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

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

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.

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 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 (P_app) 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) P_app data and basolateral to apical (B-A) P_app 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 P_app of lucifer yellow exceeds 0.5 x 10^-6 cm/s in one well, but the derived P_app 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 P_app 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 P_app 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 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)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.

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 FDA Guidance for Industry – In Vitro Drug Interaction Studies - Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions (January 2020)
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.