US20040023925A1

US20040023925A1 - Method for enhancing the effectiveness of therapies of hyperproliferative diseases

Info

Publication number: US20040023925A1
Application number: US10/408,723
Authority: US
Inventors: Yan Chang; Vodek Sasak
Original assignee: GlycoGenesys Inc
Current assignee: Prospect Therapeutics Inc; GlycoGenesys Inc
Priority date: 2001-06-21
Filing date: 2003-04-07
Publication date: 2004-02-05
Also published as: MXPA03012021A; EP1406639A4; US6680306B2; AU2002320140B2; EP1406639A2; BR0210550A; US20040043962A1; WO2003000118A2; CZ200487A3; CA2451841A1; IL159461A0; CN1543351A; JP2004535430A; WO2003000118A3; CN100558367C; US20030013681A1

Abstract

The efficacy of conventional cancer therapies such as surgery, chemotherapy and radiation is enhanced by the use of a therapeutic material which binds to and interacts with galectins. The therapeutic material can enhance apoptosis thereby increasing the effectiveness of oncolytic agents. It can also inhibit angiogenesis thereby moderating tumor growth and/or metastasis.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. Ser. No. 10/176,235 filed Jun. 20, 2002, which claims the benefit of U.S. Provisional Application No. 60/299,991, filed Jun. 21, 2001, and entitled Method for Enhancing the Effectiveness of Cancer Therapies; the specifications of each of which are hereby incorporated by reference in their entirety.[0001]

BACKGROUND OF THE INVENTION

Galectins comprise a family of proteins which are expressed by plant and animal cells and which bind β-galactoside sugars. These proteins can be found on cell surfaces, in cytoplasm, in the nucleus, and in extracellular fluids. The two most studied galectins, galectin-1 and galectin-3, have a molecular weight in the general range of 13-16 kDa and 29-35 kD, respectively; they have an affinity for β-galactoside containing materials, and have been found to play a number of important roles in biological processes including cell migration, cell-cell adhesion, angiogenesis, cell fusion and other cell-cell interactions, as well as immune-based reactions and apoptosis. As such, the role of galectins is very strongly tied to cancer and other proliferative diseases. While there are a large number of galectins which manifest the foregoing activities, galectin-3 and galectin-1 have been strongly implicated in connection with cellular processes involving cancers.

Galectin-3 is a carbohydrate binding protein having a molecular weight of approximately 30,000. It is composed of two distinct structural motifs, an amino-terminal portion containing Gly-X-Y tandem repeats which are characteristic of collagens, and a carboxyl-terminal portion containing a carbohydrate binding site. Galectin-3 is found in almost all tumors, and has a binding affinity for β-galactoside-containing glyco-conjugates. Galectin-3 is believed to play a role in mediating cell-cell interactions and thereby fostering metastasis. It has been found that cells which have high expressions of galectin-3 are more prone to metastasis and are more resistant to apoptosis induced by chemotherapy or radiation. It has also been reported in the literature that galectin-3 plays a role in promoting angiogenesis.

Galectin-1 is a highly conserved homodimer of 14-15 kD and is one of the most abundant of the galectins. It binds to laminin which has been found to exert strong regulatory effects on cellular interactions such as adhesion, proliferation, migration and differentiation. In this regard, galectin-1 has been found to strongly influence these processes in various cells. It is believed to be implicated in the secretion of a number of cellular growth factors and interleukins. Galectin-1 has been found to be expressed at very high levels in many cancer cells and is strongly implicated in metastasis.

It has been shown that galectin-3 shares the “death suppression motif” of Bcl-2, a protein involved in the regulation of apoptosis, or programmed cell death. Bcl-2 is a member of a family of proteins regulating apoptosis. Some members of the family promote apoptosis, whereas others, including Bcl-2 and Bcl-xL, counterbalance by preventing it. The latter group is called herein “anti-apoptotic Bcl-2 protein.” In chemoresistant cells, changes in the activities of Bcl family of proteins by changes in the expression levels, phosphorylation state, or intracellular localization, that prevent the induction of apoptosis are often implicated as the mechanism of such resistance. Inhibition of anti-apoptotic Bcl-2 protein, in combination with the administration of cytotoxic or cytostatic chemotherapeutic agents, may overcome chemoresistance and restore or enhance the efficacy of cytotoxic chemotherapeutic agents or may potentiate the effect of cytotoxic or cytostatic chemotherapeutic agents. Overabundance of anti-apoptotic Bcl-2 protein, which is seen in some cancerous cells, correlates with the lack of cellular response to apoptosis inducers. Galectin-3 has the ability to form a heterodimer with Bcl-2, and, through this interaction, perhaps participate in the anti-apoptotic effect of Bcl-2. There is also evidence that the signal transduction pathway for galectin-3 may share some commonality with the Bcl-2 pathway.

The Bcl-2 pathway is a target of many cancer treatment regimens. Neoplasts that develop or possess resistance to antineoplastic agents often have elevated levels of anti-apoptotic Bcl-2 proteins and are resistant to apoptosis induction by these agents. In such instances, combination of antineoplastic agents with therapeutic agents that abolish the anti-apoptotic effect mediated by the anti-apoptotic Bcl-2 proteins is an effective treatment for those patients that fail to respond to the antineoplastic agents alone.

Conventional treatment for cancers and other diseases involving unwanted cellular proliferation involves the use of chemotherapeutic agents, radiation, and surgery, either alone or in combination. The medical arts have developed a number of treatments based upon the foregoing therapies.

BRIEF SUMMARY OF THE INVENTION

Disclosed herein is a method for enhancing the efficacy of a therapeutic treatment for cancer and other hyperproliferative disorders involving unwanted cellular proliferation, such as psoriasis or rheumatoid arthritis, in a patient by conjointly administering conventional therapy and specific materials disclosed herein. The treatment being enhanced may comprise chemotherapy, radiation therapy, surgery and combinations thereof. The specific material to be conjointly administered is a therapeutically effective amount of a compound which binds to a galectin. This compound may be administered prior to, after, or concomitant with the other treatment.

A preferred class of therapeutic treatment for cancer in a patient is chemotherapy. Chemotherapy may be carried out using various classes of therapeutic agents, including but not limited to: anti-proliferative agents, anti-angiogenic agents, antimitotic agents, antimicrotubule agents, antimetabolites, anti-migratory agents, differentiation modulators, growth factor inhibitors, cell cycle inhibitors, hormone analogs, apoptosis inducers, poly(ADP-ribose) polymerase inhibitors, DNA topoisomerase inhibitors, retinoic acid receptor alpha/beta selective agonists, and antibiotics.

A class of reagent of such chemotherapy is chemotherapeutic agents that interfere with DNA replication fidelity or cell-cycle progression of neoplastic cells. Alternatively, merely to illustrate, the chemotherapeutic can be an inhibitor of chromatin function, a topoisomerase inhibitor, a microtubule inhibiting drug, a DNA damaging agent, an antimetabolite (such as folate antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), a DNA synthesis inhibitor, a DNA interactive agent (such as an intercalating agent), and/or a DNA repair inhibitor. In addition to conventional chemotherapeutics, the agent of the subject method can also be antisense RNA, RNAi or other polynucleotides to inhibit the expression of the cellular components that contribute to unwanted cellular proliferation that are targets of conventional chemotherapy.

In other embodiments, the subject method combines an agent that binds to a galectin and reduces its biological activities related to cell proliferation, angiogenesis, and anti-apoptosis (“galectin inhibitor”) with a corticosteroid, such as cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prenisolone.

Another preferred class of therapeutic treatment is radiation therapy, and more specifically radioimmunotherapy or ionizing radiation therapy.

Another aspect of the invention provides a method for reducing the rate of growth of cells undergoing unwanted proliferation which express anti-apoptotic Bcl-2 proteins comprising, (i) obtaining a sample of unwanted proliferating cells from a patient; (ii) ascertaining the status of the anti-apoptotic Bcl-2 proteins of such cell sample; and (iii) for patients having cells that express the anti-apoptotic Bcl-2 proteins, administering treatment regimen including (a) a chemotherapeutic agent whose cytotoxicity is influenced by the status of the anti-apoptotic Bcl-2 proteins of the proliferating cell for cytotoxicity, (b) a galectin inhibitor.

Another aspect of the invention provides a kit that includes (i) a chemotherapeutic agent that interferes with DNA replication fidelity or cell-cycle progression of cells undergoing unwanted proliferation, (ii) a therapeutically effective amount of a galectin inhibitor; and (iii) instructions and/or a label for conjoint administration of the chemotherapeutic agent and the galectin inhibitor.

