Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1996-06-07
2001-01-09
Houtteman, Scott W. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C514S04400A, C514S283000, C424S145100
Reexamination Certificate
active
06171786
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention is directed to methods for preventing the emergence of multidrug resistance in tumor cells during cancer chemotherapy. In particular, it relates to the use of inhibitors of particular pathways of signal transduction to prevent the induction of the multidrug resistance (MDR1) gene by chemotherapeutic drugs. MDR1 gene expression, which results in tumor cell resistance to subsequent treatment with certain chemotherapeutic drugs is shown herein to be induced in response to treatment with various cytotoxic agents. Inhibitors of protein kinases, cytoplasmic calcium antagonists and calmodulin inhibitors, phosphoinositol-dependent phospholipase C inhibitors, and substances that inhibit activation of the transcription factor NF-&kgr;B (each of which have been implicated in intracellular eukaryotic signal transduction) are also shown herein to suppress this cellular response. Therefore, such inhibitors are useful in preventing MDR1 induction by chemotherapeutic drugs in a variety of tumor cells, when administered prior to and/or simultaneously with cytotoxic drug treatment in cancer patients.
2. Summary of the Related Art
Chemotherapy is a primary form of conventional cancer treatment. However, a major problem associated with cancer chemotherapy is the ability of tumor cells to develop resistance to the cytotoxic effects of anti-cancer drugs during the course of treatment. It has been observed that tumor cells can become simultaneously resistant to several chemotherapeutic drugs with unrelated chemical structures and mechanisms of action. This phenomenon is referred to as multidrug resistance. The best documented and clinically relevant mechanism for multidrug resistance in tumor cells is correlated with the expression of P-glycoprotein, the product of the MDR1 gene.
P-glycoprotein is a broad specificity efflux pump located in the cell membrane, and functions by decreasing the intracellular accumulation of many lipophilic cytotoxic drugs, including some widely used anticancer agents such as anthracyclines, vinca alkaloids, epipodophyllotoxins, actinomycin D and taxol, thereby rendering cells resistant to these drugs (Pastan and Gottesman, 1991,
Annu. Rev. Med.
42: 277-286; Roninson (ed.), 1991,
Molecular and Cellular Biology of Multidrug Resistance in Tumor Cells
, Plenum Press, New York; Schinkel and Borst, 1991,
Sem. Cancer Biol.
2: 213-226).
Human P-glycoprotein is expressed in several types of normal epithelial and endothelial tissues (Cordon-Cardo et al., 1990,
J. Histochem. Cytochem.
38: 1277-1287; Thiebaut et al., 1989,
Proc. Natl. Acad. Sci. USA
84: 7735-7738), as well as in hematopoietic stem cells (Chaudhary and Roninson, 1991,
Cell
66: 85-94), and a subpopulation of mature lymphocytes (Neyfakh et al., 1989,
Exp. Cell Res.
185: 496-505). More importantly, MDR1 mRNA or P-glycoprotein have been detected in most types of human tumors, both before and after chemotherapeutic treatment (Goldstein et al., 1989,
J. Natl. Cancer Inst.
81: 116-124; Noonan et al., 1990,
Proc. Natl. Acad. Sci. USA
87: 7160-7164). The highest levels of MDR1 expression are usually found in tumors derived from MDR1-expressing normal tissues; e.g., renal, adrenocortical or colorectal carcinomas. In other types of solid tumors and leukemias, MDR1 expression prior to treatment is usually relatively low or undetectable, but a substantial fraction of such malignancies express high levels of MDR1 after exposure to chemotherapy (Goldstein et al., 1989, ibid.). Prior to the present invention, the increase in MDR1 expression after chemotherapy was believed to result from in vivo selection for rare, pre-existing tumor cells that were already inherently resistant to chemotherapeutic drugs due to MDR1 expression.
Even low levels of MDR1 expression have been correlated with the lack of response to chemotherapy in several different types of cancer (Chan et al., 1990,
J. Clin. Oncol.
8: 689-704; Chan et al., 1991,
N. Engl. J. Med.
325: 1608-1614; Musto et al., 1991,
Brit. J. Haematol.
