Generation of Caco-2 cells with predictable metabolism by CYP3A4, UGT1A1 and CES using the PITCh system

Area of Science:

• Pharmacology
• Cell Biology
• Biotechnology

Background:

• Caco-2 cells are a standard in vitro intestinal model but have suboptimal expression of key drug-metabolizing enzymes like CYP3A4 and UGT1A1.
• The expression pattern of carboxylesterase 1 (CES1) and CES2 in Caco-2 cells is reversed compared to the human small intestine, impacting prodrug metabolism.
• Accurate prediction of intestinal drug absorption and metabolism requires an in vitro model that better reflects in vivo enzyme expression.

Purpose of the Study:

• To engineer Caco-2 cells with an expression profile more representative of the human small intestine for drug metabolism studies.
• To enhance the expression of CYP3A4, POR, UGT1A1, and CES2 while knocking out CES1 in Caco-2 cells.
• To evaluate the utility of these genome-edited Caco-2 cells as an improved in vitro model for predicting intestinal drug disposition.

Main Methods:

• Utilized a PITCh system for precise genome editing in Caco-2 cells.
• Generated Caco-2 cell lines with knock-in of CYP3A4, POR, UGT1A1, and CES2, and knock-out of CES1.
• Performed transport assays using the ester-containing prodrug temocapril to assess drug absorption and metabolism.

Main Results:

• Genome-edited Caco-2 cells successfully expressed functional CYP3A4, POR, UGT1A1, and CES2, with complete knockout of CES1 protein.
• Transport assays showed a higher apparent permeability (Papp) of temocapril in genome-edited Caco-2 cells compared to wild-type (WT) Caco-2 cells.
• The apical accumulation of temocaprilat, the active metabolite, was lower in genome-edited cells, indicating efficient intracellular processing.

Conclusions:

• Genome-edited Caco-2 cells exhibit a more physiologically relevant expression of drug-metabolizing enzymes and esterases.
• These modified cells demonstrate enhanced capacity for metabolizing ester-containing prodrugs.
• The developed genome-edited Caco-2 cell line serves as a superior in vitro model for predicting intestinal drug absorption and metabolism, particularly for prodrugs.