It has been found that certain therapeutic materials can bind to galectins thereby inactivating them toward interaction with other carbohydrate materials and/or cells. Specifically, it has been found that treatment of galectin-bearing cells with the therapeutic materials of this invention can inhibit the interaction of those cells with other cells and/or biomolecules and thereby inhibit angiogenesis and enhance the efficacy of apoptosis-inducing therapies such as chemotherapy or radiation. Furthermore, these materials can inhibit cell-cell interactions and thereby enhance the effectiveness of surgical therapies by inhibiting metastases, which are often initiated by surgical dislodgement of cells.

As will be explained in detail herein below, the materials employed in the present invention are generally comprised of natural or synthetic polymers and oligomers. They are very low in toxicity and interact synergistically with heretofore employed cancer therapies so as to increase the effectiveness thereof. Through the method of the present invention, the dosages of potentially toxic therapies such as chemotherapies and radiation may be reduced. Likewise, the effectiveness of surgical therapies is enhanced by the method of the present invention. For example, since the methodology of the present invention acts to inhibit the post-surgery metastatic process, use of this invention allows a surgeon to implement more aggressive surgical therapies without being limited by the possibility of precipitating metastatic events.

A preferred class of therapeutic materials of the present invention comprises a polymeric backbone having side chains dependent therefrom. The side chains are terminated by a galactose, rhamnose, xylose, or arabinose unit. This material may be synthetic, natural, or semi-synthetic. In one particular embodiment, the therapeutic compound comprises a substantially demethoxylated polygalacturonic acid backbone which is interrupted with rhamnose residues. Such compounds may be prepared from naturally occurring pectin, and are referred to as partially depolymerized pectin or modified pectin.

The method of present invention may be administering such materials orally, transdermally, by injection, by pulmonary inhalation, by subcutaneous implantation, or by topical application, depending upon the specific type of cancer or hyperproliferative disorder being treated, and the adjunct therapy.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. [0019] 1A-1C depict the promotion of apoptosis in vitro by formulations comprising modified pectin GCS-100 in a dose- and time-dependent manner.

