Methods of using cytochrome P450 reductase for the...

Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai

Reexamination Certificate

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C435S320100, C435S455000, C536S023200, C536S023400

Reexamination Certificate

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06207648

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 NADPH cytochrome P450 reductase (RED) to enhance cytochrome P450-based anti-cancer gene therapy.
2. Related Art
Traditional methods for cancer treatment rely on a combination of surgery, radiation, and cytotoxic chemotherapeutic drugs. Although the treatment of tumor cells with cytotoxic chemicals 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 novel 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 tumors drug sensitivity and responsiveness. One promising strategy 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.
23:31-45 (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. In one example, herpes simplex virus thymidine kinase (HSV-TK) in combination with the prodrug ganciclovir represents a prototypic prodrug/enzyme activation system known in the art with respect to its potential applications in cancer gene therapy. 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).
In a second example, 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: 4735-4742 (1996)), and
E. coli
nitro reductase, which activates the prodrug CB1954 and related mustard prodrug analogs (Drabek, D., et al.,
Gene Ther.
4:93-100 (1997); Green, N. K., et al.,
Cancer Gene Ther.
4:229-238 (1997)), some of which may be superior to CB1954 (Friedlos, F. et al.,
J Med Chem
40:1270-1275 (1997)). The principle underlying these approaches to prodrug activation gene therapy is that transduction of a tumor cell population with the foreign gene confers upon it a unique prodrug activation capacity, and hence a chemosensitivity which is absent from host cells that do not express the gene.
Current gene therapy technologies are limited by their inability to deliver prodrug activation or other therapeutic genes to a population of tumor cells with 100% efficiency. The effectiveness of this cancer gene therapy strategy can be greatly enhanced, however, by using drugs that exhibit a strong “bystander effect” (Pope, I. M., et al.,
Eur J Cancer
33:1005-1016 (1997)). Bystander cytotoxicity results when active drug metabolites diffuse or are otherwise transferred from their site of generation within a transduced tumor cell to a neighboring, naive tumor cell. Ideally, the bystander effect leads to significant tumor regression even when a minority of tumor cells is transduced with the prodrug activation gene (e.g., Chen, L., et al.,
Hum Gene Ther.
6:1467-1476 (1995); Freeman, S., et al.,
Cancer Res.
53:5274-5283 (1993)). Bystander cytotoxic responses may also be mediated through the immune system, following its stimulation by interleukins and other cytokines secreted by tumor cells undergoing apoptosis (Gagandeep, S., et al.,
Cancer Gene Ther.
3:83-88 (1996)).
Although the ganciclovir/HSV-TK and 5-fluorocytosine/CD systems have shown promise in preclinical studies, and clinical trials are underway (Eck, S. L., et al.,
Hum Gene Ther.
7:1465-1482 (1996); Link, C. J. et al.,
Hum Gene Ther.
7:1161-1179 (1996); Roth, J. A., and Cristiano, R. J.,
J Natl Cancer Inst.
89:21-39 (1997)), several limitations restrict their efficacy and limit their application to cancer chemotherapeutics. These include: (a) the non-mammalian nature of the HSV/TK and CD genes, whose gene products may elicit immune responses that interfere with prodrug activation; (b) their reliance on drugs which were initially developed as antiviral drugs (ganciclovir) or antifungal drugs (5-fluorocytosine) and whose cancer chemotherapeutic activity is uncertain; (c) the dependence of these gene therapy strategies on ongoing tumor cell DNA replication; and (d) the requirement, in the case of HSV-TK, for direct cell-cell contact to elicit an effective bystander cytotoxic response (Mesnil, M., et al.,
Proc. Natl. Acad. Sci. USA.
93: 1831-1835 (1996)). These considerations, together with the general requirement of combination chemotherapies to achieve effective, durable clinical responses, necessitates the development of alterative strategies to treat cancers using suicide gene-based (prodrug activation) gene therapy.
More recently, a drug activation/gene therapy strategy has been developed based on a cytochrome P450 gene (“CYP” or “P450”) in combination with a cancer chemotherapeutic agent that is activated through a P450-catalyzed monoxygenase reaction (Chen, L. and Waxman, D. J.,
Cancer Research
55:581-589 (1995); Wei, M. X., et al.,
Hum. Gene Ther.
5:969-978 (1994); U.S. Pat. No. 5,688,773, issued Nov. 18, 1997). Unlike the prodrug activation strategies mentioned above, the P450-based drug activation strategy utilizes a mammalian drug activation gene (rather than a bacterially or virally derived gene), and also utilizes established chemotherapeutic drugs widely used in cancer therapy.
Many anti-cancer drugs are known to be oxygenated by cytochrome P450 enzymes to yield metabolites that are cytotoxic or cytostatic toward tumor cells. These include several commonly used cancer chemotherapeutic drugs, such as cyclophosphamide (CPA), its isomer ifosfamide (IFA), dacarbazine, procarbazine, thio-TEPA, etoposide, 2-aminoanthracene, 4-ipomeanol, and tamoxifen (LeBlanc, G. A. and Waxman, D. J.,
Drug Metab. Rev.
20:395-439 (1989); Ng, S. F. and Waxman D. J.,
Intl. J. Oncology
2:731-738 (1993); Goeptar, A. R., et al.,
Cancer Res.
54:2411-2418 (1994); van Maanen, J. M., et al.,
Cancer Res.
47:4658-4662 (1987); Dehal, S. S., et al.,
Can

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