Using MDCK-MDR1 cells (a transfected cell line overexpressing human P-glycoprotein (multidrug resistance 1; MDR1)) to evaluate whether your compound is a substrate of the P-glycoprotein (P-gp) efflux transporter is crucial in early drug discovery/development as such knowledge feeds into both:

1. A holistic mechanistic understanding around the prediction of intestinal absorption of oral drugs gained from Caco-2 permeability screens, as well as,
   
2. Enabling a prediction of central nervous system (CNS) permeability (penetration) across the tight epithelial cells of the blood-brain barrier, since such penetration is restricted to a large extent by P-gp mediated efflux^1,2.

MDCK-MDR1 permeability is included in our portfolio of in vitro ADME drug transporter screening services. Cyprotex delivers consistent, high quality data with cost-efficiency that comes from a highly automated approach. We offer permeability screening services as well as full regulatory DDI and drug transporter packages according to FDA guidance and EMA guidance.

MDCK-MDR1 permeability assay:

• Cyprotex’s MDCK-MDR1 cells originate from the transfection of Madin Darby Canine Kidney (MDCK) cells with the human MDR1 gene (ABCB1)³, the gene encoding for the efflux transporter, human P-gp.
• Assessing flux in both directions (apical to basolateral (A-B) and basolateral to apical (B-A)) across the polarized cell monolayer enables calculation of apparent permeability, as well as determination of an efflux ratio, which provides an indicator as to whether a compound undergoes P-gp mediated active efflux. As a follow-up to any observed efflux in the MDCK-MDR1 permeability assay, confirmation of the compound as a substrate of P-gp can be established using our P-gp substrate identification assay, performed with and without a known reference P-gp inhibitor.
• Due to the formation of tight junctions and overexpression of P-gp, the polarised MDCK-MDR1 cell monolayer test system is a suitable surrogate for the epithelial cells of the blood-brain barrier, and as such the MDCK-MDR1 permeability assay can be a useful predictor of CNS permeability^4,5.

The Cyprotex MDCK-MDR1 permeability assay can be performed in a range of study formats to suit your needs, our default assay conditions are listed below. If you require more information, or are interested in a bespoke study setup, please contact our team.

MDCK-MDR1 cells are grown on a semi-permeable membrane in a Transwell™ system plate and form a confluent polarized monolayer over 4 or 5 days prior to the experiment. The test compound is added to the apical side of the cell monolayer (A-B) and the flux of the compound across the monolayer is monitored after a 60-minute incubation period. To study drug efflux, it is also necessary to investigate transport of the compound from the basolateral compartment to the apical compartment (B-A) and calculate an efflux ratio.

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

Where P_app is the apparent permeability coefficient (cm/s x10^-6), dQ/dt is the rate of permeation of the drug across the cells (pmol/sec), C₀ is the donor concentration at time zero (pmol/mL) and A is the area of the cell monolayer (cm²).

There are several ways in which the data from the MDCK-MDR1 permeability assay can be used. Firstly, an indication of P-gp mediated drug efflux of compounds can be obtained (for follow up in a P-gp substrate identification assay). Secondly, the compounds can be ranked by their A-B P_app values which can give an indication of the extent of permeation across the blood-brain barrier.

Lucifer yellow, a paracellular membrane integrity marker, is co-incubated with the test compound at the start of the experiment. At the end of the study, the extent of the lucifer yellow permeation is measured against a predetermined acceptance threshold.

The % recovery can be useful in interpreting MDCK-MDR1 data. If the recovery is very low, this may indicate problems with poor solubility, non-specific binding of the compound to the assay plates⁷ or accumulation of the compound in the cell monolayer⁸.

_{1) Alavijeh, M.S et al. (2005) Drug metabolism and pharmacokinetics, the blood-brain barrier, and central nervous system drug discovery. Neurotherapeutics 2, 554–571
2) FDA Guidance for Industry – In Vitro Drug Interaction Studies - Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions (January 2020)
3) Pastan I et al. (1988) A retrovirus carrying an MDR1 cDNA confers multidrug resistance and polarized expression of P-glycoprotein in MDCK cells. Proc Natl Acad Sci USA 85; 4486-4490
4) Wang Q et al. (2005) Evaluation of the MDR-MDCK cell line as a permeability screen for the blood–brain barrier. Int J Pharmaceut 288; 349-359
5) Jiang, L. et al. (2022) Application of a high‐resolution in vitro human MDR1‐MDCK assay and in vivo studies in preclinical species to improve prediction of CNS drug penetration. Pharmacology Research & Perspectives 2022; 10: 4–12
6) Zhou L et al (2009) The effect of breast cancer resistance protein and P-glycoprotein on the brain penetration of flavopiridol, Imatinib Mesylate (gleevec), prazosin, and 2-methoxy-3-(4-(2-(5-methyl-2-phenyloxazol-4-yl)ethoxy)phenyl)propanoic acid (PF-407288) in mice. Drug Metabolism and Disposition 37: 946–955
7) Cai X et al (2012) Approach to improve compound recovery in a high-throughput Caco-2 permeability assay supported by liquid chromatography–tandem mass spectrometry. Journal of Pharmaceutical Sciences 101: 2755–2762
8) Bednarczyk D, Sanghvi MV (2019) The impact of assay recovery on the apparent permeability, a function of lysosomal trapping. Xenobiotica 50: 753–760}