DETAILED DESCRIPTION OF THE INVENTION

I. Overview [0020]
Conventional treatment for cancers and other diseases involving unwanted cellular proliferation involves the use of chemotherapeutic agents, radiation, and surgery, either alone or in combination. The medical arts have developed a number of treatments based upon the foregoing therapies. The present invention recognizes that the effectiveness of conventional therapies for cancer and other unwanted cellular proliferation, such as chemotherapy, surgery and radiation can be enhanced through the use of a therapeutic material which interacts with galectins. [0021]
Although chemotherapy has been effective in treating various types of malignancies, many antineoplastic compounds induce undesirable side effects. It has been shown that when two or more different treatments are combined, the treatments may work synergistically and allow reduction of dosage of each of the treatments, thereby reducing the detrimental side effects exerted by each compound at higher dosages. In other instances, malignancies that are refractory to a treatment may respond to a combination therapy of two or more different treatments. [0022]
A salient feature of certain aspects of the present invention relies on a relationship between anti-apoptotic Bcl-2 proteins and galectin-3 in regulating cell death, particularly that galectin-3 has a positive effect on the anti-apoptotic activity of these proteins. To further illustrate, galectin-3 expression has been implicated in sensitivity of tumor cells to certain chemotherapeutic agents, such as cisplatin and genistein. For instance, it has been observed that genistein effectively induces apoptosis in BT549 cells, a human breast epithelial cell line that does not express detectable levels of galectin-3. When galectin-3 transfected BT549 cells are treated with genistein, cell cycle arrest at the G(2)/M phase takes place without apoptosis induction. However, treatment of those cells with a galectin-3 inhibitor is sufficient to restore chemotherapeutic sensitivity. [0023]
The present invention is directed to methods and compositions for augmenting treatment of cancers and other hyperproliferative disorders such as psoriasis, rheumatoid arthritis, lamellar ichthyosis, epidermolytic hyperkeratosis, restenosis, endometriosis, or abnormal wound healing. In particular embodiments, the invention combines the administration of a galectin inhibitor with a chemotherapeutic agent so as to potentiate the cytotoxicity of the chemotherapeutic agent. In certain preferred embodiments, the conjoint therapies of the present invention can be used to improve the efficacy of those chemotherapeutic agents whose cytotoxicity is influenced by the status of an anti-apoptotic Bcl-2 protein for the treated cell. For instance, galectin inhibitors can be administered in combination with a chemotherapeutic agent that interferes with DNA replication fidelity or cell-cycle progression of cells undergoing unwanted proliferation. [0024]
Another aspect of the invention relies on the observation that galectins are involved in promoting angiogenesis. In order for a solid tumor to grow or metastasize the tumor must be vascularized. Galectin-3 in particular has been demonstrated to affect chemotaxis and morphology, and to stimulate angiogenesis in vivo. In accord with the present invention, a galectin inhibitor is administered to a patient in combination with conventional chemotherapy. [0025]
Depending on the nature of the cancer and the therapy, the galectin inhibitor may be administered prior to, contemporaneously with and/or after other therapies. When administration contemporaneously with other drugs, the galectin inhibitor may be formulated separately from, or co-formulated with, one or more of the other drugs. [0026]
II. Definitions [0027]
The terms “apoptosis” or “programmed cell death,” refers to the physiological process by which unwanted or useless cells are eliminated during development and other normal biological processes. Apoptosis is a mode of cell death that occurs under normal physiological conditions and the cell is an active participant in its own demise (“cellular suicide”). It is most often found during normal cell turnover and tissue homeostasis, embryogenesis, induction and maintenance of immune tolerance, development of the nervous system and endocrine-dependent tissue atrophy. Cells undergoing apoptosis show characteristic morphological and biochemical features. These features include chromatin aggregation, nuclear and cytoplasmic condensation, partition of cytoplasm and nucleus into membrane bound vesicles (apoptotic bodies) which contain ribosomes, morphologically intact mitochondria and nuclear material. Cytochrome C release from mitochondria is seen as an indication of mitochondrial dysfunction accompanying apoptosis. In vivo, these apoptotic bodies are rapidly recognized and phagocytized by either macrophages or adjacent epithelial cells. Due to this efficient mechanism for the removal of apoptotic cells in vivo no inflammatory response is elicited. In vitro, the apoptotic bodies as well as the remaining cell fragments ultimately swell and finally lyse. This terminal phase of in vitro cell death has been termed “secondary necrosis.”[0028]
The term “anti-apoptotic Bcl-2 protein” refers to a family of proteins related to the Bcl-2 protein and which are antagonists of cellular apoptosis. This family includes Bcl-2, Bcl-xL, Bcl-w, Mcl-1 and A-1. See, for example, Hockenbery et al., 1990, [0029] Nature 348:334-336; Boise et al., 1993, Cell 74:597-608; Gibson et al., 1996, Oncogene 13:665-675; Zhou et al., 1997, Blood 89 :630-643; and Lin et al., 1993, J. Inmunol. 151:1979-1988. This family of proteins shares four homology regions, termed Bcl homology (BH) domains, namely BH1, BH2, BH3, and BH4. A representative sequence for a human Bcl-2 coding sequence and protein are provided in GenBank Accession NM_—000657 (GI 4557356). A representative sequence for a human Bcl-xL coding sequence and protein are provided in GenBank Accession Z23115 (GI 510900). Exemplary anti-apoptotic Bcl-2 proteins are those which are at least 90 percent identical to the protein sequences set forth in GenBank Accessions NM_—000657 or Z23115, and/or which can be encoded by a nucleic acid sequence that hybridizes under stringent wash conditions of 0.2×SSC at 65 C. to a coding sequence set forth in GenBank Accessions NM_—000657 or Z23115.
The term “status of anti-apoptotic Bcl-2 proteins” includes within its meaning such quantitative measures as: the level of mRNA encoding an anti-apoptotic Bcl-2 protein; the level of the protein; the number and location of, or the absence of, phosphorylated residues or other posttranslational modifications; the intracellular localization of the protein; the status of association of anti-apoptotic Bcl-2 proteins with each other or with other proteins; and/or any other surrogate or direct measurement of anti-apoptotic activity due to an anti-apoptotic Bcl-2 protein. [0030]
More specifically, the term “status of anti-apoptotic Bcl-2 protein levels” means the amount of an anti-apoptotic Bcl-2 protein in a cell, such as may be detected by immunohistochemistry using antibodies specific to an anti-apoptotic Bcl-2 protein. [0031]
As used herein the term “animal” refers to mammals, preferably mammals such as humans. Likewise, a “patient” or “subject” to be treated by the method of the invention can mean either a human or non-human animal. [0032]
The term “antibody” as used herein, unless indicated otherwise, is used broadly to refer to both antibody molecules and a variety of antibody-derived molecules. Such antibody derived molecules comprise at least one variable region (either a heavy chain of light chain variable region), as well as individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains and other molecules, and the like. Functional immunoglobulin fragments according to the present invention may be Fv, scFv, disulfide-linked Fv, Fab, and F(ab′)2. [0033]
As used herein, the term “cancer” refers to any neoplastic disorder, including such cellular disorders as, for example, renal cell cancer, Kaposi's sarcoma, chronic leukemia, prostate cancer, breast cancer, sarcoma, pancreatic cancer, ovarian carcinoma, rectal cancer, throat cancer, melanoma, colon cancer, bladder cancer, lymphoma, mastocytoma, lung cancer, mammary adenocarcinoma, pharyngeal squamous cell carcinoma, and gastrointestinal or stomach cancer. Preferably, the cancer which is treated in the present invention is melanoma, myeloma, lung cancer, breast cancer, pancreatic cancer, prostate cancer, colon cancer, or ovarian cancer. [0034]
The “growth state” of a cell refers to the rate of proliferation of the cell and the state of differentiation of the cell. [0035]
