Anti-vascular Methods and Therapies Employing Lysyl Oxidase Inhibitors

Field of the Invention and Introduction

The present invention relates to pharmaceutical compositions and methods of treating angiogenesis related diseases, such as cancer. Specifically, the invention relates to a composition comprising a lysyl oxidase inhibitor alone or in combination with at least one chemotherapeutic agent, as well as use of the composition and/or combination in the treatment of angiogenesis related disease, such as cancer.

Background of the Invention Lysyl oxidase (LOX) (protein-6-oxidase; EC 1.4.3.13) is an extracellular copper- dependent amine oxidase that catalyses the oxidation of amines, including but not limited to primary amines and, in particular, the amino side chain of lysine in extracellular matrix proteins. Lysine oxidation catalyzed by lysyl oxidase has been observed in polypeptide chains of collagen and tropoelastin, and thus LOX is the key enzyme that controls collagen and elastin maturation. More precisely, it catalyzes the oxidative deamination of peptidyl lysine and hydroxylysine to peptidyl-α-aminoadipic-δ-semialdehyde into elastin and collagen chains. The aldehydes formed lead to a spontaneous condensation and inter- and intrachain cross-links. Crosslinked molecules, including elastin and collagen, are significant components of fully functional connective tissue. Deficiencies in lysyl oxidase, such as that found in lathyrism, a connective tissue disorder, lead to marked phenotypic changes that can compromise viability. The symptoms of lathyrism include bone deformities, weakening of the joints, and weakening of skin and cartilage. Maki, J. M. et al. (Circulation 2002, 106: 2503-2509) have generated knock out mice Lox-/-, who died at the end of gestation with structural alterations in the arterial aortic walls and abnormalities in the cardiovascular functions, thereby suggesting that LOX might play a critical role in certain cardiovascular diseases. In addition, a progressive loss of LOX expression has also been observed during prostate cancer progression in mice (Ren et al. (1998) Cancer Res. 58:1285-1290), suggesting that lysyl oxidases may function as tumor suppressors.

One of the roles of LOX includes a ras recission function, and the encoded gene has been classified in this context as a ras-recission gene or rrg. Its expression is altered to apparently reduce the oncogenic phenotype of cells expressing aberrant ras. Lysyl oxidase levels also change during differentiation and development and in response to growth factors.

Lysyl oxidase is typically made as a larger protein, which includes a collection of amino acid residues at the amino terminus of the protein. This form, termed preprolysyl oxidase, is secreted from the cell simultaneously with the cleavage of the amino-terminal region to generate prolysyl oxidase, which in turn is cleaved outside the cell to generate the mature form of the protein. Mature lysyl oxidase enzyme additionally contains copper and at least one organic cofactor. The organic cofactor has also been considered to be covalently or non-covalently bound to the enzyme. Examples of organic cofactors that have been found to be associated with oxidases include quinones, such as P. Q. Q., topaquinone (2,4,5 trihydroxyphenylalanine quinone), and tryptophan tryptophylquinone. The organic cofactor for lysyl oxidase is now known to be a quinone. It is possible that the cofactor can be supplied, generated by interaction of a quinone derivative with the depleted apo-form of the enzyme, and/or generated by protein oxidation such as that mediated by the participation of copper. There is at least one atom of copper tightly bound per one molecule of functional enzyme. However, it was experimentally demonstrated that when copper is removed by a chelating agent, the inactive apoenzyme can be restored to its former level of activity by the addition of copper.

Isolation of endogenous lysyl oxidase from, for example, mammalian tissue typically uses chemical agents that interfere with protein association, such as urea. Modest amounts of lysyl oxidase can be recovered in this way from skin. The purified material is typically maintained in solutions containing chemical agent(s), in part to minimize protein aggregation and loss of catalytic function. When prepared in this way, the enzyme displays sluggish activity, which has led to the assertion that the enzyme (or a macromolecular complex) is altered during the relatively harsh extraction procedure.

