P-glycoprotein Inhibition assay

P-glycoprotein inhibition assay protocol

Substrate 5 μM Loperamide (FDA recommended substrate) Test Compound Concentrations Single point or 8 point IC50 Direction Apical to Basolateral and Basolateral to Apical Incubation Time 60 min Growth Period 4 days Compound Requirements 100 μL of 10 mM solution Analysis Method LC-MS/MS quantification Integrity Marker Lucifer Yellow Data Delivery Papp and efflux ratios in presence and absence of inhibitor (single point) IC50

Interference at the level of ATP binding cassette (ABC) and other transporters is increasingly being identified as the mechanism behind clinically important drug–drug interactions3.

Data from the Cloe Select P-glycoprotein Inhibition assay

A set of known P-gp inhibitors were investigated in the Cloe Select P-gp Inhibition assay using loperamide as substrate.

Figure 1

Cloe Select P-gp Inhibition data from single point assay illustrating efflux ratio of loperamide with and without known P-gp inhibitors.

Graph shows the inhibitory effect of known P-gp inhibitors (10 μM cyclosporin A; 50 μM ketoconazole; verapamil; tacrolimus; GF120918) and a negative control (50 μM phenelzine) on the efflux of 5 μM loperamide. Error bars represent the standard deviation from 3 replicates.

 

Figure 2

Cloe Select P-gp Inhibition data from the IC₅₀ assay determined by measuring the efflux ratio of loperamide in the presence of known P-gp inhibitors.

Graph shows the inhibitory effect of known P-gp inhibitors on the efflux ratio of 5 μM loperamide. Error bars represent the standard deviation of 9 replicates.

 

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Question and answers on P-glycoprotein inhibition

P-glycoprotein (P-gp) is one of the most well recognised 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 interactions. 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 clearance¹.

Please provide an overview of the Cloe Select P-gp Inhibition assay.

Bidirectional transport studies are the preferred ‘industry standard’ methodology used to identify drugs as substrates and/or inhibitors of P-gp and are the current recommended approach indicated in the draft FDA guidelines⁴. The MDR1-MDCK cell line is a commonly used in vitro model for investigating Pgp inhibition. The cells line originates from transfection of Madin Darby canine kidney (MDCK) cells with the MDR1 gene, the gene encoding for the efflux protein, P-glycoprotein (P-gp)². The cells are seeded on a Multiscreen™ plate (Millipore, MA, USA) and form a confluent monolayer over 4 days prior to the experiment. On day 4, the FDA recommended⁴ P-glycoprotein substrate, loperamide, is added to either the apical or basolateral side of a confluent monolayer of MDR1-MDCK cells and permeability is measured by monitoring its appearance on the opposite side of the membrane using LC-MS/MS. The permeability is assessed in the presence and absence of the test compound to investigate P-glycoprotein inhibition.

How is the single point inhibition and IC₅₀ determined from the bidirectional MDR1-MDCK data?

For the single point P-gp inhibition, the apparent permeability coefficient (P_app) of loperamide 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.

An efflux ratio is calculated from the mean apical to basolateral (A-B) P_app data and basolateral to apical (B-A) P_app data.

The efflux ratios in the presence and absence of test compound are compared to assess whether the test compound is an inhibitor of P-gp.

For the IC₅₀ determination; the degree of inhibition is calculated from the following equation;

Where i and α are the percentage of loperamide transport in the presence and absence of test compound respectively.

The IC₅₀ is calculated from the degree of inhibition using the curve fitting software XLfit v4.3, model 200.

Why should I investigate P-gp inhibition?

Although many P-gp-mediated interactions are not considered clinically relevant, inhibition of P-gp is of great concern for drugs such as digoxin which have a narrow therapeutic window. It is well recognised that P-gp plays an important role in determining digoxin disposition^1,5 and even a 25% increase in exposure is viewed as clinically relevant because untoward toxicity may occur as a result of increased drug levels⁶.

The FDA Draft Guidance for Industry (Drug Interaction Studies – Study Design, Data Analysis, and Implications for Dosing and Labeling, Sept 2006)⁴ recommends that investigational drugs are evaluated in vitro to determine if they are P-gp
substrates or inhibitors. This FDA document provides decision trees for guidance on whether a clinical drug interaction study is necessary. For P-gp inhibition, the guidance recommends that a clinical drug-drug interactions trial with digoxin should be performed if the systemic inhibitor concentration [I] to inhibitory potency measure (K_i or IC₅₀) ratio is >0.1.

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, loperamide, based on time linearity and substrate concentration kinetics. An incubation time of 60 minutes was chosen as this was within the linear phase and in accordance with MDR1-MDCK studies in the literature. The K_m for loperamide transport was determined to be approximately 36 μM. Therefore a loperamide concentration of 5 μM was chosen as this was below the K_m and within the concentration range recommended in the draft FDA guidelines⁴.

What controls do you include in the Cloe Select P-gp inhibition assay?

We typically evaluate two positive control inhibitors for the Cloe Select P-gp inhibition assay: cyclosporin A and ketoconazole. Additional controls can be included if required. 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?

Establishing the inhibition of P-gp at a single concentration is more appropriate as an initial screening assay. If inhibition is observed in the single point point assay and the compound is considered important as a potential drug candidate then a more detailed analysis (eg IC₅₀ or K_i determination) is necessary. The decision tree in the FDA draft guidance⁴ provides recommendations on whether a clinical drug interaction study is necessary based on the IC₅₀ or K_i value.

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