As used herein, “hyperproliferative disease” or “hyperproliferative disorder” refers to any disorder which is caused by or is manifested by unwanted proliferation of cells in a patient. Hyperproliferative disorders include but are not limited to cancer, psoriasis, rheumatoid arthritis, lamellar ichthyosis, epidermolytic hyperkeratosis, restenosis, endometriosis, and abnormal wound healing. [0036]
As used herein, “proliferating” and “proliferation” refer to cells undergoing mitosis. [0037]
As used herein, “unwanted proliferation” means cell division and growth that is not part of normal cellular turnover, metabolism, growth, or propagation of the whole organism. Unwanted proliferation of cells is seen in tumors and other pathological proliferation of cells, does not serve normal function, and for the most part will continue unbridled at a growth rate exceeding that of cells of a normal tissue in the absence of outside intervention. A pathological state that ensues because of the unwanted proliferation of cells is referred herein as a “hyperproliferative disease” or “hyperproliferative disorder.”[0038]
As used herein, “transformed cells” refers to cells that have spontaneously converted to a state of unrestrained growth, i.e., they have acquired the ability to grow through an indefinite number of divisions in culture. Transformed cells may be characterized by such terms as neoplastic, anaplastic and/or hyperplastic, with respect to their loss of growth control. For purposes of this invention, the terms “transformed phenotype of malignant mammalian cells” and “transformed phenotype” are intended to encompass, but not be limited to, any of the following phenotypic traits associated with cellular transformation of mammalian cells: immortalization, morphological or growth transformation, and tumorigenicity, as detected by prolonged growth in cell culture, growth in semi-solid media, or tumorigenic growth in immuno-incompetent or syngeneic animals. [0039]
III. Exemplary Embodiments [0040]
A. Galectin Inhibitors [0041]
While galectins are known to bind galactose and other such simple sugars in vitro, those simple sugars are not therapeutically effective in moderating galectin mediated cellular processes in vivo. Preferred materials for the practice of the present invention generally comprise molecules which contain an active galectin binding sugar site, but which have somewhat higher molecular weights than simple sugars. [0042]
One group of materials falling within this general class comprises a substantially demethoxylated polygalacturonic acid backbone having rhamnose, galactose, arabinose, or other sugar residues pendent therefrom. It is believed that in materials of this type, the terminal galactose or arabinose units pendent from the backbone bind to galectin proteins. The remaining bulk of the molecule potentiates the compound's action in moderating immune system response. Materials of this general type are described by formulas I and II below, and it is to be understood that yet other variants of this general compound may be prepared and utilized in accord with the principles of the present invention. [0043]
1. Homogalacturonan[0044]
-[α-Galpa-(1→4)-α-GalpA]_n- (I)
[0045]
(Where m, n, o≧1, X can be either α-GalpA or α-Rhap; Y can be any of the following sugars (and can represent different sugars within a branch) such as α-Galp, β-Galp, β-Apif, β-Rhap, α-Rhap, α-Fucp, β-GlcpA, α-GalpA, β-GalpA, β-DhapA, Kdop, β-Acef, α-Galp, α-Arap, β-Araf, α-Xylp, not limited to these sugars. The abbreviated monomer names each corresponds to the following: GalA: galacturonic acid, Rha: rhamnose, Gal: galactose, Api: erythro-apiose, Fuc: fucose, GlcA: glucuronic acid, DhaA: 3-deoxy-D-lyxo-heptulosaric acid, Kdo: 3-deoxy-D-manno-2-octulosonic acid, Ace: aceric acid (3C-carboxy-5-deoxy-L-lyxose), Ara: arabinose. Italicized p stands for pyranose and italicized f stands for furanose.) [0046]
Pectin is a complex carbohydrate having a highly branched structure comprised of a polygalacturonic backbone with numerous branching side chains dependent therefrom. The branching creates regions which are characterized as being “smooth” and “hairy.” It has been found that pectin can be modified by various chemical, enzymatic or physical treatments to break the molecule into smaller portions having a more linearized, substantially demethoxylated, polygalacturonic backbone with pendant side chains of rhamnose residues having decreased branching. The resulting partially depolymerized pectin is known in the art as modified pectin, and its efficacy in treating cancer has been established; although galectin blocker materials of this type have not been used in conjunction with surgery, chemotherapy or radiation. [0047]
U.S. Pat. No. 5,895,784, the disclosure of which is incorporated herein by reference, describes modified pectin materials, techniques for their preparation, and use of the material as a treatment for various cancers. The material of the '784 patent is described as being prepared by a pH based modification procedure in which the pectin is put into solution and exposed to a series of programmed changes in pH which results in the breakdown of the molecule to yield therapeutically effective modified pectin. The material in the '784 patent is most preferably prepared from citrus pectin; although, it is to be understood that modified pectins may be prepared from pectin starting material obtained from other sources, such as apple pectin and the like. Also, modification processes may be accomplished by enzymatic treatment of the pectin, or by physical processes such as heating. Further disclosure of modified pectins and techniques for their preparation and use are also disclosed in U.S. Pat. No. 5,834,442 and U.S. patent application Ser. No. 08/024,487, the disclosures of which are incorporated herein by reference. Modified pectins of this type generally have molecular weights less than 100 kilodalton. A group of such materials has an average molecular weight of less than 3 kilodalton. Another group has an average molecular weight in the range of 1-15 kilodalton, with a specific group of materials having a molecular weight of about 10 kilodalton. [0048]
In certain embodiments, the modified pectin preparation is a substantially ethanol-free product suitable for parenteral administration. By substantially free of ethanol, it is meant that the compositions of the invention contain less than 5% ethanol by weight. In preferred embodiments the compositions contain less than 2%, and more preferably less than 0.5% ethanol by weight. In certain embodiments, the compositions further comprise one or more pharmaceutically acceptable excipients. Such compositions include aqueous solutions of the modified pectin of the invention. In certain embodiments of such aqueous solutions, the pectin modification occurs at a concentration of at least 7 mg/mL, and preferably at least 10 or even 15 or more mg/ml. Any of such compositions are also substantially free of organic solvents other than ethanol. [0049]
The apoptosis-promoting activity of a modified pectin material is illustrated in Example 1, below. [0050]
As disclosed in the prior art, such modified pectin materials have therapeutic efficacy against a variety of cancers and other hyperproliferative disorders. These materials interact with galectins, including galectin-1 and galectin-3, and in that regard also have efficacy against immune-based diseases. In accord with the present invention, the effect of conventional therapies for cancer and other hyperproliferative disorders is enhanced by use of pectin materials and other materials which interact with galectins. These materials may be administered orally; or by intravenous injection; or by injection directly into an affected tissue, as for example by injection into a tumor site. In some instances the materials may be applied topically at the time surgery is carried out. Also, other techniques such as transdermal delivery systems, inhalation, intramuscular injection, or subcutaneous implantation may be employed. [0051]
B. Chemotherapeutic Agents [0052]
Pharmaceutical agents that may be used for the subject combination chemotherapy include, merely to illustrate: aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine. [0053]

These chemotherapeutic agents may be categorized by their mechanism of action into, for example, following groups: anti-metabolites/anti-cancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, teniposide, triethylenethiophosphoramide and etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazinine (DTIC); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, COX-2 inhibitors, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; chromatin disruptors.