The applicants have now discovered that certain inhibitors of lysyl oxidase are useful in the treatment of diseases and conditions associated with abnormal angiogenesis.

Angiogenesis is the generation of new blood vessels from preexisting vessels in a tissue or organ. Angiogenesis is required and normally observed under normal physiological conditions, such as for example wound healing, fetal and embryonic development, female reproduction, Le_., formation of the corpus luteum, endometrium and placenta, organ formation, tissue regeneration and remodeling (Risau W et al., Nature, 1997, 386, 671-674). Angiogenesis begins with local degradation of the basement membrane of capillaries, followed by invasion of stroma by underlying endothelial cells in the direction of nn angiogenic stimulus. Subsequent to migration, endothelial cells proliferate at the leading edge of a migrating column and then organize to form new capillary tubes.

Persistent, unregulated angiogenesis occurs in a multiplicity of pathological conditions, including tumor metastasis and abnormal growth by endothelial cells, and supports the pathological damage seen in these conditions. The diverse pathological disease states in which unregulated angiogenesis is present have been grouped together as angiogenic dependent or angiogenic associated diseases. Outgrowth of new blood vessels under pathological conditions can lead to the development and progression of diseases such as tumor growth, diabetic retinopathy, tissue and organ malformation, obesity, macular degeneration, rheumatoid arthritis, psoriasis, and cardiovascular disorders.

Several studies have produced direct and indirect evidence that tumor growth and metastasis are angiogenesis-dependent (Brooks et al, Cell, 1994, 79, 1 154-1 164; Kim KJ et al., Nature, 1993, 362, 841-844). Expansion of the tumor volume requires the induction of new capillary blood vessels. Tumor cells promote angiogenesis by the secretion of angiogenic factors, in particular basic fibroblast growth factor (bFGF)

(Kandel J. et al, Cell, 1991 , 66, 1095-1 104) vascular endothelial growth factor (VEGF) (Ferrara et al, Endocr. Rev., 1997, 18: 4-25) and platelet derived growth factor (PDGF).

Tumors may produce one or more of these angiogenic peptides that can synergistically stimulate tumor angiogenesis (Mustonen et al, JCell Biol, 1995, 129, 865-898).

Current anti-angiogenic therapies or chemotherapies target tumors where there is immature development or growth of the vasculature. It is known that such immature

vasculature, which mainly comprises endothelial cells without perivasculature, Le_., mural cell support, are influenced by the tumor's production of pro-angiogenic factors and are especially sensitive to anti-angiogenic therapy. Yet, the majority of the vasculature found within the tumors of mammals comprises perivasculature, Le., mural cell support to the endothelial cells, including pericytes, smooth muscle cells, and fibroblasts. Such perivasculature can be arterioles, which are endothelial cell tubes surrounded by pericytes/smooth muscle cells. It has been shown that endothelial cells within this context become resistant to chemotherapy or anti-angiogenic therapy, and they no longer respond to the pro-angiogenic factor produced by the tumor, so the tumor remains nourished by a mature vasculature network {see, for example, Minasi et al., Development 129: 2773 (2002), specifically incorporated herein by reference).

Thus, there is a need for a treatment capable of not only preventing the development of new vasculature, but also capable of abrogating mature perivasculature in and/or supporting tumors and tumors that are refractory or resistant to standard therapy or treatments.

It is thus one aspect or object of the present invention to provide an anti-vascular therapy capable of efficiently abrogating mature vasculature, as found in patients' tumors where arterioles of endothelial cell tubes surrounded by pericytes or smooth muscle cells, are already established. Particularly, such mature vasculature has been shown, until now, to be resistant to current and/or standard chemotherapy treatments. In this regard, experimental designs have not adequately represented higher order vessels as they are present in human tumors.

