Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2000-07-14
2004-08-03
Guzo, David (Department: 1636)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C424S093200, C435S320100, C435S455000, C435S456000, C435S458000, C435S459000
Reexamination Certificate
active
06770632
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is directed to the killing of neoplastic cells. More specifically, the present invention relates to the use of folylpolyglutamyl synthetase (FPGS) gene transfer to enhance antifolate drug sensitivity.
2. Related Art
Because of the critical role of folate coenzymes in the synthesis of DNA precursors, folate antagonists (antifolates) have found widespread use as chemotherapeutic agents. Methotrexate (MTX), a 4-aminofolate analogue, has been in clinical use for the treatment of various human malignancies, especially leukemias and breast cancer, for about 40 years. For a review, see Chu, E. and Allegra, C, “Antifolates,” in
Cancer Chemotherapy and Biotherapy: Principles and Practice
, Chabner et al, eds., Lippincott-Raven, Philadelphia (1996), pp. 109-148.
MTX is a potent inhibitor of dihydrofolate reductase (DHFR). Inhibition of this enzyme prevents the reduction of dihydrofolate that accumulates in cells actively synthesizing thymaidylate via the thymidylate synthetase reaction. The cells subsequently become depleted of reduced folate cofactors, needed for synthesis of thymidylate and for de novo purine synthesis. The ensuing disruption of DNA replication leads to cell death in actively replicating cells found in tumors and some normal tissues.
Naturally occurring folates and some antifolates, including MTX, possess a single terminal benzoylglutamate residue and are converted intracellularly from monoglutamates into polyglutamates through the action of an enzyme, folylpolyglutamyl synthetase (FPGS), that attaches up to six glutamyl groups in &ggr;-peptide linkage to the terminal benzoylgutamate. Polyglutamylation of folates and antifolates causes two effects. First, polyglutamylation causes intracellular accumulation of folates and antifolates because the highly ionized polyglutamylated forms are not readily transported across cell membranes. For example, MTX polyglutamates efflux out of cells 70 times slower than the monoglutamylated drug (Balinska, M., et al.,
Cancer Research
41:2751-2756 (1981)). Second, polyglutamylation enhances the affinity of folates for the enzymes that utilize them as cofactors, and increases the affinity (and inhibitory effect) of antifolates for their target enzymes, as well as expanding the range of enzymes which antifolates inhibit.
Because MTX is polyglutamylated much more inefficiently than naturally occurring folates, reductions in FPGS activity that have little effect on folate polyglutamate pools can have marked effects on the level of MTX polyglutamates and thus, on the cytotoxicity of MTX. The ability to generate MTX polyglutamates correlates directly with sensitivity to MTX for both human and murine tumor cells (Samuels, L. L., et al.,
Cancer Research
45:1488 (1985)). Human leukemia cell lines that have become resistant to clinically relevant antifolate doses through mutations in FPGS have been described (Pizzorno, G., et al.,
Cancer Research
48:2149 (1988); Roy, K., et al.,
Journal of Biological Chemistry
270:26918-26922 (1995); Roy, K., et al.,
Journal of Biological Chemistry
272:6903-6908 (1997); Takemura, Y., et al.,
British Journal of Cancer
75 (
suppl
. 1):31 (1997)). Human soft tissue sarcomas have been found to be intrinsically resistant to MTX as a result of low FPGS activity (Li, W. W., et al.,
Cancer Research
52:1434-1438 (1992)). Leukemias that have developed MTX resistance have been removed from patients and found to have impaired drug polyglutamylation (Rodenhuis, S., et al.,
Cancer Research
46:6513-6519(1986)). In addition, antifolates exhibiting the most therapeutic selectivity in murine tumor models consistently display a greater differential in accumulation of the polyglutamylated drug in tumor compared to normal proliferative tissues (Rumberger, S. et al.,
Cancer Research
50:4639-4643 (1990)).
Transfection of mutant CHO cells lacking FPGS activity with an FPGS expression cassette has been shown to increase the sensitivity of these cells to 4 hour MTX pulses in cell culture (Kim, J. S., et al.,
Journal of Biological Chemistry
268:21680-21685 (1993)). However, the question of whether tumor cells which already possess intermediate FPGS activity will show a similar enhancement of MTX sensitivity after FPGS gene delivery has not been previously addressed.
