|Product||Transporter||Technology||Probe Substrate||Reference Inhibitor||Barrier|
|MultiDrugQuant™ Assay Kit||MDR1/P-gp
|Multi-drug Resistance Determination
For the functional determination of MDR1, MRP1 and BCRP transporter-related multidrug resistance
Kit for 10 independent MDR1 / MRP1 / BCRP measurements carried out in triplicates
SOLVO MDQTM assay is designed as a routine laboratory flow cytometry method for the measurement of the functional activity of clinically relevant MDR1, MRP1 and BCRP efflux transporters in viable cells, offering several advantages:
- Quantitative flow cytometry: results are expressed in harmonized MDR activity factor (MAF) values
- Ready-to-use reagents
- Functional assay
- Compatible with cell surface markers (see Figure 1.)
- Highly selective inhibitors
- Different probe substrate for MDR1/MRP1 and BCRP
- Required specimen: Cell suspension, blood, bone marrow (etc: 6 hours stability before testing) and cell cultures
- Research Use Only
In the past few years, it has become widely accepted that resistance to chemotherapy and disease-modifying antirheumatic drugs (DMARDs) correlates with the overexpression of ATP-binding cassette (ABC) transporters, such as ABCB1 (MDR1 or P-gp), ABCC1 (MRP1), and ABCG2 (MXR or BCRP). These drug efflux pumps are actively extruding xenobiotics including anticancer drugs and DMARDs at the expense of ATP hydrolysis. Concordant clinical data show that the incidence of multidrug resistance (MDR) in previously untreated cases is approximately 40%, making it the most frequent type of resistance.
Inter-individual variability in drug response and the emergence of adverse drug reactions are the main causes of treatment failure in cancer therapy. Therefore, chemotherapies are effective only in a proportion of cancer patients, whereas most of them are likely to suffer from adverse side effects. Measuring membrane transporter activity in the target cells helps predict the resistance of these cells to particular therapeutic agents, and not only the high expenses of inefficient treatments can be saved, but also the adverse effects suffered by the patients. Furthermore, choosing an alternative therapy can improve the chances of recovery. A performance evaluation study of the MultiDrugQuantTM assay kit for the prediction of therapy response is under way. The following figure demonstrates the process in which an MDR transporter causes multidrug resistance.
Figure 1. MDR activities on the CD19+ cells of a CLL patient
a) without MDR-MRP inhibitors,
b) with selective MRP inhibitor,
c) with MDR+MRP inhibitor,
d) without selective BCRP inhibitor,
e) with selective BCRP inhibitor
Principle of the test
For the quantitative measurement of MDR1 and MRP1 activities in viable cells, SOLVO MDQTM Kit applies the Calcein-assay technology patented by SOLVO Biotechnology. The Calcein-assay utilizes the fluorogenic dye calcein-acetoxymethyl ester (calcein-AM), which is a hydrophobic, non-fluorescent compound, which readily penetrates the cell membrane. After entering the living cell, calcein AM is rapidly hydrolyzed by endogenous esterases. As a result of the cleavage, highly fluorescent free acid derivative of the dye is formed, which becomes trapped in the cytoplasm due to its hydrophilic character. Since calcein-AM is an excellent substrate of both MDR1 and MRP1, activity of these efflux transporters results in a lower cellular accumulation of the fluorescent calcein (Figure, Left Panel).
Adding selective inhibitors of MDR1 and MRP1 in excess blocks the dye extrusion activity of the relevant transporter and increases calcein accumulation in the cells. The activities of MDR1 and MRP1 transporters are reflected by the difference between the amount of calcein accumulated in the presence or absence of the selective inhibitors. This difference is normalized to the dye uptake measured in the presence of the inhibitor, and the results of the test are expressed in MDR activity factor (MAF) values.
Thus, the result of the test becomes independent of the factors influencing the cellular accumulation of calcein other than the activity of the multidrug transporters. These variables include the differences in the cellular properties (membrane composition, intracellular esterase activity, cell size, cell surface, etc.); and the methodological differences (e.g. use of different equipment, amplification, and individual variables).
Since the influence of these factors is diminished by the simple normalization approach mentioned above, the intra- and inter-laboratory comparison of MAF values is possible.
BCRP activity is measured using a similar principle: intracellular accumulation of the fluorescent BCRP-specific reporter substrate is measured in the presence and absence of the selective BCRP-inhibitor (Figure, Right Panel). However, the BCRP-specific reporter substrate is directly fluorescent and does not require cleavage by the intracellular esterases.
If you have any questions about our MultiDrugQuantTM Kit, please contact us!
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