As described below, it is now been found that the use of lysyl oxidase inhibitors provides a very beneficial effect on abrogating immature and mature vasculature, and thus are useful for treating pathological angiogenesis. Lysyl oxidase inhibitor alone or in combination with a chemotherapeutic agent is particularly active in abrogating immature and mature vasculature on chemotherapy refractory tumors.

Summary of the Invention

In a first aspect of the invention, there is provided a method of inhibiting angiogenesis and/or inhibiting unwanted angiogenesis in a mammal, including administering to said mammal an effective amount of an inhibitor of lysyl oxidase.

In a second aspect of the invention, there is provided a method of treating pathological angiogenesis in a mammal, including administering to said mammal an effective amount of an inhibitor of lysyl oxidase.

In a third aspect of the invention, there is provided a pharmaceutical composition including an effective amount of a lysyl oxidase inhibitor.

In a fourth aspect of the invention, there is provided a combination including an effective amount of a lysyl oxidase inhibitor and a therapeutically effective amount of at least one chemotherapeutic agent for use in therapy.

In a fifth aspect of the invention, there is provided a method of using a pharmaceutical composition including therapeutically effective amounts of a lysyl oxidase inhibitor in preparing a medicament for the treatment of angiogenesis related diseases.

In a sixth aspect of the invention, there is provided a method of using a pharmaceutical combination including an effective amount of a lysyl oxidase inhibitor and an effective amount of an anti-angiogenic and/or chemotherapeutic agent for cancer treatment.

In a seventh aspect, the invention relates to a method for screening for a compound capable of abrogating mature vasculature in a patient comprising (i) introducing tumor cells to a collection of microvascular cells including mural cells and pericytes; (ii) administering to the tumor cells a lysyl oxidase inhibitor; (iii) administering to the tumor cells one or more anti-cancer agents; and (iv) measuring one or more of tumor volume, mean vessel density, endothelial cell proliferation, division, and migration, or endothelial cell apoptosis in the cells compared to a control, whereby a difference between the control and the cells administered the lysyl oxidase inhibitor alone or in combination with one or more chemotherapeutic agents can be detected.

In any of these aspects of the invention, any one or more lysyl oxidase inhibitors, agents, or compounds as mentioned herein can be selected for use. In addition, for any of the combinations including one or more lysyl oxidase inhibitors, any of the chemotherapeutic agents noted herein, any available cancer drugs, or any available anti- angiogeneic agents can be selected, alone or in combination. Furthermore, the methods of using the lysyl oxidase inhibitors of the invention, and combinations including them, can also be combined with other known or available cancer treatment methods, such as radiation therapy and/or surgical or tissue ablation methods, in mammals.

In general, the lysyl oxidase inhibitors of the invention can be any compound, organic molecule, nucleic acid, antibody, fusion protein, chelating agent, or binding agent or the like that specifically binds to a lysyl oxidase and/or that inhibits one or more enzymatic functions of lysyl oxidase and/or that is capable of inhibiting microtubule formation and/or abrogating mature vasculature. Inhibition can be reversible or irreversible.

Brief Description of the Sequences

SEQ ID No: 1 represents the amino acid sequence of the human lysyl oxidase. SEQ ID NO: 2 is the conserved 9 amino acid copper coordination domain of Lox proteins, isozymes, and Lox-like proteins. SEQ ID NO: 3 is the cDNA sequence of human Lox, Accession number NM 002317.

Detailed Description of Exemplary Embodiments

Throughout this disclosure, applicants refer to journal articles, patent documents, published references, web pages, sequence information available in databases, and other sources of information. One skilled in the art can use the entire contents of any of the cited sources of information to make and use aspects of this invention. Each and every cited source of information is specifically incorporated herein by reference in its entirety. Portions of these sources may be included in this document as allowed or required. However, the meaning of any term or phrase specifically defined or explained in this disclosure shall not be modified by the content of any of the sources. The description and

examples that follow are merely exemplary of the scope of this invention and content of this disclosure. One skilled in the art can devise and construct numerous modifications to the examples listed below without departing from the scope of this invention.

