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ADME PK

P-glycoprotein Inhibition assay

Understand the potential drug-drug interaction liabilities of your compound by using our P-glycoprotein (P-gp) inhibition (IC50) assay.

P-gp inhibition is one of our portfolio of in vitro experimental transporter services. Cyprotex deliver consistent, high quality data for either your preclinical development candidate selection stage project or your later stage regulatory studies.

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 draft 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 draft 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)

Protocol

P-glycoprotein inhibition assay protocol

Substrate 5 μM [3H]-Digoxin (clinically relevant substrate)
Test Article Concentrations Seven point IC50 (triplicate wells)
Direction Unidirectional (basolateral to apical)
Inhibitor Preincubation Time 30 min
Incubation Time 90 min
Growth Period 4 days
Analysis Method Liquid scintillation counting
Integrity Marker Lucifer Yellow
Data Delivery IC50 (derived from corrected B-A Papp)

Data

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.

 
InhibitorMean IC50 ± Standard Deviation (n=3)
Cyclosporin A (positive control) 0.931 ± 0.0574
Ketoconazole 8.83 ± 4.09
Verapamil 54.7 ± 10.3
Elacridar 0.284 ± 0.0452
Table 1: Inhibition of P-gp-mediated digoxin transport by literature inhibitors.

Q&A

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 draft 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 Multiscreen™ plate (Millipore, MA, USA) 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 Draft Guidance for Industry (In vitro metabolism- and transporter-mediated drug-drug interaction studies, 2017)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 draft 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 draft 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.

References

1 The International Transporter Consortium (2010) Membrane transporters in drug development. Nat Rev Drug Disc 9(3); 215–236
2 Wakasugi H et al. (1998) Effect of clarithromycin on renal excretion of digoxin: Interaction with P-glycoprotein. Clin Pharmacol Ther 64(1); 123 -128
3 Draft FDA Guidance for Industry – In vitro metabolism- and transporter-mediated drug-drug interaction studies (2017)
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 Ther 85(2); 173–181

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