TABLE 1


Exemplary conventional combination cancer chemotherapy

Name	Therapeutic agents

ABV	Doxorubicin, Bleomycin, Vinblastine
ABVD	Doxorubicin, Bleomycin, Vinblastine, Dacarbazine
AC (Breast)	Doxorubicin, Cyclophosphamide
AC (Sarcoma)	Doxorubicin, Cisplatin
AC	Cyclophosphamide, Doxorubicin
(Neuro-
blastoma)
ACE	Cyclophosphamide, Doxorubicin, Etoposide
ACe	Cyclophosphamide, Doxorubicin
AD	Doxorubicin, Dacarbazine
AP	Doxorubicin, Cisplatin
ARAC-DNR	Cytarabine, Daunorubicin
B-CAVe	Bleomycin, Lomustine, Doxorubicin, Vinblastine
BCVPP	Carmustine, Cyclophosphamide, Vinblastine,
	Procarbazine, Prednisone
BEACOPP	Bleomycin, Etoposide, Doxorubicin, Cyclophosphamide,
	Vincristine, Procarbazine, Prednisone, Filgrastim
BEP	Bleomycin, Etoposide, Cisplatin
BIP	Bleomycin, Cisplatin, Ifosfamide, Mesna
BOMP	Bleomycin, Vincristine, Cisplatin, Mitomycin
CA	Cytarabine, Asparaginase
CABO	Cisplatin, Methotrexate, Bleomycin, Vincristine
CAF	Cyclophosphamide, Doxorubicin, Fluorouracil
CAL-G	Cyclophosphamide, Daunorubicin, Vincristine,
	Prednisone, Asparaginase
CAMP	Cyclophosphamide, Doxorubicin, Methotrexate,
	Procarbazine
CAP	Cyclophosphamide, Doxorubicin, Cisplatin
CaT	Carboplatin, Paclitaxel
CAV	Cyclophosphamide, Doxorubicin, Vincristine
CAVE ADD	CAV and Etoposide
CA-VP16	Cyclophosphamide, Doxorubicin, Etoposide
CC	Cyclophosphamide, Carboplatin
CDDP/VP-16	Cisplatin, Etoposide
CEF	Cyclophosphamide, Epirubicin, Fluorouracil
CEPP(B)	Cyclophosphamide, Etoposide, Prednisone, with or
	without/Bleomycin
CEV	Cyclophosphamide, Etoposide, Vincristine
CF	Cisplatin, Fluorouracil or Carboplatin Fluorouracil
CHAP	Cyclophosphamide or Cyclophosphamide, Altretamine,
	Doxorubicin, Cisplatin
ChlVPP	Chlorambucil, Vinblastine, Procarbazine, Prednisone
CHOP	Cyclophosphamide, Doxorubicin, Vincristine, Prednisone
CHOP-BLEO	Add Bleomycin to CHOP
CISCA	Cyclophosphamide, Doxorubicin, Cisplatin
CLD-BOMP	Bleomycin, Cisplatin, Vincristine, Mitomycin
CMF	Methotrexate, Fluorouracil Cyclophosphamide
CMFP	Cyclophosphamide, Methotrexate, Fluorouracil,
	Prednisone
CMFVP	Cyclophosphamide, Methotrexate, Fluorouracil,
	Vincristine, Prednisone
CMV	Cisplatin, Methotrexate, Vinblastine
CNF	Cyclophosphamide, Mitoxantrone, Fluorouracil
CNOP	Cyclophosphamide, Mitoxantrone, Vincristine,
	Prednisone
COB	Cisplatin, Vincristine, Bleomycin
CODE	Cisplatin, Vincristine, Doxorubicin, Etoposide
COMLA	Cyclophosphamide, Vincristine, Methotrexate,
	Leucovorin, Cytarabine
COMP	Cyclophosphamide, Vincristine, Methotrexate,
	Prednisone
Cooper	Cyclophosphamide, Methotrexate, Fluorouracil,
Regimen	Vincristine, Prednisone
COP	Cyclophosphamide, Vincristine, Prednisone
COPE	Cyclophosphamide, Vincristine, Cisplatin, Etoposide
COPP	Cyclophosphamide, Vincristine, Procarbazine,
	Prednisone
CP	Chlorambucil, Prednisone
(Chronic
Lymphocytic
Leukemia)
CP (Ovarian	Cyclophosphamide, Cisplatin
Cancer)
CT	Cisplatin, Paclitaxel
CVD	Cisplatin, Vinblastine, Dacarbazine
CVI	Carboplatin, Etoposide, Ifosfamide, Mesna
CVP	Cyclophosphamide, Vincristine, Prednisome
CVPP	Lomustine, Procarbazine, Prednisone
CYVADIC	Cyclophosphamide, Vincristine, Doxorubicin,
	Dacarbazine
DA	Daunorubicin, Cytarabine
DAT	Daunorubicin, Cytarabine, Thioguanine
DAV	Daunorubicin, Cytarabine, Etoposide
DCT	Daunorubicin, Cytarabine, Thioguanine
DHAP	Cisplatin, Cytarabine, Dexamethasone
DI	Doxorubicin, Ifosfamide
DTIC/	Dacarbazine, Tamoxifen
Tamoxifen
DVP	Daunorubicin, Vincristine, Prednisone
EAP	Etoposide, Doxorubicin, Cisplatin
EC	Etoposide, Carboplatin
EFP	Etoposie, Fluorouracil, Cisplatin
ELF	Etoposide, Leucovorin, Fluorouracil
EMA 86	Mitoxantrone, Etoposide, Cytarabine
EP	Etoposide, Cisplatin
EVA	Etoposide, Vinblastine
FAC	Fluorouracil, Doxorubicin, Cyclophosphamide
FAM	Fluorouracil, Doxorubicin, Mitomycin
FAMTX	Methotrexate, Leucovorin, Doxorubicin
FAP	Fluorouracil, Doxorubicin, Cisplatin
F-CL	Fluorouracil, Leucovorin
FEC	Fluorouracil, Cyclophosphamide, Epirubicin
FED	Fluorouracil, Etoposide, Cisplatin
FL	Flutamide, Leuprolide
FZ	Flutamide, Goserelin acetate implant
HDMTX	Methotrexate, Leucovorin
Hexa-CAF	Altretamine, Cyclophosphamide, Methotrexate,
	Fluorouracil
ICE-T	Ifosfamide, Carboplatin, Etoposide, Paclitaxel, Mesna
IDMTX/6-MP	Methotrexate, Mercaptopurine, Leucovorin
IE	Ifosfamide, Etoposie, Mesna
IfoVP	Ifosfamide, Etoposide, Mesna
IPA	Ifosfamide, Cisplatin, Doxorubicin
M-2	Vincristine, Carmustine, Cyclophosphamide, Prednisone,
	Melphalan
MAC-III	Methotrexate, Leucovorin, Dactinomycin,
	Cyclophosphamide
MACC	Methotrexate, Doxorubicin, Cyclophosphamide,
	Lomustine
MACOP-B	Methotrexate, Leucovorin, Doxorubicin,
	Cyclophosphamide, Vincristine, Bleomycin, Prednisone
MAID	Mesna, Doxorubicin, Ifosfamide, Dacarbazine
m-BACOD	Bleomycin, Doxorubicin, Cyclophosphamide,
	Vincristine, Dexamethasone, Methotrexate, Leucovorin
MBC	Methotrexate, Bleomycin, Cisplatin
MC	Mitoxantrone, Cytarabine
MF	Methotrexate, Fluorouracil, Leucovorin
MICE	Ifosfamide, Carboplatin, Etoposide, Mesna
MINE	Mesna, Ifosfamide, Mitoxantrone, Etoposide
mini-BEAM	Carmustine, Etoposide, Cytarabine, Melphalan
MOBP	Bleomycin, Vincristine, Cisplatin, Mitomycin
MOP	Mechlorethamine, Vincristine, Procarbazine
MOPP	Mechlorethamine, Vincristine, Procarbazine, Prednisone
MOPP/ABV	Mechlorethamine, Vincristine, Procarbazine, Prednisone,
	Doxorubicin, Bleomycin, Vinblastine
MP (multiple	Melphalan, Prednisone
myeloma)
MP (prostate	Mitoxantrone, Prednisone
cancer)
MTX/6-MO	Methotrexate, Mercaptopurine
MTX/6-MP/VP	Methotrexate, Mercaptopurine, Vincristine, Prednisone
MTX-	Methotrexate, Leucovorin, Cisplatin, Doxorubicin
CDDPAdr
MV (breast	Mitomycin, Vinblastine
cancer)
MV (acute	Mitoxantrone, Etoposide
myelocytic
leukemia)
M-VAC	Vinblastine, Doxorubicin, Cisplatin
Methotrexate
MVP	Vinblastine, Cisplatin
Mitomycin
MVPP	Mechlorethamine, Vinblastine, Procarbazine, Prednisone
NFL	Mitoxantrone, Fluorouracil, Leucovorin
NOVP	Mitoxantrone, Vinblastine, Vincristine
OPA	Vincristine, Prednisone, Doxorubicin
OPPA	Add Procarbazine to OPA.
PAC	Cisplatin, Doxorubicin
PAC-I	Cisplatin, Doxorubicin, Cyclophosphamide
PA-CI	Cisplatin, Doxorubicin
PC	Paclitaxel, Carboplatin or Paclitaxel, Cisplatin
PCV	Lomustine, Procarbazine, Vincristine
PE	Paclitaxel, Estramustine
PFL	Cisplatin, Fluorouracil, Leucovorin
POC	Prednisone, Vincristine, Lomustine
ProMACE	Prednisone, Methotrexate, Leucovonn, Doxorubicin,
	Cyclophosphamide, Etoposide
ProMACE/	Prednisone, Doxorubicin, Cyclophosphamide, Etoposide,
cytaBOM	Cytarabine, Bleomycin, Vincristine, Methotrexate,
	Leucovorin, Cotrimoxazole
PRoMACE/	Prednisone, Doxorubicin, Cyclophosphamide, Etoposide,
MOPP	Mechlorethamine, Vincristine, Procarbazine,
	Methotrexate, Leucovorin
Pt/VM	Cisplatin, Teniposide
PVA	Prednisone, Vincristine, Asparaginase
PVB	Cisplatin, Vinblastine, Bleomycin
PVDA	Prednisone, Vincristine, Daunorubicin, Asparaginase
SMF	Streptozocin, Mitomycin, Fluorouracil
TAD	Mechlorethamine, Doxorubicin, Vinblastine, Vincristine,
	Bleomycin, Etoposide, Prednisone
TCF	Paclitaxel, Cisplatin, Fluorouracil
TIP	Paclitaxel, Ifosfamide, Mesna, Cisplatin
TTT	Methotrexate, Cytarabine, Hydrocortisone
Topo/CTX	Cyclophosphamide, Topotecan, Mesna
VAB-6	Cyclophosphamide, Dactinomycin, Vinblastine,
	Cisplatin, Bleomycin
VAC	Vincristine, Dactinomycin, Cyclophosphamide
VACAdr	Vincristine, Cyclophosphamide, Doxorubicin,
	Dactinomycin, Vincristine
VAD	Vincristine, Doxorubicin, Dexamethasone
VATH	Vinblastine, Doxorubicin, Thiotepa, Flouxymesterone
VBAP	Vincristine, Carmustine, Doxorubicin, Prednisone
VBCMP	Vincristine, Carmustine, Melphalan, Cyclophosphamide,
	Prednisone
VC	Vinorelbine, Cisplatin
VCAP	Vincristine, Cyclophosphamide, Doxorubicin, Prednisone
VD	Vinorelbine, Doxorubicin
VelP	Vinblastine, Cisplatin, Ifosfamide, Mesna
VIP	Etoposide, Cisplatin, Ifosfamide, Mesna
VM	Mitomycin, Vinblastine
VMCP	Vincristine, Melphalan, Cyclophosphamide, Prednisone
VP	Etoposide, Cisplatin
V-TAD	Etoposide, Thioguanine, Daunorubicin, Cytarabine
5 + 2	Cytarabine, Daunorubicin, Mitoxantrone
7 + 3	Cytarabine with/, Daunorubicin or Idarubicin or
	Mitoxantrone
“8 in 1”	Methylprednisolone, Vincristine, Lomustine,
	Procarbazine, Hydroxyurea, Cisplatin, Cytarabine,
	Dacarbazine

