Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2000-01-19
2001-07-10
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S005000, C435S006120, C435S007100, C435S007900, C435S174000, C435S177000, C435S287100, C436S809000
Reexamination Certificate
active
06258526
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to an ex-vivo test kit for testing the effectiveness of reversers of multidrug resistance, to methods for the use thereof and to methods for the preparation of the components thereof. More particularly, the present invention relates to an ex-vivo test kit for testing the effectiveness of reversers of multidrug resistance in the blood, serum or plasma of a patient containing said reversers.
DESCRIPTION OF THE RELATED ART
Most people who die as a result of having cancer do so because their tumors have either developed resistance to chemotherapy or were resistant to begin with. Therefore, the development of drug resistance in a patient is still a major barrier to the effectiveness of chemotherapy treatment of cancer. Until ways to entirely prevent cancer are found, overcoming drug resistance will remain the main avenue for saving millions of lives. Great efforts have been invested in the development of accessory drugs which can block multidrug resistance and hence restore effectiveness to the chemotherapeutic regime.
A certain cell membrane protein, P-glycoprotein, is an important contributor to multidrug resistance in a substantial number of tumors. There are other ways by which cancers develop resistance to chemotherapeutic drugs, e.g., the presence in their cell membranes of the Multidrug Resistance-associated Protein, the Mitoxantrone Resistance protein and Lung Resistance Protein systems, as well as other, non-membrane systems. All these membrane proteins have a similar action, i.e. they are able to pump out anti-cancer drugs and many other drugs from the cells in which they are found. In this way, a low concentration of such anti-cancer drugs are retained in the cancer cell, thus defeating the aim of the clinician which is to keep the anti-cancer drug in as high a concentration as possible within the cancer cell. It has been postulated and it is believed that these pump proteins appear in the membranes of the cancer cells as a typical response of an aggressive cancer cell which tries all ways to survive against the efforts of the clinician. In this case, the cancer cell evolves the ability to express the pump proteins in its membrane. Great efforts by major pharmaceutical are being made in order to find clinically-useful reversers of multidrug resistance, i.e. agents that can block its action, allowing formerly resistant cells to renew the accumulation of drugs. Many reversers have been identified and a few are now in clinical trial which are used in combination with an appropriate anti-cancer drug.
It has been found that many of the reversers that have been used in clinical trials have not been effective simply because they are bound in the patient's plasma and are not able to enter the membrane of the cancer cells and therefore are not in a position to counteract the pump that is pumping out the anti-cancer drug.
The present inventor has previously reported the development of an assay, using living P-glycoprotein-containing cells grown outside the body in cell culture (Ayesh, S. E. Lyubimov, N. Algour, and W. D. Stein. 1996.) Reversal of P-glycoprotein is greatly reduced by the presence of plasma but can be monitored by an ex vivo clinical assay. (Anti-Canc. Drugs 7:678686), which enabled the immediate measurement in a sample of the plasma taken from a patient of the effectiveness of any reverser of the membrane pumps. This assay, while reliable and useful proved to be cumbersome, and required that there be available at all times a supply of these cultured cells. Hence it is not suitable as a routine clinical assay for the hospital laboratory.
BRIEF SUMMARY OF THE INVENTION
With this state of the art in mind, there have now been developed according to the present invention, an ex vivo test, in kit form, which can be used to test the effectiveness of reversers, directly in the plasma of patients that are receiving the combined therapy. With the presently proposed kit, which is designed as an ex vivo assay to be readily usable in standard clinical laboratories, clinicians will be able to treat patients with reversers of multidrug resistance and, simultaneously, assay in plasma, serum or blood samples from such patients the effectiveness of the reversers in the plasma, serum or blood of the patient. The clinician will be able to try different reversers and test the effectiveness of each of these, and choose the most effective, before the patient even receives the chemotherapy. In addition, the pharmokinetics of the drug can be readily and safely studied in the patient so as to ascertain the time/dose requirements for the reverser in question. Before the patient receives chemotherapy, he/she will be treated with the reverser, over a twenty-four hour period, and blood samples will be taken for measuring how much of the reverser is available, free to block the patient's multidrug resistance pump. In this way, the doctor looking after the patient will be able to know in one day whether his/her patient has received an effective dose of the blocker rather than waiting months until it can be determined whether or not the tumor has been destroyed. If, by the present test, the doctor can show that the dose of reverser received was not effective, a higher dose or another reverser can be immediately substituted and the effectiveness of this second treatment again checked by the serum assay. All this will be able to be ascertained before the patient is treated with the chemotherapeutic agent, with all of its attendant dangers and discomforts. Use of the presently proposed assay should therefore greatly increase the success of cancer treatments, increasing the health and well-being of many cancer sufferers all over the world.
In addition, the assay of the present invention can be used by the pharmacological companies in testing and developing new blockers for other multidrug resistance systems and can be used in high-throughput searches for such blockers.
Furthermore, the present kit enables the effectiveness of P-glycoprotein reversers to be tested against both of the two sites for substrate or reverser binding that Shapiro and Ling have identified as being involved in drug pumping by P-glycoprotein (Shapiro, A. B. and V. Ling. 1995. Reconstitution of drug transport by purified P-glycoprotein. J. Biol. Chem. 270:16167-16165). These two sites have different affinities for the various ligands of P-glycoprotein and the kit of the present invention can enable the effectiveness of any reversers to be ascertained in serum against each of these two sites.
DETAILED DESCRIPTION OF THE INVENTION
Thus, according to the present invention there is now provided an ex-vivo test kit for testing the effectiveness of reversers of multidrug resistance in the blood, serum or plasma of a patient containing said reversers, said kit comprising, a plurality of cell membranes from highly drug-resistant cells, said cell membranes containing proteins which pump drugs and said membranes being respectively attached to a plurality of support surfaces, and a dye which provides a light signal, which dye is pumped by said proteins contained in said membranes.
In preferred embodiments of the present invention said dye is a fluorescent dye which provides a fluorecence signal.
The assay of the present invention uses preparations of cell membranes from highly drug-resistant cells, grown in cell culture and is designed to allow the measurement of the clearance, or the absorption, of fluorescent dyes from such membranes. The membranes are affixed to paper discs or to the sides and bottoms of, the wells of 96-well or 384-well plastic cell culture dishes.
Thus, in a first preferred embodiment of the present invention said surfaces are the surfaces of a plurality of wells.
In a second preferred embodiment of the present invention said surfaces are a plurality of plastic, comb-like teeth.
In a third preferred embodiment of the present invention said support surfaces are a plurality of filter paper elements and preferably said filter paper elements are sized to
Kott Miriam Viviana
Stein Wilfred Donald
Ladas & Parry
Le Long V.
M.D.R. Test LTD
Padmanabhan Kartic
LandOfFree
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