Understand the suitability of your compound for oral dosing or for use as a CNS therapeutic by using our MDCK-MDR1 permeability assay to identify intestinal or CNS permeability and to investigate drug efflux.
MDCK-MDR1 permeability is one of our portfolio of in vitro ADME screening services. Cyprotex deliver consistent, high quality data with cost-efficiency that comes from a highly automated approach.
P-gp and BCRP are not expected to impact the oral bioavailability of highly permeable and highly soluble drugs.
1Draft FDA Guidance for Industry – In Vitro Metabolism- and Transporter-mediated Drug-Drug Interaction Studies, (October 2017).
|Test Article Concentration||10 μM|
|Passage Number||6 - 30|
|Period of Cell Culture||4 days|
|Number of Replicates||2|
|Incubation Time||60 min|
|Test Article Requirements||100µL of 10mM DMSO solution|
|Integrity Marker||Lucifer Yellow|
|Control Compounds||Propranolol and prazosin|
|Analysis Method||LC-MS/MS quantification|
Cyprotex's MDCK-MDR1 permeability assay is able to identify compounds which are substrates of P-gp (see Figure 1 below) and distinguish between compounds which are CNS negative and CNS positive as shown in Table 1.
|Drug||A-B Papp (cm/s x 10-6)||Brain Uptake Classification|
Please provide an overview of the Cyprotex's MDCK-MDR1 permeability assay.
MDCK-MDR1 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)2. The cells are seeded on a multiwell-insert plate and form a confluent monolayer over 4 days prior to the experiment. On day 4, the test compound is added to the apical side of the membrane and the transport of the compound across the monolayer is monitored over a 60 min time period. To study drug efflux, it is also necessary to investigate transport of the compound from the basolateral compartment to the apical compartment and calculate an efflux ratio.
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.
How do I interpret data from the MDCK-MDR1 permeability assay?
There are several ways in which the data from the MDCK-MDR1 permeability assay can be used. Firstly, the compounds can be ranked by their Papp (apical to basolateral) values. This will give an indication of the extent of permeation across cells which express P-gp (e.g., in the gastrointestinal tract and the blood brain barrier). Secondly, an indication of drug efflux by P-gp can be determined by calculating an efflux ratio. To confirm the role of P-gp in the efflux, a reference P-gp inhibitor such as elacridar or cyclosporin A is included to inhibit active efflux (see Cyprotex's P-gp substrate identification assay).
How is the efflux ratio calculated, and how do I interpret this value?
The efflux ratio (i.e. Papp(B-A)/Papp(A-B)) is calculated by performing a bidirectional MDCK-MDR1 permeability assay where the transport of the compound is measured in the apical to basolateral direction as well as the basolateral to apical direction. If the efflux ratio is greater than or equal to 2 then this indicates drug efflux is occurring. Prazosin, a known P-gp substrate, is screened as a positive control substrate to confirm that the cells are expressing functional P-gp efflux transporter protein.
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 the lucifer yellow exceeds 0.5 x 10-6 cm/s in one well, but the derived Papp result for the test compound or control compound 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 and 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.
Two control compounds, propranolol (passive transcellular transport) and prazosin (P-gp substrate) are screened alongside the test compounds.
How and why is the % recovery calculated?
The % recovery can be useful in interpreting 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 MDCK-MDR1 test system.
What is the relationship between MDCK-MDR1 permeability and human intestinal absorption?
The relationship between Cyprotex's MDCK-MDR1 permeability and human intestinal absorption is displayed in Figure 2. There is good correlation between human intestinal absorption and MDCK-MDR1 permeability although there are only a limited number of compounds displayed in the plot. The intestinal absorption values used in this plot are taken from Zhao et al., 200112.
1Draft FDA Guidance for Industry – In Vitro Metabolism- and Transporter-mediated Drug-Drug Interaction Studies, October 2017
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10 Murakami H et al. (2000) Comparison of blood-brain barrier permeability in mice and rats using in situ brain perfusion technique. Am J Physiol Heart Circ Physiol 279; H1022-1028
11 Liu X et al. (2004) Development of a computational approach to predict blood-brain barrier permeability. Drug Metab Dispos 32; 132-139
12 Zhao YH et al. (2001) Evaluation of human intestinal absorption data and subsequent derivation of a quantitative structure-activity relationship (QSAR) with the Abraham descriptors. J Pharmaceut Sci 90; 749-784
Learn more about permeability and drug transporters in Chapter 4 of our popular Everything you need to know
about ADME guide.