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ADME PK

MDR1-MDCK Permeability Assay (P-glycoprotein substrate identification) for Screening and Regulatory Reporting Purposes

Understand the suitability of your compound for oral dosing or for use as a CNS therapeutic by using our MDR1-MDCK permeability assay to identify intestinal or CNS permeability, characterize P-gp substrates or investigate P-gp efflux.

MDR1-MDCK 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.

Use of MDR1-MDCK permeability in the identification of P-gp substrates

  • MDR1-MDCK 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 MDR1-MDCK permeability assay is a valuable tool for the identification and characterization of P-gp substrates and inhibitors.
  • Using MDR1-MDCK cells avoids the complexities of multiple transporters by focusing specifically on P-gp, one of the most well-recognized efflux transporters in many tissues including the brain, kidney and intestine.
  • MDR1-MDCK helps to gain a greater understanding of the mechanism of drug efflux, and highlights early potential issues with drug permeability.
  • MDR1-MDCK has been found to be a useful predictor of blood brain barrier permeability.
All investigational drugs should be evaluated in vitro to determine whether they are a potential substrate of P-gp or BCRP.

1FDA Draft Guidance for Industry - Drug Interaction Studies - Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations (February 2012).

Protocol

MDR1-MDCK permeability assay protocol for screening (1 concentration) or regulatory type studies (4 concentrations & single concentration plus inhibitor)

Test Article Concentration Screening study - 10 μM (different concentrations available)
Regulatory study - 1, 10, 50 and 100 µM (different concentrations available) plus inhibition at a single substrate concentration
Direction Apical to Basolateral and/or Basolateral to Apical
Number of Replicates 2
Incubation Time 60 min
Growth Period 4 days
Analysis Method LC-MS/MS quantification
Integrity Marker Lucifer Yellow
Data Delivery Papp
Efflux Ratio of Bidirectional Assessment

Data

Data from Cyprotex's MDR1-MDCK permeability assay

Cyprotex's MDR1-MDCK 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. Figure 2 below illustrates how the P-gp inhibitor cyclosporin A was used to confirm the role of P-gp in the efflux of 3 known P-gp substrates.

 
DrugPapp A-B (x 10-6 cm/s)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 MDR1-MDCK Permeability.
Cyprotex's MDR1-MDCK distinguishes between CNS positive and CNS negative compounds based on their Pappvalues.
Figure 1
Net flux ratio for a set of 20 compounds (calculated using the efflux ratios of the wild type and MDR1-MDCK bidirectional assays).

By performing a bidirectional study in both the wild type and MDR1-MDCK assay, the net flux ratio can be calculated to identify compounds which are substrates of human P-glycoprotein.
Figure 2
Graph shows the effect of cyclosporin A (10 μM) on the efflux of prazosin, quinidine and talinolol.

Cyclosporin A is an inhibitor of P-glycoprotein and inhibits the efflux of prazosin, quinidine and talinolol in Cyprotex's MDR1-MDCK bidirectional assay.

Q&A

Questions and answers on MDR1-MDCK permeability

Please provide an overview of the Cyprotex's MDR1-MDCK Permeability assay.

MDR1-MDCK 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. This cell line is ideal for identifying substrates and inhibitors of P-gp, and quantifying the extent of the interaction. The cells are seeded on a Multiscreen™ plate (Millipore, MA, USA) 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 compartment 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 the data from the MDR1-MDCK permeability assay?

There are several ways in which the data can be used. Firstly, the compounds can be ranked in terms of their MDR1-MDCK 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, the extent 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 P-gp inhibitor such as cyclosporin A is included to suppress the efflux. Thirdly, the test compound can be evaluated for its inhibitory effects on P-gp by investigating the effect of the test compound on the permeability of a known P-gp substrate (see Cyprotex's P-gp Inhibition assay). Finally, the efflux by human P-gp should be confirmed by assessing the efflux in the wild type cell line and a net flux ratio can be calculated. The FDA Draft Guidance for Industry (Drug Interaction Studies – Study Design, Data Analysis, Implications for Dosing and Labeling Recommendations, Feb 2012)1 recommends that investigational drugs are evaluated to determine if they are P-gp substrates or inhibitors. This FDA document provides decision trees for guidance on whether an in vivo drug interaction study may be necessary.

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 MDR1-MDCK 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 control compound to confirm that the cells are expressing functional P-gp efflux proteins. To confirm P-gp is responsible for the efflux of the test compound, cyclosporin A (10 µM), a P-gp inhibitor, can be included in the test compound incubation. The efflux ratio should decrease in the presence of cyclosporin A if the compound is a P-gp substrate. The role of human P-gp in the efflux should be confirmed by assessing the efflux in the native MDCK cell line and calculating a net flux ratio.

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 (MDR1-MDCK) or 1 x 10-6 cm/s (wild type MDCK) then it is assumed that the formation of the cell monolayer has been unsuccessful and the compound is re-screened. If both lucifer yellow Papp values fail for the same compound on 2 separate occasions then it is assumed that the compound exhibits cytotoxic effects against the MDR1-MDCK cells.

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 the MDR1-MDCK 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 MDR1-MDCK screen.

What is the relationship between MDR1-MDCK permeability and human intestinal absorption?

The relationship between Cyprotex's MDR1-MDCK permeability and human intestinal absorption is displayed in Figure 3. There is good correlation between human intestinal absorption and MDR1-MDCK 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 3
Relationship between Cyprotex's MDR1-MDCK permeability (apical to basolateral) and % human intestinal absorption.

References

1 Draft FDA Guidance for Industry - Drug Interaction Studies - Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations (February 2012)
2 Pastan I et al. (1988) Proc Natl Acad Sci USA 85; 4486-4490
3 Wang O et al. (2005) Int J Pharmaceut 288; 349-359
4 Di L et al. (2003) Eur J Med Chem 38; 223-232
5 Seelig A et al. (1994) Proc Natl Acad Sci USA 91; 68-72
6 Thomas RC et al. (1975) J Pharm Sci 64; 1360-6
7 Piovan D et al. (1986) Pharmacol Res Commun 18; 739-745
8 Yang H et al. (1996) Pharm Res 13; 1570-5
9 Courad JP et al. (2001) Life Sci 69; 1455-64
10 Murakami H et al. (2000) Am J Physiol Heart Circ Physiol 279; H1022-1028
11 Liu X et al. (2004) Drug Metab Dispos 32; 132-139
12 Zhao YH et al. (2001) J Pharmaceut Sci 90; 749-784

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