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ADME PK

P-glycoprotein (P-gp) Substrate Identification Assay for Screening and Regulatory Reporting Purposes

Understand the potential of your compound to be a substrate of the P-gp efflux transporter using our P-gp substrate identification assay.
 
P-gp substrate identification is in our portfolio of in vitro transporter services. Cyprotex deliver consistent, high quality data for either your early stage screening projects or your later stage regulatory studies.

In vitro P-gp substrate identification

  • P‑gp (P‑glycoprotein; MDR1, ABCB1) is an important efflux transporter. It is expressed in the gastrointestinal tract, liver, kidney and brain endothelium1.
  • The ITC1, the EMA guideline2 and the draft FDA guidance3 recommend investigating P‑gp due to the clinical importance of P‑gp in the absorption and disposition of drugs.
  • Madin Darby canine kidney (MDCK) cells transfected with the human MDR1 gene overexpress P‑gp. The EMA2 and draft FDA3 regulatory guidelines recommend polarised MDCK‑MDR1 cell monolayers as one of the preferred methods for evaluating the role of P‑gp in the efflux of new chemical entities.
  • The assay investigates bidirectional transport across the cell monolayer in the presence and absence of a P‑gp reference inhibitor, elacridar (screening) or cyclosporin A (regulatory), to determine if active efflux is occurring, and whether this efflux is mediated by P‑gp.
  • Where MDCK‑MDR1 cell assays indicate a compound has inherently low passive permeability, then P‑gp membrane vesicles can be used as an alternate in vitro test system to identify P‑gp substrates (assay available upon request).
P-gp has an important role in limiting entry of various drugs into the central nervous system. In addition, it also plays a part in the intestinal absorption and in the biliary and urinary excretion of drugs.

1The International Transporter Consortium (2010) Nat Rev Drug Disc 9; 215-236

Protocol

P‑gp substrate identification assay protocol for screening  (1 concentration) or regulatory type studies (4 concentrations &  2 concentrations plus inhibitor)

Test Article Concentration Screening study - 10 μM plus/minus reference inhibitor (different concentrations available)

Regulatory study - 1, 10, 50 and 100 µM (different concentrations available) plus inhibition at two substrate concentrations (1 and 10 µM)
Assay Conditions Apical to basolateral and basolateral to apical in presence and absence of elacridar (2 µM; screening) or cyclosporin A (10 µM; regulatory)
Number of Replicates 2 (screening) or 3 (regulatory)
Incubation Time 60 min (screening) or 90 min (regulatory)
Analysis Method LC-MS/MS quantification
Integrity Marker Lucifer Yellow
Data Delivery Papp

Efflux ratio in presence and absence of reference inhibitor

% Recovery

Data

Data from Cyprotex's P-gp substrate identification assay

Functional activity of P-gp in MDCK-MDR1 polarized cell monolayers was demonstrated by investigating the inhibition of efflux of the P-gp substrate prazosin by the reference inhibitor cyclosporin. 

 
Figure 1
Graph showing effect of the P‑gp inhibitor, cyclosporin A (10 µM) on the efflux of the P‑gp substrate prazosin. Data show the mean ± standard deviation.

Q&A

Question and answers on P-gp substrate identification assay

Why is it important to identify substrates of P-gp?

P‑glycoprotein (P‑gp; ABCB1, MDR1) is an ATP-binding cassette drug efflux transporter which is apically expressed in the gastrointestinal tract, liver, kidney and brain endothelium1. Consequently, P‑gp plays an important role in the oral bioavailability, CNS distribution and biliary and renal elimination of drugs which are substrates of this transporter.
 
Interactions with respect to P‑gp have been shown to be responsible for clinical drug-drug interactions. For example, the P‑gp inhibitor AZD5672 dose-dependently inhibits the transport of the narrow therapeutic index P‑gp substrate digoxin resulting in a significant increase in plasma AUC4

The International Transporter Consortium1, the EMA guideline2 and the draft FDA guidance3 recommend investigating P‑gp due to the clinical importance of P‑gp in the absorption and disposition of drugs.

Please provide an overview of Cyprotex's P-gp substrate identification assay.

Bidirectional transport studies are one of the preferred ‘industry standard’ methodologies used to identify drugs as substrates 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 identifying P‑gp substrates. The cells are seeded on a multiwell-insert plate and form a confluent monolayer over 4 days prior to the experiment. The test article is then added to either the apical or 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 LC-MS/MS.

The permeability coefficient (Papp) is calculated from the following equation:

Where dQ/dt is the rate of permeation of the drug across the cells, C0 is the donor 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 permeability is assessed in the presence and absence of a reference P-gp inhibitor to confirm the test article is a substrate of P-gp.

How do you know if the cells have formed a confluent monolayer?

Transepithelial electrical resistance (TEER) measurement is used to determine tight-junction formation between cells. In addition, lucifer yellow, a membrane integrity marker, is co-incubated with the test compound at the start of the experiment. If the Papp of lucifer yellow exceeds 0.5 x 10-6 cm/s in one well, but the derived Papp result for the test compound or positive control substrate in that well is qualitatively similar to that determined in the remaining replicate well(s) (within the lucifer yellow threshold) then, based upon the scientific judgement of the responsible scientist, the cell monolayer may be considered acceptable. If this is not the case, then the result from the affected monolayer is excluded. For regulatory studies, if lucifer yellow Papp values are above the threshold in all replicate wells for a particular test compound concentration, then the data for that concentration is excluded with a comment that toxicity or inherent fluorescence of the test compound is assumed. No further experiments are performed. For screening studies, if one replicate well is excluded, then an n=1 result is reported, or the compound may be re-tested. If both replicates are affected then the compound is re-screened. If both lucifer yellow Papp values fail for the same compound on two separate occasions then it is assumed that the compound exhibits either cytotoxic effects against the MDCK‑MDR1 cells or inherent fluorescence.

A positive control substrate (prazosin) is evaluated alongside the test compound in the presence and absence of reference inhibitor.

How and why is the % recovery calculated?

The % recovery can be useful in interpreting the MDCK‑MDR1 data. If the recovery is very low, this may indicate problems with binding of the compound to the plate or accumulation of the compound in the cell monolayer. However, poor solubility is the most common reason for unexpected recoveries in the test system.

How do I decide if a clinical study is required?

The FDA Draft Guidance for Industry (Drug Interaction Studies – In Vitro Metabolism- and Transporter-mediated Drug-Drug Interaction Studies, October 2017)3 and The European Medicines Agency (EMA) Guideline on the Investigation of Drug Interactions (2012)2 recommend that all investigational drugs are evaluated in vitro to determine if they are P‑gp substrates or inhibitors. For P‑gp substrates, the draft FDA guidance recommends that there should be an assessment of nonclinical and clinical information to determine if an in vivo DDI study is warranted if the efflux ratio is ≥ 2 and the efflux is reduced towards unity by a P‑gp reference inhibitor.

References

1 The International Transporter Consortium (2010) Membrane transporters in drug development.  Nat Rev Drug Disc 9: 215-236
2 The European Medicines Agency (EMA) Guideline on the Investigation of Drug Interactions (Adopted 2012)
3 Draft FDA Guidance for Industry – Drug Interaction Studies – In Vitro Metabolism and Transporter-mediated Drug-Drug Interaction Studies, October 2017
4 Elsby R et al., (2011) The utility of in vitro data in making accurate predictions of human P-glycoprotein mediated drug-drug interactions: A case study for AZD5672. Drug Metab Dispos 39(2): 275-282.

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