Chemistry: analytical and immunological testing – Involving immune complex formed in liquid phase
Reexamination Certificate
2001-04-23
2002-05-21
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving immune complex formed in liquid phase
C435S006120, C435S007800, C435S007100, C435S007210, C435S029000, C435S034000, C435S968000, C435S975000, C435S005000, C435S188000, C435S184000, C435S810000, C435S004000, C436S536000, C436S546000, C436S800000, C436S808000
Reexamination Certificate
active
06391656
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to methods of screening test substances for their effect on multi-drug resistance in cells, and of characterizing the type and degree of multi-drug resistance exhibited by cells. The present invention utilizes a quantitative in vitro diagnostic assay method for determining the activity and concentration of transport proteins that cause multi-drug resistance in tumors (MDR proteins). The invention further relates to reagent kits for performing said assay method.
BACKGROUND OF THE INVENTION
The efficiency of chemotherapy of tumors is seriously affected or hindered by the onset of the so called multi-drug resistance that, with respect to human tumor cells, is caused by the over expression of certain membrane bound proteins relative to normal cells. These membrane bound proteins or MDR proteins include the MDR1 protein (alternatively termed P-glycoprotein), MRP and their functional analogs. The MDR proteins extrude the applied cytostatic agents (e.g. Vinca alkaloids, anthracycline derivatives and other clinically used effective anti cancer agents) by active membrane transport to the exterior of the cell. These over expressed proteins make use of the energy obtained from ATP hydrolysis, thus making practically impossible for the applied agents to produce their cytostatic effect.
Hydrophobic cell permeable ester derivatives of some fluorescent dyes are actively extruded from the cells by MDR proteins present in the cell membranes, before the ester derivatives can reach the cytosol. However, once the ester derivatives reach the cytosol, intracellular esterases cleave the esters from the fluorescent dyes, and the MDR proteins can not extrude the resulting free dye compounds [Homolya, L. et al, J. Biol. Chem., 268, 21493 (1993)]. It has been demonstrated that the commercially available compound, calcein-AM (calcein-acetoxy-methylester), generally used in cell viability assays, is—unlike free calcein—an excellent activator of the MDR dependent ATPase (K
a
≦1 &mgr;mol/l). It has also been shown that calcein accumulation in the cell following calcein-AM uptake is reduced by the presence of MDR activity.
Calcein-AM is practically non fluorescent and highly lipid soluble (hydrophobic) and rapidly penetrates through the cell membrane. Intracellular esterases rapidly cleave the ester bond present in the compound, producing a highly fluorescent water soluble (hydrophilic) compound. When living cells are contacted with calcein-AM, calcein-AM molecules continuously penetrate the cells because of the established concentration gradient and cleavage products accumulate within the cell. The use of calcein-AM in cell viability assays is also based on this principle, since in damaged or dead cells entering calcein-AM molecules are not be transformed to a fluorescent product [Handbook of fluorescent probes and research chemicals, pp. 172-173, ed. Haugland, R. P., Molecular Probes Inc., Eugene, Oreg. (1992-94)].
It has been demonstrated that calcein-AM is useful for the qualitative functional analysis of the presence of multi-drug resistance in cells (Holló, Zs. et al, Biochim. Biophys. Acta 1191, 384 (1994); application Ser. No. 08/928,528 by Sarkadi et al, filed Sep. 12, 1997, hereby incorporated by reference; International PCT Publication No. WO 96/06945). If any of the over expressed proteins that exhibit MDR-type cell transport are present in the cell membrane, the cell extrudes the penetrating calcein-AM molecules via an active transport mechanism, and thus the rate of transformation of calcein-AM to fluorescent calcein (or other fluorescent calcein derivatives) and of accumulation of the fluorescent product(s) within the cells will be significantly reduced, relative to wild type cells. The resulting in vitro clinical diagnostic assay method makes possible, by applying a relatively simple and inexpensive measurement, the reliable pre-estimation of the measure of multi-drug resistance of different tumor types.
Clinicians are especially interested in learning the drug resistance profile, and the substrate specificity and drug extrusion activity of the various multi-drug resistance proteins in a given tumor sample. The demonstration of the transport activity of various multi-drug resistance proteins (e.g. MDR1, MRP1, cMOAT) in the plasma membrane requires a sensitive and reproducible in vitro multi-drug resistance assay.
The major requirements concerning current methods are the following: a) high sensitivity for the detection of relatively low levels of various MDR proteins; b) standardization, which is suitable for inter-laboratory comparison; c) functional characterization of the actual transport activity and substrate specificity of a given tumor sample; and d) straightforward evaluation of potential inhibitors of MDR transport activity.
The principles underlying the calcein assay method have permitted the formulation of highly advantageous assays that meet the above requirements. The assays of the invention are directed to quantitative measurement of both the degree of multi-drug resistance being exhibited (to what extent MDR-type transport is occurring in the sample cells) as well as the nature of the multi-drug resistance being exhibited (what type of expressed proteins are responsible and transport pathways utilized) in a single assay.
In addition, the instant assay permits the evaluation of potential mediators and effectors of multi-drug resistance, such as anti-cancer drugs, multi-drug resistance inhibitors, or environmental toxins. These screening assays are well-suited for high-throughput methods in a clinical setting.
REFERENCES:
patent: 4281061 (1981-07-01), Zuk et al.
patent: 5314805 (1994-05-01), Haugland et al.
patent: 5872014 (1999-02-01), Sarkadi et al.
Homolya et al., “Fluorescent cellular indicators are Extruded by the Multidrug resistance Protein”, The Journal of Biological Chemistry, vol. 268, No. 29, Oct. 15, 1993, pp. 21493-21496.*
Hollo et al., “Calcein accumulation as a fluorometric functional assay of the multidrug transporter”, Biochimica et Biophysica Acta 1191 (1994) pp. 384-388.*
Liminga et al., “Microfluorometric Evaluation of calcein Acetoxymethyl ester as a probe for p-glycoprotein-mediated resistance . . . ”, Experimental Cell Research, vol. 212, pp. 291-296, 1994.*
Kessel et al., Characterization of Multidrug resistance by Fluorescent Dyes, Cancer Research 51, pp. 4665-4670, Sep. 1991.*
Neyfakh, “Use of Fluorescent dyes as molecular probes for the study of multidrug resistance,” Experimntal Cell Research, vol. 174, 1988, pp. 168-176.
Holló Zsolt
Homolya László
Sarkadi Balász
Cook Lisa V
Kolisch, Hartwell, Dickinson, McCormack & Heuser, P.C.
Le Long V.
SOLVO Biotechnology
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