Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2000-11-21
2004-10-12
Shukla, Ram R. (Department: 1635)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S006120, C435S091100, C435S091310, C435S458000, C536S023100, C536S024500
Reexamination Certificate
active
06803360
ABSTRACT:
BACKGROUND OF THE INVENTION
The failure of a significant number of tumors to respond to drug and/or radiation therapy is a serious problem in the treatment of cancer. While the genetic basis of this resistance in mammalian cells is still poorly understood, evidence has been obtained in recent years linking proto-oncogenes and oncogenes to the phenomenon of cellular radiation resistance.
The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References.
The earliest report of such a possible link was that of FitzGerald et al. (1985), who found that transfection of NIH 3T3 cells with a human N-ras oncogene was able to increase the radiation resistance level of the recipient cell line. Expanding upon this was the report by Sklar (1988) that NIH 3T3 cells transformed not only by N-ras but also by mutated Ha- and Ki-ras were more radiation resistant than the parent cell line. Additionally, we demonstrated a similar effect on the radiation resistance level of NIH 3T3 cells by both the mutated form of Ha-ras and the overexpression of the Ha-ras proto-oncogene (Pirollo et al., 1989). A synergistic increase in the radiation resistance level of primary rat embryo cells was also seen after cotransfection of ras and myc oncogenes (Ling and Endlich, 1989; McKenna et al., 1990). All documents cited herein supra and infra are hereby incorporated in their entirety by reference thereto.
The association was extended to include other oncogenes when transfection of NIH 3T3 cells by high molecular weight DNA from both radiation-resistant cells derived from a human laryngeal squamous carcinoma (Kasid et al., 1987) and radiation-resistant noncancerous skin fibroblast (NSF) cell lines from members of a family with Li-Fraumeni syndrome (Pirollo et al., 1989; Chang et al., 1987) led to the identification of an activated human raf-1 oncogene in the resulting radiation-resistant transformants. Transfections not only of the raf-1 oncogene but also of other protein-serine kinase encoding oncogenes, mos and cot, have been shown to confer the radiation-resistant phenotype on the recipient human Beas-2B (Kasid et al., 1989), mouse NIH 3T3 (Pirollo et al., 1989), and hamster SHOK cells (Suzuki et al., 1992), respectively. The effect of activated oncogenes on the radiation resistance level of NIH 3T3 cells is not a generalized phenomenon but is particular to specific oncogenes, as was clearly shown by Sklar (Sklar, 1988; Sklar, 1986) for abl and fms and by our laboratory (Pirollo et al., 1989) for myc, fes, and abl.
Evidence continues to accumulate which indicates that the normal counterpart of many of the known oncogenes (proto-oncogenes) are involved in vital, normal cellular functions (Bishop, 1991; Cantley et al., 1991; Hunter, 1991). They have also been shown to interact with one another as components of a proposed signal transduction pathway which involves transmission of messages from the membrane to the nucleus directing the cells to divide or to differentiate. On the basis of antibody-blocking experiments, it has been proposed that raf-1 is downstream of ras in this pathway (Morrison, 1990; Rapp et al., 1988; Smith et al., 1986; Weinstein, 1988).
Part of the signal transduction pathway leading to raf-1 expression is HER-2 (c-erb B-2
eu) which encodes a transmembrane protein tyrosine kinase with extensive homology to the epidermal growth factor receptor (EGF-R). Elevation of HER-2 in cancer cells has been shown to correlate with failure to respond to radiation therapy and there is significant evidence that expression of HER-2 affects the response of breast cancer tumors to endocrine therapy with Tamoxifen, and chemotherapy using drugs such as cisplatin, carboplatin, 5-fluorouracil, mitoxantrone, cyclophosphamide, methotrexate, doxorubicin, carmustine, melphalan, mitomycin, etoposide and combinations of these drugs (Pegram et al., 1993; Wright et al., 1992; Allred et al., 1992; Gusterson et al., 1992; Van Diest et al., 1992; Muss et al., 1994; Tsai et al., 1993).