77: 50-53), indicating that P-glycoprotein-mediated multidrug resistance represents an important component of clinical drug resistance. Whereas many clinical and pre-clinical studies have addressed pharmacological strategies for inhibiting P-glycoprotein function (Ford and Hait, 1990,
Pharmacol. Rev.
42: 155-199), prior to the present invention, little was known about the factors that are responsible for the induction or up-regulation of P-glycoprotein expression in tumor cells under conditions relevant to cancer chemotherapy. Understanding such factors provides insight into the development of methods for preventing the appearance of P-glycoprotein in human tumors, thus reducing the incidence of multidrug resistance in cancer, and leading to more effective chemotherapy of cancer.
Numerous gene transfer studies have demonstrated that elevated expression of the MDR1 gene is sufficient to confer the multidrug resistance phenotype (Roninson, 1991, ibid.). For instance, mouse NIH 3T3 cells infected with a recombinant retrovirus carrying human MDR1 cDNA became multidrug-resistant in proportion to the density of human P-glycoprotein on their surface; the correlation was not affected by the presence or absence of cytotoxic selection (Choi et al., 1991,
Proc. Natl. Acad. Sci. USA
88: 7386-7390).
Nevertheless, consistent association of other biochemical changes with multidrug-resistant cells suggested that these alterations may also play a role in multidrug resistance, possibly by affecting the expression or function of P-glycoprotein. The most prominent of such changes is the increased activity of protein kinase C (PKC), found in many, but not all, multidrug-resistant cell lines obtained after multiple steps of cytotoxic selection (Aquino et al., 1990,
Cancer Commun.
2: 243-247; Fine et al., 1988,
Proc. Natl. Acad. Sci. USA
85: 582-586; O'Brian et al., 1989,
FEBS Lett.
246: 78-82; Posada et al., 1989,
Cancer Commun.
1: 285-292). PKC activation has been shown to increase the level of drug resistance in some drug-sensitive and multidrug-resistant cells lines (Ferguson and Cheng, 1987,
Cancer Res.
47: 433-441; Fine et al., 1988, ibid.; Yu et al., 1991,
Cancer Commun.
3: 181-189). Although PKC is reported to be capable of phosphorylating P-glycoprotein (Chambers et al., 1990,
Biochem. Biophys. Res. Commun.
169: 253-259; Chambers et al., 1990,
J. Biol. Chem.
265: 7679-7686; Hamada et al., 1987,
Cancer Res.
47: 2860-2865), it is not known whether such phosphorylation is responsible for the observed changes in drug resistance. While it has been shown that certain PKC inhibitors reversed multidrug resistance in some P-glycoprotein expressing cell lines (O'Brian et al., 1989, ibid.; Posada et al., 1989,
Cancer Commun.
1: 285-292; Palayoor et al., 1987,
Biochem. Biophys. Res. Commun.
148: 718-725), the available evidence suggests that at least some of the observed effects were due to direct inhibition of P-glycoprotein function by the tested compounds rather than inhibition of PKC-mediated phosphorylation (Ford et al. 1990,
Cancer Res.
50: 1748-1756; Sato et al., 1990,
Biochem. Biophys. Res. Commun.
173: 1252-1257). These studies have provided no indication that PKC-interactive agents could have an effect on expression, rather than phosphorylation or function, of P-glycoprotein.
Several laboratories have investigated the factors that regulate MDR1 gene expression in normal and malignant cells. One example of normal physiological regulation of an MDR1 homolog was found in mouse uterine endometrium, where the expression of a mouse mdr gene was induced by steroid hormones at the onset of pregnancy (Arceci et al., 1990,
Molec. Repro. Dev.
25: 101-109; Bates et al., 1989,
Molec. Cell. Biol.
9: 4337-4344). In rat liver, the expression of an mdr gene was found to be inducible by several carcinogenic or cytotoxic xenobiotics; similar induction was also observed during liver regeneration (Fairchild et al., 1987,
Proc. Natl. Acad. Sci.
Roninson Igor B.
Shtil Alexander A.
Board of Trustees of University of Illinois
Houtteman Scott W.
McDonnell & Boehnen Hulbert & Berghoff
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