The effect of P-glycoprotein inhibition and its measurement in vitro
P-gp is one of the most well-recognized efflux transporters in many tissues including the intestine, brain and kidney1.
Inhibition of P-gp has shown to be responsible for several clinical drug-drug interactions. For example, clarithromycin can inhibit the transport of the P-gp substrate digoxin resulting in a clinically significant elevation of plasma exposure and a decrease in renal clearance2.
The International Transporter Consortium1, the FDA guidance 3 and the EMA guideline4 recommend investigating P-gp due to P-gp’s clinical importance in the absorption and disposition of drugs.
Cyprotex use MDCK-MDR1 cells to identify P-gp inhibitors using a range of test inhibitor concentrations in the presence of the clinically relevant probe substrate digoxin. This method conforms with the recommended methods within the International Transporter Consortium white paper1, the FDA drug interactions guidance3 and the EMA drug interactions guideline4.
In vitro inhibition studies are recommended to investigate whether the investigational drug inhibits any of the transporters known to be involved in clinically relevant in vivo drug interactions.
4The European Medicines Agency (EMA) Guideline on the Investigation of Drug Interactions (Adopted 2012)
P-glycoprotein inhibition assay protocol
5 μM [3H]-Digoxin (clinically relevant substrate)
Test Article Concentrations
Seven point IC50 (triplicate wells)
Unidirectional (basolateral to apical)
Inhibitor Preincubation Time
Liquid scintillation counting
IC50 (derived from corrected B-A Papp)
Data from Cyprotex's P-gp inhibition assay
A set of known P-gp inhibitors were investigated in Cyprotex's P-gp inhibition assay using [3H]-digoxin as substrate.
Mean IC50 ± Standard Deviation (µM, n=3)
Cyclosporin A (positive control)
0.931 ± 0.0574
8.83 ± 4.09
54.7 ± 10.3
0.284 ± 0.0452
Table 1: Inhibition of P-gp-mediated digoxin transport by literature inhibitors.
Questions and answers on P-glycoprotein inhibition
Why is it important to investigate P-gp inhibition?
P-gp is one of the most well-recognized efflux transporters. It is present in many tissues including the intestine, brain, liver and kidney. Inhibition of P-gp has shown to be responsible for several clinical drug-drug interactions1. For example, clarithromycin can inhibit the transport of the P-gp substrate, digoxin, resulting in an elevation of plasma levels and a decrease in renal clearance2.
The International Transporter Consortium1, the FDA guidance3 and the EMA guideline4 recommend investigating P-gp due to P-gp’s clinical importance in the absorption and disposition of drugs.
Please provide an overview of Cyprotex's P-gp Inhibition assay
Unidirectional (B-A) transport studies are the preferred ‘industry standard’ methodology used to identify drugs as inhibitors of P-gp and are the current recommended approach indicated by the regulatory authorities. The MDCK-MDR1 cell line is a commonly used in vitro model for investigating P-gp inhibition. The cells are seeded on a multiwell-insert plate and form a confluent monolayer over 4 days prior to the experiment. The P-gp substrate, [3H]-digoxin, is then added to the basolateral side of a confluent monolayer of the cells and permeability is measured by monitoring its appearance on the opposite side of the membrane using liquid scintillation counting. The permeability is assessed in the presence and absence of the test compound to investigate P-gp inhibition.
How is the IC50 determined from the unidirectional MDCK-MDR1 data?
The apparent permeability coefficient (Papp) of digoxin in the absence and presence of test compound is calculated from the following equation:
Where dQ/dt is the rate of permeation of the drug across the cells, C0 is the donor compartment concentration at time zero and A is the area of the cell monolayer.
The determined Papp is then converted to percentage control transport activity as follows:
% control activity = ([(Papp (+test compound) – Papp (passive)) / (Papp (vehicle control) – Papp (passive))] × 100)
where : Papp (+test compound) is the Papp of [3H]-digoxin in the presence of test compound Papp (vehicle control) is the mean Papp of [3H]-digoxin in the absence of test compound Papp (passive) is the mean Papp of [3H]-digoxin in the presence of the highest concentration of positive control inhibitor.
Percentage control transport activity values are then plotted against test compound/positive control inhibitor concentration and subsequently fitted to calculate an IC50 value (concentration which produces 50% inhibition of vehicle control transport activity) using a four parameter logistic equation.
How do I decide if a clinical study is required?
The FDA Guidance for Industry – In Vitro Drug Interaction Studies - Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions (January 2020)3 and The European Medicines Agency (EMA) Guideline on the Investigation of Drug Interactions (2012)4 recommend that investigational drugs are evaluated in vitro to determine if they are P-gp substrates or inhibitors. For P-gp inhibition, the FDA guidance recommends that a clinical drug-drug interactions trial with a P-gp substrate (for example, digoxin) should be performed if [Igut]/IC50 (or Ki) ≥ 10, where [Igut] represents the theoretical intestinal concentration (dose of the inhibitor (in mol)/250mL).
How did you decide on the incubation conditions for P-gp inhibition?
The incubation conditions have been fully characterised for our chosen P-gp substrate, digoxin, based on time linearity and chosen substrate concentration being at least ten-times lower than the reported Km.
What controls do you include in Cyprotex's P-gp Inhibition assay?
We evaluate the positive control inhibitor cyclosporin A for Cyprotex’s P-gp inhibition assay. The integrity of the monolayers throughout the experiment is checked by monitoring lucifer yellow permeation using fluorimetric analysis.
At what stage should I perform an IC50 evaluation?
As intestinal P-gp-mediated DDIs are important for the absorption of the narrow therapeutic index drug digoxin5, establishing the inhibitory potential (IC50) of your compound versus P-gp during preclinical candidate selection may assist in shortlisting based on favourable DDI risk assessment profile. Such knowledge during preclinical or early clinical development will help towards clinical trial design and therefore patient recruitment. The FDA guidance3 and the EMA guideline4 on drug interactions provides recommendations on whether a clinical drug interaction study is necessary based on the IC50 or Ki value.
1 The International Transporter Consortium (2010) Membrane transporters in drug development. Nat Rev Drug Disc9(3); 215–236 2 Wakasugi H et al. (1998) Effect of clarithromycin on renal excretion of digoxin: Interaction with P-glycoprotein. Clin Pharmacol Ther64(1); 123 -128 3 FDA Guidance for Industry – In Vitro Drug Interaction Studies - Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions (January 2020) 4 The European Medicines Agency (EMA) Guideline on the Investigation of Drug Interactions (Adopted 2012) 5 Fenner KS et al. (2009) Drug-drug interactions mediated through P-glycoprotein: clinical relevance and in vitro-in vivo correlation using digoxin as a probe drug. Clin Pharmacol Ther85(2); 173–181
Learn more about permeability and drug transporters in our popular Everything you need to know about ADME guide.