| Please provide an overview of the Cloe Screen 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) (Pastan et al ., 1988). 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 Cloe Select P-gp Inhibition). 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 The FDA Draft Guidance for Industry (Drug Interaction Studies – Study Design, Data Analysis, and Implications for Dosing and Labeling, Sept 2006) recommends that investigational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 (10µM), 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. This is detailed in the FDA Draft Guidance for Industry (Drug Interaction Studies – Study Design, Data Analysis, and Implications for Dosing and Labeling, Sept 2006). 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 Cloe Screen 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 ., 200111. Figure 3.
Relationship between Cloe Screen MDR1-MDCK permeability (apical to basolateral) and % human intestinal absorption.
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