Quintara Discovery

P-gp Assays

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.

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Assay Methodology

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)
Assay Buffer HBSS, pH 7.4, and other buffer systems are available
Plate Format 24-well or 96-well Transwell plates
Transport Period 1 or 2 hour at 37C
Detection Method LC-MS/MS
Control Substrate Digoxin
Control Inhibitor GF120918
Deliverables Papp value for A-B and B-A directions, efflux ratio
Turnaround Time 5-7 Days
Compound requirements 50 μL 10 mM DMSO stock