Compound profiling for P-glycoprotein at the blood-brain barrier using a microplate screening system

Area of Science:

• Pharmacology
• Neuroscience
• Biochemistry

Background:

• P-glycoprotein (P-gp) is a key efflux transporter at the blood-brain barrier.
• Understanding drug interactions with P-gp is crucial for predicting drug efficacy and CNS penetration.
• Existing methods for P-gp interaction screening can be time-consuming and complex.

Purpose of the Study:

• To develop a rapid, fluorescent dye-based assay for screening compound interactions with P-gp.
• To evaluate the utility of this assay for kinetic analysis of P-gp transport.
• To utilize porcine brain capillary endothelial cells (PBCECs) for P-gp studies.

Main Methods:

• Isolated PBCECs were cultured in 96-well plates.
• Cells were incubated with calcein-acetoxymethylester (calcein-AM) in the presence of various ABC transporter substrates and inhibitors.
• Intracellular fluorescence was measured using a fluorescence plate reader to assess calcein-AM efflux.

Main Results:

• PBCECs demonstrated stable P-gp expression, leading to calcein-AM extrusion.
• P-gp substrates and inhibitors significantly increased intracellular fluorescence, indicating reduced calcein-AM efflux.
• The observed increase in fluorescence correlated with the P-gp affinity of the tested compounds.
• Kinetic analysis revealed competitive, mixed, and non-competitive inhibition patterns.

Conclusions:

• The calcein-AM assay using PBCECs is a viable microplate screening system for drug-P-gp interactions at the blood-brain barrier.
• This assay serves as a valuable tool for drug profiling and understanding drug interactions.
• The assay facilitates convenient kinetic studies to elucidate the mode of P-gp inhibition.