In addition to conventional chemotherapeutics, the agent of the subject method can also be compounds and antisense RNA, RNAi or other polynucleotides to inhibit the expression of the cellular components that contribute to unwanted cellular proliferation that are targets of conventional chemotherapy. Such targets are, merely to illustrate, growth factors, growth factor receptors, cell cycle regulatory proteins, transcription factors, or signal transduction kinases. [0055]
An antisense nucleic acid can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes a target gene. Alternatively, the construct is an oligonucleotide which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences encoding a target gene. Such oligonucleotide are optionally modified oligonucleotide which are resistant to endogenous nucleases, e.g., exonucleases and/or endonucleases, and is therefore stable in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in nucleic acid therapy have been reviewed, for example, by van der Krol et al., 1988, [0056] Biotechniques 6:958-976; and Stein et al., 1988, Cancer Res. 48:2659-2668.
In another embodiment, the invention relates to the use of RNA interference (RNAi) to effect knockdown of expression of a target gene. RNAi constructs comprise double stranded RNA that can specifically block expression of a target gene. “RNA interference” or “RNAi” is a term initially applied to a phenomenon observed in plants and worms where double-stranded RNA (dsRNA) blocks gene expression in a specific and post-transcriptional manner. RNAi provides a useful method of inhibiting gene expression in vitro or in vivo. As used herein, the term “RNAi construct” is a generic term including small interfering RNAs (siRNAs), hairpin RNAs, and other RNA species which can be cleaved in vivo to form siRNAs. RNAi constructs herein also include expression vectors (also referred to as RNAi expression vectors) capable of giving rise to transcripts which form dsRNAs or hairpin RNAs in cells, and/or transcripts which can produce siRNAs in vivo. [0057]
RNAi constructs can comprise either long stretches of dsRNA identical or substantially identical to the target nucleic acid sequence or short stretches of dsRNA identical to substantially identical to only a region of the target nucleic acid sequence. [0058]
Optionally, the RNAi constructs contain a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the mRNA transcript for the gene to be inhibited (i.e., the “target” gene). The double-stranded RNA need only be sufficiently similar to natural RNA that it has the ability to mediate RNAi. Thus, the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism or evolutionary divergence. The number of tolerated nucleotide mismatches between the target sequence and the RNAi construct sequence is no more than 1 in 5 basepairs, or 1 in 10 basepairs, or 1 in 20 basepairs, or 1 in 50 basepairs. Mismatches in the center of the siRNA duplex are most critical and may essentially abolish cleavage of the target RNA. In contrast, nucleotides at the 3′ end of the siRNA strand that is complementary to the target RNA do not significantly contribute to specificity of the target recognition. Sequence identity may be optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the portion of the target gene is preferred. Alternatively, the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed by washing). [0059]
The double-stranded structure may be formed by a single self-complementary RNA strand or two complementary RNA strands. RNA duplex formation may be initiated either inside or outside the cell. The RNA may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of double-stranded material may yield more effective inhibition, while lower doses may also be useful for specific applications. Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition. [0060]
The subject RNAi constructs can be “small interfering RNAs” or “siRNAs.” These nucleic acids are around 19-30 nucleotides in length, and even more preferably 21-23 nucleotides in length. The siRNAs are understood to recruit nuclease complexes and guide the complexes to the target mRNA by pairing to the specific sequences. As a result, the target mRNA is degraded by the nucleases in the protein complex. In a particular embodiment, the 21-23 nucleotides siRNA molecules comprise a 3′ hydroxyl group. In certain embodiments, the siRNA constructs can be generated by processing of longer double-stranded RNAs, for example, in the presence of the enzyme dicer. In one embodiment, the Drosophila in vitro system is used. In this embodiment, dsRNA is combined with a soluble extract derived from Drosophila embryo, thereby producing a combination. The combination is maintained under conditions in which the dsRNA is processed to RNA molecules of about 21 to about 23 nucleotides. The siRNA molecules can be purified using a number of techniques known to those of skill in the art. For example, gel electrophoresis can be used to purify siRNAs. Alternatively, non-denaturing methods, such as non-denaturing column chromatography, can be used to purify the siRNA. In addition, chromatography (e.g., size exclusion chromatography), glycerol gradient centrifugation, affinity purification with antibody can be used to purify siRNAs. [0061]
Production of RNAi constructs can be carried out by chemical synthetic methods or by recombinant nucleic acid techniques. Endogenous RNA polymerase of the treated cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vitro. The RNAi constructs may include modifications to either the phosphate-sugar backbone or the nucleoside, e.g., to reduce susceptibility to cellular nucleases, improve bioavailability, improve formulation characteristics, and/or change other pharmacokinetic properties. For example, the phosphodiester linkages of natural RNA may be modified to include at least one of an nitrogen or sulfur heteroatom. Modifications in RNA structure may be tailored to allow specific genetic inhibition while avoiding a general response to dsRNA. Likewise, bases may be modified to block the activity of adenosine deaminase. The RNAi construct may be produced enzymatically or by partial/total organic synthesis, any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis. Methods of chemically modifying RNA molecules can be adapted for modifying RNAi constructs (see, e.g., Heidenreich et al., 1997, [0062] Nucleic Acids Res. 25:776-780; Wilson et al., 1994, J. Mol. Recog. 7:89-98; Chen et al., 1995, Nucleic Acids Res. 23:2661-2668; Hirschbein et al., 1997, Antisense Nucleic Acid Drug Dev. 7:55-61). Merely to illustrate, the backbone of an RNAi construct can be modified with pbosphorothioates, phosphoramidate, phosphodithioates, chimeric methylphosphonate-phosphodiesters, peptide nucleic acids, 5-propynyl-pyrimidine containing oligomers or sugar modifications (e.g., 2′-substituted ribonucleosides, a-configuration).
In some cases, at least one strand of the siRNA molecules has a 3′ overhang from about 1 to about 6 nucleotides in length, though may be from 2 to 4 nucleotides in length. More preferably, the 3′ overhangs are 1-3 nucleotides in length. In certain embodiments, one strand having a 3′ overhang and the other strand being blunt-ended or also having an overhang. The length of the overhangs may be the same or different for each strand. In order to further enhance the stability of the siRNA, the 3′ overhangs can be stabilized against degradation. In one embodiment, the RNA is stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine nucleotide 3′ overhangs by 2′-deoxythyinidine is tolerated and does not affect the efficiency of RNAi. The absence of a 2′ hydroxyl significantly enhances the nuclease resistance of the overhang in tissue culture medium and may be beneficial in vivo. [0063]
The RNAi construct can also be in the form of a long double-stranded RNA. In certain embodiments, the RNAi construct is at least 25, 50, 100, 200, 300 or 400 bases. In certain embodiments, the RNAi construct is 400-800 bases in length. The double-stranded RNAs are digested intracellularly, e.g., to produce siRNA sequences in the cell. However, use of long double-stranded RNAs in vivo is not always practical, presumably because of deleterious effects which may be caused by the sequence-independent dsRNA response. In such embodiments, the use of local delivery systems and/or agents which reduce the effects of interferon or PKR are preferred. [0064]
Alternatively, the RNAi construct is in the form of a hairpin structure (named as hairpin RNA). The hairpin RNAs can be synthesized exogenously or can be formed by transcribing from RNA polymerase III promoters in vivo. Examples of making and using such hairpin RNAs for gene silencing in mammalian cells are described in, for example, Paddison et al., 2002, [0065] Genes Dev. 16:948-58; McCaffrey et al., 2002, Nature 418:38-9; McManus et al., 2002, RNA 8:842-50; Yu et al., 2002, Proc. Nat'l Acad. Sci. USA 99:6047-52). Preferably, such hairpin RNAs are engineered in cells or in an animal to ensure continuous and stable suppression of a desired gene. It is known in the art that siRNAs can be produced by processing a hairpin RNA in the cell.
PCT application WO 01/77350 describes an exemplary vector for bi-directional transcription of a transgene to yield both sense and antisense RNA transcripts of the same transgene in a eukaryotic cell. Accordingly, in certain embodiments, the present invention provides a recombinant vector having the following unique characteristics: it comprises a viral replicon having two overlapping transcription units arranged in an opposing orientation and flanking a transgene for an RNAi construct of interest, wherein the two overlapping transcription units yield both sense and antisense RNA transcripts from the same transgene fragment in a host cell. [0066]
In another embodiment, the invention relates to the use of ribozyme molecules designed to catalytically cleave a target mRNA transcripts to prevent translation of the mRNA (see, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al., 1990, [0067] Science 247:1222-1225; and U.S. Pat. No. 5,093,246). While ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy particular mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5′-UG-3′. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, 1988, Nature 334:585-591. The ribozymes of the present invention also include RNA endoribonucleases (hereinafter “Cech-type ribozymes”) such as the one which occurs naturally in Tetrahymena thermophila (known as the IVS or L-19 IVS RNA) and which has been extensively described (see, e.g., Zaug et al., 1984, Science 224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug et al., 1986, Nature 324:429-433; published International patent application No. WO88/04300 by University Patents Inc.; Been and Cech, 1986, Cell 47:207-216).
In a further embodiment, the invention relates to the use of DNA enzymes to inhibit expression of a target gene which encodes a cellular component relevant to cellular proliferation. DNA enzymes incorporate some of the mechanistic features of both antisense and ribozyme technologies. DNA enzymes are designed so that they recognize a particular target nucleic acid sequence, much like an antisense oligonucleotide, however much like a ribozyme they are catalytic and specifically cleave the target nucleic acid. Briefly, to design an ideal DNA enzyme that specifically recognizes and cleaves a target nucleic acid, one of skill in the art must first identify the unique target sequence. Preferably, the unique or substantially sequence is a G/C rich of approximately 18 to 22 nucleotides. High G/C content helps insure a stronger interaction between the DNA enzyme and the target sequence. When synthesizing the DNA enzyme, the specific antisense recognition sequence that will target the enzyme to the message is divided so that it comprises the two arms of the DNA enzyme, and the DNA enzyme loop is placed between the two specific arms. Methods of making and administering DNA enzymes can be found, for example, in U.S. Pat. No. 6,110,462. [0068]
The method of present invention is advantageous over combination therapies known in the art because it allows conventional chemotherapeutic agent to exert greater effect at lower dosage. In preferred embodiment of the present invention, the effective dose (ED50) for a chemotherapeutic agent or combination of conventional chemotherapeutic agents when used in combination with the compound of present invention is at least 5 fold less than the ED50 for the chemotherapeutic agent alone. Conversely, the therapeutic index (TI) for such chemotherapeutic agent or combination of such chemotherapeutic agent when used in combination with the compound of present invention is at least 5 fold greater than the TI for conventional chemotherapeutic regimen alone. [0069]
C. Radiation Therapy [0070]
Radiation therapy, which includes gamma radiation as well as particle beams, and chemotherapeutic agents, for cancer and other hyperproliferative disorders, are cytotoxic, and their effectiveness in treating a disorder is based upon the fact that unwanted proliferating cells are generally more sensitive to such cytotoxic therapies than are normal cells either because of their rapid metabolism, or because they employ biochemical pathways not employed by normal cells. It is believed that these therapies exert their cytotoxic effects by activating programmed cell death, also referred to as apoptosis. Cells undergo apoptosis when they undergo a critical level of damage. A balance between the activities of apoptotic and anti-apoptotic intracellular signal transduction pathways is important toward a cell's decision of whether to undergo apoptosis or to attempt internal repair. It has been demonstrated that galectins, and specifically galectin-3, are involved in both apoptosis resistance and tumor progression. [0071]
D. Administration [0072]
The therapeutic materials of the present invention may be administered orally, by injection, transdermally, by pulmonary inhalation, by subcutaneous implantation, or by topical application, depending upon the specific type of cancer being treated, and the adjunct therapy. [0073]
In accord with the present invention, a galectin binding therapeutic material is administered to a patient, in combination with conventional therapies such as surgery, radiation or chemotherapy. The material is most preferably administered prior to the administration of the conventional therapy, so as to allow it sufficient time to interact with and bind to galectins in the tumor or in non-cancerous cells. Depending on the nature of the cancer and the therapy, administration of the galectin binding therapeutic material may be continued while the other therapy is being administered and/or thereafter. Administration of the galectin binding material may be made in a single dose, or in multiple doses. In some instances, administration of the therapeutic material is commenced at least several days prior to the conventional therapy, while in other instances, administration is begun either immediately before or at the time of the administration of the conventional therapy. [0074]
In some instances, particularly with regard to surgical therapies, the carbohydrate material may be advantageously administered. both before, during and after the therapy. [0075]
E. Exemplary Targets for Treatment [0076]
The method of present invention is effective in treatment of various types of cancers, including but not limited to: pancreatic cancer, renal cell cancer, Kaposi's sarcoma, chronic leukemia, breast cancer, sarcoma, ovarian carcinoma, rectal cancer, throat cancer, melanoma, colon cancer, bladder cancer, lymphoma, mastocytoma, lung cancer, mammary adenocarcinoma, pharyngeal squamous cell carcinoma, gastrointestinal cancer, stomach cancer, myeloma or prostate cancer. [0077]
The present invention is also effective against other diseases related to unwanted cell proliferation. Such hyperproliferative diseases include but are not limited to: psoriasis, rheumatoid arthritis, lamellar ichthyosis, epidermolytic hyperkeratosis, restenosis, endometriosis, or abnormal wound healing. [0078]
Because galectin-3 and Bcl-2 or Bclx interact and because the compounds of present invention are especially useful to treat cells with elevated anti-apoptotic Bcl-2 protein activities, it is beneficial to determine the level of active anti-apoptotic Bcl-2 proteins in a tumor or in leukemic cells in a patient. The presence of elevated levels of anti-apoptotic Bcl-2 proteins in a tumor can be determined by immunodetection using antibodies specific to each of these proteins, either through enzyme-linked immunosolvent assays, or immunohistochemistry of solid tumor samples. The immunohistochemistry will also allow determination of the intracellular localization of anti-apoptotic Bcl-2 protein in a tumor sample. By using monoclonal antibodies specific to phosphorylated proteins, the phosphorylation state of anti-apoptotic Bcl-2 proteins can also be determined by the same techniques. The expression of anti-apoptotic Bcl-2 proteins can be determined by detecting the levels of mRNA in Southern blots, using probes specific to the nucleotide sequence of each of the anti-apoptotic Bcl-2 proteins. Alternatively, quantitative polymerase chain reaction may be done, using a pair of primers specific to each of the anti-apoptotic Bcl-2 protein. Once the expression levels and the status of the anti-apoptotic Bcl-2 protein are determined, a patient with cancerous growth which have elevated levels of anti-apoptotic Bcl-2 protein activities are treated with the compounds of present invention along with other anti-cancer therapies as necessary. [0079]