In addition to conventional adjuvant therapies, a new molecular therapy has shown promise in the treatment of breast cancer and is now in Phase III clinical trials. This new therapeutic, Herceptin (trastuzumab), is a humanized mouse antibody against the HER-2 receptor. HER-2 is a member of the epidermal growth factor family. Overexpression of HER-2 is associated with poor prognosis in breast cancer. The combination of Herceptin and chemotherapy has demonstrated increased response duration, time to progression and survival in clinical trials (Shak, 1999; Burris, 2000) and has been termed receptor-enhanced chemosensitivity. However, despite the promise of this new strategy, there are drawbacks. Overexpression of HER-2 is only found in approximately 25% of breast cancer patients, and it has been documented that Herceptin has no effect on cells that do not overexpress HER-2 (Pegram and Slamon, 1999), the majority of breast cancers. Moreover, some HER-2 positive tumors have not responded to Herceptin (McNeil, 2000). Most significant is the increase in cardiac toxicity observed when patients received the combination of Herceptin and either doxorubicin or cyclophosphamide, two drugs routinely used in the treatment of breast cancer (McNeil, 2000; Jerian and Keegan, 1999; Gilewski et al, 2000; Schaller, 1999).
Recent studies indicate that the radiation resistant (RR) phenotype appears to be linked to the activation of specific protooncogenes in a signal transduction pathway involving HER-2 as an upstream member of the pathway and Ha-ras and raf-1 downstream of HER-2, analogous to that described for cell growth and differentiation (Pirollo et al., 1993). We hypothesized that disruption of the pathway therefore should lead to reversal of this phenotype and increased sensitivity of resistant cells to drug/radiation therapy which would have far-reaching clinical implications in the treatment of drug and radiation resistant tumors.
SUMMARY OF THE INVENTION
A specific strategy to interfere with the signaling is to modulate the expression of specific genes in the pathway at the RNA level using antisense oligonucleotides (ASO). Short antisense DNA oligonucleotides selectively bind to cellular mRNA targets through complementary sequence-specific Watson-Crick base pairing. The hydrogen-bonded antisense molecule can modulate the expression of the targeted gene product (Uhlmann and Peyman, 1990). We and others have demonstrated the ability of antisense oligonucleotides and their modified analogues to specifically inhibit ras p21 protein synthesis in in vitro translation, in cell culture, and in tumorigenesis in nude mice (Yu et al., 1989; Brown et al., 1989; Chang et al., 1991; Ts'o et al., 1992; Plenat, 1996). Additionally, ASO against genes such as c-myb, c-myc, c-fos, BCR-ABL and the IGF receptor, have also been shown to suppress human tumor cell growth in vitro and in some cases are currently in clinical trials as anti-cancer therapeutics (Stein et al., 1988; Mercola and Cohen, 1995; Scanlon et al., 1995).
The serine/threonine kinase Raf-1 protein appears to be a central component of multiple signal transduction pathways in the cell (reviewed in Campbell et al., 1995; Daum et al., 1994) including that for radiation resistance. Consequently, the use of ASO against raf-1 itself, or against upstream effectors of raf-1 such as Ha-ras and HER-2, to impede signaling through this gene should result in increased drug and radiation sensitivity, which would have-far-reaching clinical implications in the treatment of radioresistant tumors.
Therefore, the present invention relates to a method for reversing the drug and radiation resistance phenotype of cells, more specifically tumor cells which have acquired drug and/or radiation resistance. The method of the present invention employs antisense oligonucleotides targeted against specific proto-oncogenes in the signal transduct
Chang Esther H.
Pirollo Kathleen F.
Georgetown University
Rothwell Figg Ernst & Manbeck
Shukla Ram R.
Zara Jane
LandOfFree
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