Traditional methods for cancer treatment rely on a combination of surgery, radiation, and cytotoxic chemotherapeutic drugs. Although the treatment of tumor cells with chemotherapeutic drugs is well-known in the art, presently, the therapeutic activity of many cytotoxic anti-cancer drugs is limited by a moderate therapeutic index associated with nonspecific toxicity toward normal host tissues, such as bone marrow, and the emergence of drug-resistant tumor cell sub-populations. One recent approach to enhancing the selectivity of cancer chemotherapeutics, and thereby reducing the toxicity of treatment, involves the application of gene therapy technologies to cancer treatment. See, Roth, J. A. and Cristiano, R. J.,
J. Natl. Cancer Inst
. 89:21-39 (1997); Rosenfeld, M. E. and Curiel, D. T.,
Curr. Opin. Oncol
. 8:72-77 (1996).
In one such therapy known in the art, the phenotype of the target tumor cells is genetically altered to increase the tumor's drug sensitivity and responsiveness. One strategy being actively investigated involves directly transferring a “chemosensitization” or “suicide” gene encoding a prodrug activation enzyme to malignant cells, in order to confer sensitivity to otherwise innocuous agents (Moolten, F. L.,
Cancer Gene Therapy
1:279-287 (1994); Freeman, S. M., et al.,
Semin. Oncol
. 23:3145 (1996); Deonarain, M. P., et al.,
Gene Therapy
2: 235-244 (1995)).
Several prodrug activation genes have been studied for application in cancer gene therapy. The two most extensively investigated prodrug-activating enzymes are herpes simplex virus thymidine kinase (HSV-TK), which activates the prodrug ganciclovir, and
E. coli
cytosine deazninase (CD), which activates the prodrug 5-fluorocytosine (Roth, J. A., Cristiano, R. J.,
Journal of the National Cancer Institute
89:21-39 (1997); Aghi, M., et al.,
Journal of the National Cancer Institute
90:370-380 (1998)).
HSV-TK phosphorylates the prodrug ganciclovir and generates nucleoside analogs that induce DNA chain termination and cell death in actively dividing cells. Tumor cells transduced with HSV-TK acquire sensitivity to ganciclovir, a clinically proven agent originally designed for treatment of viral infections. Moolten, F. L. and Wells, J. M.,
J. Natl. Cancer Inst
. 82:297-300 (1990); Ezzeddine, Z. D., et al.,
New Biol
. 3:608-614 (1991).
The bacterial gene cytosine deaminase (CD) is a prodrug/enzyme activation system that has been shown to sensitize tumor cells to the antifungal agent 5-fluorocytosine as a result of its transformation to 5-flurouracil, a known cancer chemotherapeutic agent (Mullen, C. A., et al.,
Proc. Natl. Acad. Sci. USA
89: 33-37 (1992); Huber, B. E., et al.,
Cancer Res
. 53:4619-4626 (1993); Mullen, C. A., et al.,
Cancer Res
. 54:1503-1506 (1994)). Recent studies using these drug susceptibility genes have yielded promising results. See, e.g., Caruso, M., et al.,
Proc. Natl. Acad. Sci. USA
90:7024-7028 (1993); Oldfield, E., et al.,
Hum. Gene Ther
. 4: 39 (1993); Culver, K,
Clin. Chem
40: 510 (1994); O'Malley, Jr., B. W., et al.,
Cancer Res
. 56:1737-1741 (1996); Rainov, N. G., et al.,
Cancer Gene Therapy
3:99-106 (1996).
Several other prodrug-activating enzyme systems have also been investigated (T. A. Connors,
Gene Ther
. 2:702-709 (1995)). These include the bacterial enzyme carboxypeptidase G2, which does not have a mammalian homolog, and can be used to activate certain synthetic mustard prodrugs by cleavage of a glutamic acid moiety to release an active, cytotoxic mustard metabolite (Marais, R., et al.,
Cancer Res
. 56: 47354742 (1996)), and
E. coli
nitro reductase, which activates the prodrug CB 1954 and related mustard prodrug analogs (Drabek, D., et al.,
Gene
Aghi Manish
Breakefield Xandra O.
Kramm Christof M.
Guzo David
Nguyen Quang
Sterne Kessler Goldstein & Fox PLLC
The General Hospital Corporation
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