F. EXAMPLES

Example 1

Promotion of Apoptosis by a Modified Pectin [0080]
Experiments were performed to demonstrate the ability of a modified pectin to promote apoptosis in a cell line with high Bcl-2 expression and chemoresistance. [0081]
Cell line DoHH2 is a spontaneously growing EBV-negative B-cell line, established from the pleural fluid cells of a patient with centroblastic/centrocytic non-Hodgkin's lymphoma, that had transformed into an immunoblastic lymphoma. Kluin-Nelemans et al., “A new non-Hodgkin's B-cell line (DoHH2) with a chromosomal translocation t(14;18)(q32;q21),” [0082] Leukemia Mar. 5, 1991 (3):221-4. The expression of Bcl-2 is upregulated in DoHH2 due to chromosomal translocation, and the cell line is known to have high chemoresistance that is dependent on the status of Bcl-2. When treated with a Bcl-2 antisense polynucleotide, DoHH2 proceeds to apoptosis, indicating the overexpression of Bcl-2 is a cause of lack of apoptosis.
DoHH2 cells were exposed to modified pectin GCS-100 in three different formulations, V1, V2, and V3. Formulation VI contained 12.6% ethanol, V2 contained 15% ethanol, and V3 contained 0.2% ethanol. In vitro apoptosis was quantitated by DioC6(3) stain as a measure of mitochondrial depolarization at 4, 24, 48, and 72 hours after 0, 40, 80, 160, or 320 μg /ml of each formulation was added to cell culture. See FIGS. [0083] 1A-1C. All samples demonstrated increased apoptosis over time, but the addition of GCS-100 increased the number of cells undergoing apoptosis in a dose-dependent manner. The three formulations performed similarly at the highest dose of 320 μg/ml, but at lower dosages of 40, 80, or 160 μg /ml, formulation V3, which contained the least amount of ethanol, was more effective in inducing apoptosis at earlier time points compared to formulation V1 or V2.
The foregoing discussion has been primary directed toward modified pectin materials and materials which interact with galectins-1 and 3; however, it is to be understood that other galectins are also known to be involved in the progress of various cancers, and both the modified pectin material as well as the other therapeutic materials discussed hereinabove interact with galectins. Therefore, other materials and methods may be employed in the practice of the present invention. The foregoing discussion and description is illustrative of specific embodiments, but is not meant to be a limitation upon the practice thereof. It is the following claims, including all equivalents, which define the scope of the invention. [0084]

Claims

We claim:

1. A method for enhancing the efficacy of a therapeutic treatment for cancer and unwanted proliferation of cells in a patient, said therapeutic treatment one which the degree of cytotoxicity is influenced by the status of an anti-apoptotic Bcl-2 protein of the tumor cell, said method further comprising the conjoint administration to said patient of a therapeutically effective amount of a partially depolymerized pectin having a galacturan backbone which may be disrupted by rhamnose and low branched neutral sugars dependent from said backbone.

2. A method for enhancing the pro-apoptotic effect of a chemotherapeutic agent that interferes with DNA replication fidelity or cell-cycle progression of cells undergoing unwanted proliferation in a patient, comprising the conjoint administration to said patient of a therapeutically effective amount of a partially depolymerized pectin having a galacturan backbone which may be disrupted by rhamnose and low branched neutral sugars dependent from said backbone.

3. A method for treating chemoresistant tumors in a patient, wherein said tumors have elevated levels of anti-apoptotic Bcl-2 proteins (relative to wild-type), comprising the conjoint administration to said patient of

(i) a therapeutically effective amount of a partially depolymerized pectin having a galacturan backbone which may be disrupted by rhamnose and low branched neutral sugars dependent from said backbone, and

(ii) a chemotherapeutic agent that induces apoptosis in a manner influenced by the anti-apoptotic Bcl-2 protein status of the tumor cell.

4. A method for enhancing the cytotoxicity of a chemotherapeutic for killing cells undergoing unwanted proliferation in a patient, comprising the conjoint administration to said patient of a therapeutically effective amount of a partially depolymerized pectin having a galacturan backbone which may be disrupted by rhamnose and low branched neutral sugars dependent from said backbone.

5. A method for rendering a cytostatic agent cytotoxic for cells undergoing unwanted proliferation, wherein said agent's cytotoxicity is influenced by the status of an anti-apoptotic Bcl-2 protein, comprising the conjoint administration of a therapeutically effective amount of a partially depolymerized pectin having a galacturan backbone which may be disrupted by rhamnose and low branched neutral sugars dependent from said backbone with said cytostatic agent.

6. The method of claim 2, 3 or 4, wherein the chemotherapeutic agent induces mitochondrial dysfunction and/or caspase activation.

7. The method of claim 2, 3 or 4, wherein the chemotherapeutic agent induces cell cycle arrest at G2/M in the absence of said partially depolymerized pectin.

8. The method of claim 2, 3 or 4, wherein said chemotherapeutic is an inhibitor of chromatin function.

9. The method of claim 8, wherein said chemotherapeutic is a topoisomerase inhibitor.

10. The method of claim 9, wherein said topoisomerase inhibitor is selected from the group consisting of adriamycin, amsacrine, camptothecin, daunorubicin, dactinomycin, doxorubicin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone.

11. The method of claim 8, wherein said inhibitor is a microtubule targeting drug.

12. The method of claim 11, wherein said microtubule targeting drug is a taxane.

13. The method of claim 11, wherein said microtubule targeting drug is selected from the group consisting of paclitaxel, docetaxel, vincristin, vinblastin, nocodazole, epothilones and navelbine.

14. The method of claim 2, 3 or 4, wherein said chemotherapeutic is a DNA damaging agent.

15. The method of claim 14, wherein said DNA damaging agent is selected from the group consisting of actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, paclitaxel, plicamycin, procarbazine, teniposide, triethylenethiophosphoramide and etoposide (VP16).

16. The method of claim 2, 3 or 4, wherein said chemotherapeutic is an antimetabolite.

17. The method of claim 16, wherein said antimetabolite is selected from the group consisting of folate antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs.

18. The method of claim 2, 3 or 4, wherein said chemotherapeutic is a DNA synthesis inhibitor.

19. The method of claim 18, wherein said DNA synthesis inhibitor is a thymidilate synthase inhibitors, such as 5-fluorouracil.

20. The method of claim 18, wherein said DNA synthesis inhibitor is a dihydrofolate reductase inhibitor, such as methoxtrexate.

