|P-gp (P-glycoprotein, MDR1/ABCB1) is an ATP-powered efflux pump known to transport structurally unrelated hydrophobic amphipathic compounds, including therapeutic drugs, peptides and lipid-like compounds.This 170 kDa transmembrane protein plays a crucial physiological role in protecting tissues from toxic xenobiotics and endogenous metabolites, and also affects the uptake and distribution of many clinically important drugs.P-gp forms a major component of the blood-brain barrier and restricts the uptake of drugs from the intestine.* A substrate of P-gp may have a limited CNS penetration, reduced oral absorption and potentially increased excretion. Therefore Pgp substrate assay is an integral part of drug discovery.
* An inhibitor of P-gp may have a significant DDI risk during clinical development due to its effects on drugs that are Pgp substrates. Determinations of IC50 and Ki may be necessary during discovery and preclinical development.
The efflux ratios are determined using Caco-2 or MDCK-MDR1 cells, by measuring the apparent permeability (Papp) values for both apical to basal (A>B) and basal to apical (B>A) transport in a bidirectional assay. Compound concentrations are determined by LC-MS/MS. Various in-house assay conditions have been developed for some specific purposes, such as evaluation of BBB penetration, ranking of oral absorption, and testing of compounds with low solubility.
P-gp Substrate: Standard Assay Conditions (Customizable)
Testing Compound Concentration
0.1, 1, 10 μM (substrate assay); 1, 10, 100 μM (inhibition assay)
HBSS, pH 7.4, and other buffer systems are available
24-well or 96-well Transwell plates
1 or 2 hour at 37C
Papp value for A-B and B-A directions, efflux ratio
50 μL 10 mM DMSO stock