilizers. The applicability of these stabilizers was subsequently confirmed in primary human brain tissue, where they enabled the successful determination of CYP2D6 activity. This highlights the stabilizing buffer's utility for enhancing CYP functionality in diverse tissue types, including the brain, which plays a critical role in cerebral detoxification and drug metabolism. These findings suggest that specific enzyme stabilization can enable comprehensive evaluations of CYP function in ex vivo tissue samples, advancing the development of organoid human tissue models and supporting drug metabolism research. •New buffer formulation preserves CYP2D6 activity in human brain samples, advancing cerebral CYP metabolism research.•CYP stabilization: Distinct profiles highlight the need for tailored buffer formulations to enhance enzyme stability.•CYP stabilization: Distinct profiles highlight the need for tailored buffer formulations to enhance enzyme stability.•Findings support human tissue models for pharmacogenetics and preclinical drug testing, reducing reliance on animal studies.