21. The method of claim 18, wherein said DNA synthesis inhibitor is a DNA polymerase inhibitor, such as fludarabine.

22. The method of claim 2, 3 or 4, wherein said chemotherapeutic is a DNA binding agent.

23. The method of claim 22, wherein said DNA binding agent is an intercalating agent.

24. The method of claim 2, 3 or 4, wherein said chemotherapeutic is a DNA repair inhibitor.

25. The method of any of claims 2-5, wherein said chemotherapeutic is a corticosteroid.

26. The method of claim 25, wherein said corticosteroid is selected from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prenisolone.

27. The method of any of claims 2-5, wherein said chemotherapeutic is a combinatorial therapy selected from the group consisting of ABV, ABVD, AC (Breast), AC (Sarcoma), AC (Neuroblastoma), ACE, ACe, AD, AP, ARAC-DNR, B-CAVe, BCVPP, BEACOPP, BEP, BIP, BOMP, CA, CABO, CAF, CAL-G, CAMP, CAP, CaT, CAV, CAVE ADD, CA-VP16, CC, CDDP/VP-16, CEF, CEPP(B), CEV, CF, CHAP, ChlVPP, CHOP, CHOP-BLEO, CISCA, CLD-BOMP, CMF, CMFP, CMFVP, CMV, CNF, CNOP, COB, CODE, COMLA, COMP, Cooper Regimen, COP, COPE, COPP, CP—Chronic Lymphocytic Leukemia, CP—Ovarian Cancer, CT, CVD, CVI, CVP, CVPP, CYVADIC, DA, DAT, DAV, DCT, DHAP, DI, DTIC/Tamoxifen, DVP, EAP, EC, EFP, ELF, EMA 86, EP, EVA, FAC, FAM, FAMTX, FAP, F-CL, FEC, FED, FL, FZ, HDMTX, Hexa-CAF, ICE-T, IDMTX/6-MP, IE, IfoVP, IPA, M-2, MAC-III, MACC, MACOP-B, MAID, m-BACOD, MBC, MC, MF, MICE, MINE, mini-BEAM, MOBP, MOP, MOPP, MOPP/ABV, MP—multiple myeloma, MP—prostate cancer, MTX/6-MO, MTX/6-MP/VP, MTX-CDDPAdr, MV—breast cancer, MV—acute myelocytic leukemia, M-VAC Methotrexate, MVP Mitomycin, MVPP, NFL, NOVP, OPA, OPPA, PAC, PAC-I, PA-CI, PC, PCV, PE, PFL, POC, ProMACE, ProMACE/cytaBOM, PRoMACE/MOPP, Pt/VM, PVA, PVB, PVDA, SMF, TAD, TCF, TIP, TTT, Topo/CTX, VAB-6, VAC, VACAdr, VAD, VATH, VBAP, VBCMP, VC, VCAP, VD, VelP, VIP, VM, VMCP, VP, V-TAD, 5+2, 7+3, “8 in 1”.

28. The method of claim 1, wherein said therapeutic treatment includes ionizing radiation.

29. The method of claim 5, wherein cytostatic agent is selected from the group consisting of tamoxifen, 4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline, 4-(3-ethynylphenylamino)-6,7-bis(2-metho-xyethoxy)quinazoline, hormone, steroids, steroid synthetic analogs, 17a-Ethinylestradiol, Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone, Dromostanolone propionate, Testolactone, Megestrolacetate, Methylprednisolone, Methyl-testosterone, Prednisolone, Triamcinolone, chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate, Leuprolide, Flutamide, Toremifene, Zoladex, antiangiogenics, matrix metalloproteinase inhibitors, VEGF inhibitors, ZD6474, SU6668, SU11248, anti-Her2 antibodies (ZD1839 and OS1774), EGFR inhibitors, EKB-569, Imclone antibody C225, src inhibitors, bicalutamide, epidermal growth factor inhibitors, Her-2 inhibitors, MEK-1 kinase inhibitors, MAPK kinase inhibitors, P13 inhibitors, PDGF inhibitors, combretastatins, MET kinase inhibitors, MAP kinase inhibitors, inhibitors of non-receptor and receptor tyrosine kinases (imatinib), inhibitors of integrin signaling, and inhibitors of insulin-like growth factor receptors.

30. The method of any of claims 2-4, wherein the chemotherapeutic is selected from the group consisting of aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine

31. The method of claim 1, used to inhibit growth of a tumor cell selected from the group consisting of a pancreatic tumor cell, a lung tumor cell, a prostate tumor cell, a breast tumor cell, a colon tumor cell, a liver tumor cell, a brain tumor cell, a kidney tumor cell, a skin tumor cell and an ovarian tumor cell.

32. The method of claim 1, wherein said tumor cell is selected from the group consisting of a squamous cell carcinoma, a non-squamous cell carcinoma, a glioblastoma, a sarcoma, an adenocarcinoma, a myeloma, a melanoma, a papilloma, a neuroblastoma and a leukemia cell.

33. The method of claim 1, used in the treatment of a proliferative disorder selected from the group consisting of a pancreatic cancer, renal cell cancer, Kaposi's sarcoma, chronic leukemia, breast cancer, sarcoma, ovarian carcinoma, rectal cancer, throat cancer, melanoma, colon cancer, bladder cancer, lymphoma, mastocytoma, lung cancer, mammary adenocarcinoma, pharyngeal squamous cell carcinoma, gastrointestinal cancer, stomach cancer, myeloma, or prostate cancer.

34. The method of claim 1, wherein said partially depolymerized pectin is a substantially demethoxylated polygalacturonic acid which is interrupted with rhamnose residues.

35. The method of claim 1, wherein said partially depolymerized pectin comprises a pH modified pectin, an enzymatically modified pectin, and/or a thermally modified pectin.

36. The method of claim 35, wherein said modified pectin comprises a modified citrus pectin.

37. The method of claim 1, wherein said partially depolymerized pectin is administered simultaneously with said therapeutic treatment, chemotherapeutic or cytostatic agent.

38. The method of claim 1, wherein said partially depolymerized pectin is administered before treatment with said therapeutic treatment, chemotherapeutic or cytostatic agent.

39. The method of claim 1, wherein said partially depolymerized pectin is administered after treatment with said therapeutic treatment, chemotherapeutic or cytostatic agent.

40. The method of claim 1, wherein said partially depolymerized pectin is administered by intravenous infusion.

41. The method of claim 1, wherein said partially depolymerized pectin is administered orally.

42. The method of claim 1, wherein said partially depolymerized pectin is administered by intramuscular or intraperitoneal injection or infusion.

43. The method of claim 1, wherein said partially depolymerized pectin is administered by inhalation.

44. The method of claim 1, wherein said partially depolymerized pectin is administered by topically.

45. The method of claim 1, wherein said partially depolymerized pectin is administered by subcutaneous injection.

46. The method of claim 1, wherein the effective dose (ED50) for said therapeutic treatment, chemotherapeutic or cytostatic agent used in combination with said partially depolymerized pectin is at least 5 fold less than the ED50 for said therapeutic treatment, chemotherapeutic or cytostatic agent alone.

47. The method of claim 1, wherein the therapeutic index (TI) for said therapeutic treatment, chemotherapeutic or cytostatic agent used in combination with said partially depolymerized pectin is at least 5 fold greater than the TI for said therapeutic treatment, chemotherapeutic or cytostatic agent alone.

48. A kit comprising (i) a chemotherapeutic agent that interferes with DNA replication fidelity or cell-cycle progression of cells undergoing unwanted proliferation, (ii) a therapeutically effective amount of a partially depolymerized pectin having a galacturan backbone which may be disrupted by rhamnose and low branched neutral sugars dependent from said backbone; and (iii) instructions and/or a label for conjoint administration of the chemotherapeutic agent and the partially depolymerized pectin.

49. A packaged pharmaceutical comprising (i) a therapeutically effective amount of a partially depolymerized pectin having a galacturan backbone which may be disrupted by rhamnose and low branched neutral sugars dependent from said backbone; and (ii) instructions and/or a label for conjoint administration of the partially depolymerized pectin with a chemotherapeutic agent.

50. A method for treating a tumor comprising: (i) obtaining a sample of tumor cells from a patient; (ii) ascertaining the anti-apoptotic Bcl-2 protein status of the tumor cell sample; and (iii) for patients having a wild-type or elevated level or anti-apoptotic Bcl-2 proteins, treating the patient with a regimen including administering a therapeutically effective amount of a partially depolymerized pectin having a galacturan backbone which may be disrupted by rhamnose and low branched neutral sugars dependent from said backbone.

51. The method of claim 50, wherein said treatment regimen further includes treating the patient with a chemotherapeutic agent that interferes with DNA replication fidelity or cell-cycle progression of cells undergoing unwanted proliferation.