MDCK-MDR1 Permeability Assay

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

MDCK-MDR1 permeability assay to investigate intestinal or CNS permeability

MDCK-MDR1 cells originate from transfection of Madin Darby canine kidney (MDCK) cells with the MDR1 gene (ABCB1), the gene encoding for the efflux protein, P-glycoprotein (P-gp)².
Assessing transport in both directions (apical to basolateral (A-B) and basolateral to apical (B-A)) across the cell monolayer enables an efflux ratio to be determined which provides an indicator as to whether a compound undergoes active efflux (mediated by P‑gp).
MDCK-MDR1 helps to gain an understanding of the mechanism of drug efflux, and highlights early potential issues with drug permeability.
In addition to intestinal permeability, MDCK-MDR1 permeability has also been found to be a useful predictor of blood brain barrier permeability.

Bidirectional assays evaluate whether an investigational drug is a substrate or inhibitor of efflux transporters such as P-gp or BCRP.

¹FDA Guidance for Industry – In Vitro Drug Interaction Studies - Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions (January 2020)

Protocol

MDCK-MDR1 permeability assay protocol

Test Article Concentration	10 μM
Passage Number	6 - 30
Period of Cell Culture	4 days
Number of Replicates	2
Incubation Time	60 min
Temperature	37°C
Test Article Requirements	100µL of 10mM DMSO solution
Integrity Marker	Lucifer Yellow
Control Compounds	Propranolol and prazosin
Analysis Method	LC-MS/MS quantification
Data Delivery	P_app Efflux Ratio % Recovery

Data

Data from Cyprotex's MDCK-MDR1 permeability assay

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 P_app (cm/s x 10^-6)	Brain Uptake Classification
Atenolol	0.204	CNS Negative³
Methotrexate	0.234	CNS Negative³
Ranitidine	0.369	CNS Negative³
Vinblastine	0.521	CNS Negative³
Cimetidine	0.522	CNS Negative⁴
Sulfasalazine	0.535	CNS Negative³
Quinidine	1.49	CNS Negative³
Loperamide	1.82	CNS Negative⁵
Minoxidil	2.77	CNS Negative⁶
Flecainide	3.50	CNS Positive⁷
Fluconazole	9.50	CNS Positive⁸
Acetaminophen	17.4	CNS Positive⁹
Desipramine	31.1	CNS Positive³
Indomethacin	35.6	CNS Positive³
Warfarin	40.7	CNS Positive¹⁰
Chlorpromazine	53.4	CNS Positive³
Propranolol	63.9	CNS Positive¹¹
Carbamazepine	64.5	CNS Positive³
Antipyrine	67.7	CNS Positive³

Table 1
Classification of brain uptake using Cyprotex's MDCK-MDR1 permeability assay.
Cyprotex's MDCK-MDR1 assay distinguishes between CNS positive and CNS negative compounds based on their P_appvalues.

Figure 1
Net flux ratio for a set of 20 compounds (calculated using the efflux ratios of the wild type and MDCK-MDR1 bidirectional assays).

By performing a bidirectional study in both the wild type and MDCK-MDR1 assay, the net flux ratio can be calculated to identify compounds which are substrates of human P-glycoprotein.

Q&A

Questions and answers on MDCK-MDR1 permeability

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 (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.

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

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.

Figure 2
Relationship between Cyprotex's MDCK-MDR1 permeability (apical to basolateral) and % human intestinal absorption.

References

¹FDA Guidance for Industry – In Vitro Drug Interaction Studies - Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions (January 2020)
² 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
³ 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
⁴ Di L et al. (2003) High throughput artificial membrane permeability assay for blood–brain barrier. Eur J Med Chem 38; 223-232
⁵ Seelig A et al. (1994) A method to determine the ability of drugs to diffuse through the blood-brain barrier. Proc Natl Acad Sci USA 91; 68-72
⁶ Thomas RC et al. (1975) Metabolism of minoxidil, a new hypotensive agent I: absorption, distribution, and excretion following administration to rats, dogs, and monkeys. J Pharm Sci 64; 1360-6
⁷ Piovan D et al. (1986) Plasma and tissue levels of flecainide in rats. Pharmacol Res Commun 18; 739-745
⁸ Yang H et al. (1996) Fluconazole distribution to the brain: a crossover study in freely-moving rats using in vivo microdialysis. Pharm Res 13; 1570-5
⁹ Courad JP et al. (2001) Acetaminophen distribution in the rat central nervous system. Life Sci 69; 1455-64
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