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ADME PK

P-glycoprotein Inhibition assay

Understand the potential drug-drug interaction liabilities of your compound by using our P-gp inhibition assay.

P-glycoprotein (P-gp) inhibition is one of our portfolio of in vitro experimental ADME services. Cyprotex deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.

The effect of P-glycoprotein inhibition and its measurement in vitro

  • P-glycoprotein is one of the most well-recognized efflux transporters in many tissues including the intestine, brain 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 clearance2.
  • MDR1-MDCK cells originate from transfection of Madin Darby canine kidney (MDCK) cells with the MDR1 gene, the gene encoding for the efflux protein, P-glycoprotein (P-gp)3.
  • The MDR1-MDCK cell line is a recommended in vitro model for determining the extent of P-gp inhibition of a compound.
  • Cyprotex's P-gp Inhibition assay meets criteria set in the FDA guidelines.
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.

1The European Medicines Agency (EMA) Guideline on the Investigation of Drug Interactions (Adopted 2012)

Protocol

P-glycoprotein inhibition assay protocol

Substrate 5 μM Loperamide (FDA recommended substrate)
Test Article Concentrations Single point or 8 point IC50
Direction Apical to Basolateral and Basolateral to Apical
Incubation Time 60 min
Growth Period 4 days
Test Article 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

Data

Data from Cyprotex's P-glycoprotein inhibition assay

A set of known P-gp inhibitors were investigated in Cyprotex's P-gp Inhibition assay using loperamide as substrate.

 
Figure 1
Cyprotex's 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
Cyprotex's 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.

Q&A

Questions and answers on P-glycoprotein inhibition

Please provide an overview of Cyprotex's 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)3. 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.

Why is it important to investigate P-gp inhibition?

P-glycoprotein (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 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 clearance2.

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 (Papp) 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) Papp data and basolateral to apical (B-A) Papp 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 Papp values for loperamide 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-recognized that P-gp plays an important role in determining digoxin disposition2,5 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, Implications for Dosing, and Labeling Recommendations, Sept 2012)4 and the European Medicines Agency (EMA) Guideline on the Investigation of Drug Interactions (Adopted 2012)1 recommend that investigational drugs are evaluated in vitro to determine if they are P-gp substrates or inhibitors. The draft 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 a P-gp substrate (for example, digoxin) should be performed if [I]1/IC50 (or Ki)≥ 0.1 or the [I]2/IC50 (or Ki) ≥ 10 where [I]1 represents the mean steady state total (free and bound) Cmax following administration of the highest proposed clinical dose and [I]2 represents the dose of the inhibitor (in mol)/250mL (if IC50 is molar unit).

How did you decide on the incubation conditions for P-gp inhibition?

The incubation conditions have been fully characterized 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 Km for loperamide transport was determined to be approximately 36 μM. Therefore a loperamide concentration of 5 μM was chosen as this was below the Km.

What controls do you include in Cyprotex's P-gp inhibition assay?

We typically evaluate two positive control inhibitors for the Cyprotex's 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 Ki 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 Ki value.

References

1 The European Medicines Agency (EMA) Guideline on the Investigation of Drug Interactions (Adopted 2012)
2 Wakasugi H et al. (1998) Clin Pharmacol Ther 64; 123 -128
3 Pastan I et al. (1988) Proc Natl Acad Sci USA 85; 4486-4490
4 Draft FDA Guidance for Industry. Drug Interaction Studies – Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations (2012)
5 Fromm MF et al. (1999) Circulation 99; 552-557
6 Fenner KS et al. (2009) Clin Pharmacol Ther 85(2); 173–181

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