Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing
Reexamination Certificate
2002-02-28
2003-03-25
Riley, Jezia (Department: 1637)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
C514S04400A, C514S410000, C514S411000, C514S415000
Reexamination Certificate
active
06537964
ABSTRACT:
BACKGROUND OF THE INVENTION
a) Field of the Invention
The present invention describes the use of fumitremorgin A, B and C and a series of diketopiperazines of Formula (I) to resensitize multidrug resistant (MDR) cancer cells to the cytotoxic effects of chemotherapeutic drugs.
b) Description of the Prior Art
Resistance of tumor cells to chemotherapeutic drugs is a serious problem in the clinical management of cancer and reversal of resistance is a major goal in cancer chemotherapy. One type of resistance is characterized by the cross resistance to a wide variety of chemotherapeutic agents having no major structural similarities or similar modes of action. This phenomenon is termed multiple drug resistance. Multiple drug resistance (MDR) may be due to the presence of P-glycoprotein (P-gp), a transmembrane protein member of the ATP-binding cassette transporter (ABC transporter) family of proteins. P-gp acts as a drug efflux pump and actively transports chemotherapeutic agents out of the cells and thereby lowers the intracellular concentrations of such drugs to non-toxic, i.e., ineffective, levels. Chemotherapeutic drugs or agents (also used interchangeably herein as anticancer agent, antitumor agent and chemotherapy, in clinical use for the treatment of cancer to which multiple drug resistance has been observed include mitoxantrone, doxorubicin, vinblastine, viricristine, paclitaxel, daunomycin, etoposide, teniposide and actinomycin D. Inhibition of this drug efflux pump by pharmacological agents has been shown to reverse resistance and resensitize resistant cells to antitumor agents. In the early 1980's it was discovered that some multiple drug resistant tumor cells could be resensitized to chemotherapeutic agents with verapamil and with trifluoroperizine. Although P-gp is expressed in up to 50% of human tumors, several lines of evidence in the laboratory and in cancer patients suggest that P-gp alone does not account for all forms of multidrug resistance.
Mitoxantrone is an important anticancer drug with therapeutic efficacy similar to that of standard induction and salvage regimens in non-Hodgkin's lymphoma, a variety of leukemias, advanced breast cancer, and for palliative therapy in advanced prostate cancer. Like doxorubicin, it is a multi-ring planar molecule that intercalates with DNA. However, it can be distinguished from doxorubicin based on its mechanism of action, its reduced cardiotoxicity compared to anthracyclines, and its mechanism of resistance. In particular, resistance to doxorubicin and mitoxantrone involve transporters with overlapping but distinct specificities. For example, cells expressing P-gp are often cross-resistant to mitoxantrone and doxorubicin, and both molecules are efficiently transported by this protein. However, a second ABC transporter, the multidrug resistance protein (MRP), is induced by, and can mediate resistance to, doxorubicin but not mitoxantrone.
Selection of cells in mitoxantrone, doxorubicin or the podophyllotoxins often result in drug resistant sublines in which P-gp levels remain low or absent. Here, MDR is associated with decreased drug accumulation, altered intracellular trafficking, alterations in the activity/levels of topoisomerase II or changes in glutathione metabolism. In addition, multiple changes may exist together within the drug resistant cell, and a switch in the resistance mechanism from non P-gp to P-gp mediated MDR may occur as a cell line is cultured in increasing concentration of the selective agent.
Selection of tumor cells for resistance to mitoxantrone frequently results in a characteristic phenotype that has been found in human tumors derived from diverse tissue types, including colon (WiDr and LS174T), breast (MCF-7), myeloid (8226), and gastric (EGP85). The primary phenotypic feature is profound resistance to mitoxantrone, moderate resistance to related anthracycline molecules (doxorubicin and bisantrene), and little or no cross-resistance to camptothecin or to the microtubule-active drugs vinblastine, paclitaxel, and colchicine. Overexpression of two putative drug efflux pumps, P-gp or MRP, is generally not observed. Recently, a third ABC transporter, known as breast cancer resistance protein (BCRP), mitoxantrone resistance gene (MXR), or placenta-specific ABC gene (ABCP), has been reported to induce, and mediate resistance to, mitoxantrone, doxorubicin, and topotecan.
Fumitremorgin A, B and C are tremorgenic mycotoxins isolated from
Aspergillus fumigatus
and have been reported as interesting synthetic targets(T. Hino and M. Nakagawa, Heterocycles 46, 673-704(1997); C. Cui, H. Kakeya and H. Osada, J. of Antibiotics, 49(6), 534-540(1996); M. Nakagawa et al, Tetrahedron Let. 27(28), 3235-3238(1986); S. Kodato et al, Tetrahedron, 44(2), 359-377(1988); and D. M. Harrison and R. B. Sharma, Tetrahedron Let., 27, 521(1986)) and have not been previously reported to resensitize multidrug resistant (MDR) cancer cells to the cytotoxic effects of anticancer agents.
SUMMARY OF THE INVENTION
By selecting human colon carcinoma S1 cells, a subclone of LS 174T cells, which survive in increasing concentrations of up to 3.2 &mgr;M of mitoxantrone, the multiple drug resistant human colon carcinoma cell line S1-M1-3.2 was obtained. This multiple drug resistant phenotype which is not reversed by verapamil or the potent P-gp inhibitor &agr;-(3,4-dimethoxyphenyl)-3,4-dihydro-6,7-dimethoxy-&agr;-[(4-methylphenyl)thio]-2(1H)-isoquinolineheptanenitrile (U.S. Pat. No. 5,387,685) is dependent in part upon an energy-dependent drug efflux mechanism. P-gp and the multiple drug resistance protein (MRP) are not elevated in the mitoxantrone resistant cells relative to the drug sensitive parent, suggesting that resistance is mediated by a novel pathway of drug transport.
One aspect of this invention is to provide a cell-based assay with S1-M1-3.2 cells which can be used as a method to identify agents capable of circumventing non P-gp
on-MRP mediated MDR and identify chemosensitizing compounds which can resensitize cells to the cytotoxic effects of the chemotherapeutic drugs listed hereinabove.
An important aspect of this invention is the capability to identify test compounds as chemosensitizing agents following evaluation in an assay of this invention.
A further aspect of this invention is a method for identifying chemosensitizing compounds that reverse non P-gp
on MRP multiple drug resistance in cancer cells exhibiting non P-gp
on MRP drug resistance phenotype comprising administration of a test compound and a chemotherapeutic agent to which cancer cells are resistant and measuring cancer cell survival.
An additional feature of this invention is a method for resensitizing non P-gp
on MRP multiple drug resistant cancer cells to treatment with chemotherapeutic agents to which cancer cells have developed resistance comprising administration of an effective amount of a chemosensitizing reversal agent and a chemotherapeutic agent.
Fumitremorgin A (FTA), fumitremorgin B (FTB), fumitremorgin C (FTC) and compounds of Formula (I) were found to be active agents in a cell based assay with S1-M1-3.2 cells. Mycotoxin fumitremorgin C (FTC) identified in this assay is extremely effective in reversing resistance to mitoxantrone or doxorubicin in multiple drug-selected cell lines showing this novel phenotype. The toxic amount of FTC which kills more than 20% of the S1-M1-3.2 cells is greater than 80.0 &mgr;M. At a level of 0.3 &mgr;M, FTC plus 3.2 &mgr;M mitoxantrone causes a 50% cell kill (IC
50
0.3 &mgr;M). In addition, FTC also reverses topotecan resistance. Reversal of resistance was associated with an increase in drug accumulation in the cell. FTC also reverses resistance, and increases drug accumulation in cells made resistant by transfection with the BCRP gene.
Another aspect of the invention is a method for identifying chemosensitizing compounds that reverse BCRP-mediated multiple drug resistance in cancer cells which exhibit BCRP-mediated multiple drug resistance comprising administration of a test compound and a chemother
Greenberger Lee Martin
He Haiyin
Moran Daniel B.
Riley Jezia
Wyeth Holdings Corporation
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