The present invention relates to a method of inhibiting angiogenesis and/or inhibiting unwanted angiogenesis in a mammal, including administering to said mammal a therapeutically effective amount of at least one lysyl oxidase inhibitor.

The present invention also relates to a method of treating cancer in a mammal, including administering to said mammal a therapeutically effective amount of at least one lysyl oxidase inhibitor alone, or a combination of at least one lysyl oxidase inhibitor with at least one chemotherapeutic agent.

Such compositions can be used to disrupt the association of subtypes of endothelial vessel cells required for an angiogenesis process and can act to advantageously inhibit angiogenesis in an improved and more effective manner. The beneficial effect of such composition can exist in the control or inhibition of tumor cell growth and/or metastasis.

In one embodiment of the present invention, classes of therapeutic or active compounds to be selected include one or more of those that interact with or inhibit lysyl oxidase enzymatic activity.

In accordance with the present invention, there is provided a method of abrogating mature vasculature in a patient suffering from a pathological angiogenesis characterized by such proliferation comprising the administration to the patient of an inhibitory effective amount of one or more inhibitors of lysyl oxidase enzymatic activity. A number of active compounds that inhibit lysyl oxidase enzymatic activity are known or available. According to a preferred embodiment, such inhibitors are as described in US Patents Nos: US 5,120,764; US 5,021,456; US 5,059,714; US 4,943,593; and US 4,965,288. In addition, U.S. Pat. No. 4,997,854 discusses a method for inhibiting enzymatic activity of lysyl oxidase using 1,2-diamine compounds, and U.S. Pat. Nos. 5, 182,297 and 5,252,608 discuss a method of treating fibrotic conditions with 3,3-dihalo- 2-propenylamine compounds and their salts, which are inhibitors of lysyl oxidase. Nagan, et al., Frontiers in Bioscience 3, 23-26 (1998), also mention inhibitor compounds.

Each of these references related to lysyl inhibitors is specifically incorporated herein by reference and can be relied upon to provide or generate one or more lysyl oxidase inhibitors as used and referred to herein. In addition, Raposa, et. al., Atherosclerosis, 177(1): 1-8, (2004), discusses homocysteine as a repressor of Lox mRNA levels and Lox gene activity. Accordingly, the Lox inhibitors of the invention include available agents that effect levels of homocysteine or agents used to treat hyperhomocysteinemia.

Inhibitors of Lox may be selected from primary amines that react with the carbonyl group of the active site of the Lox, and more particularly those which produce, after binding with the carbonyl, a product stabilized by resonance, such as for example, ethylenediamine, hydrazine, phenylhydrazine, and their derivatives, semicarbazide, and urea derivatives, aminonitriles, such as β-aminopropionitrile, or 2-nitroethylamine, unsaturated or saturated haloamines, such as 2-bromo-ethylamine, 2-chloroethylamine, 2- trifluoroethylamine, 3-bromopropylamine, p-halobenzylamines, and selenohomocysteine lactone. Inhibitors of lysyl oxidase may also include copper chelators, such as bathocuproine or a hydrophobic derivative thereof. Chelators having affinity for copper may be selected from the group consisting of polyamine chelating agents, ethylendiamine, diethylenetriamine, triethylenetetramine, triethylenediamine, tetraethylenepentamine, aminoethylethanolamine, aminoethylpiperazine, pentaethylenehexamine, triethylenetetramine -hydrochloride, tetraethyllene pentamine- hydrochloride, pentaethylenehexamine-hydrochloride, tetraethylpentamine, captopril, penicilamine, N,N'-bis(3-aminopropyl)-l,3-propanediamine, N,N,Bis (2-animoethyl) 1,3 propane diamine, l,7-dioxa-4,10-diazacyclododecane, 1,4,8,1 1-tetraaza cyclotetradecane- 5,7-dione, 1,4,7-triazacyclononane trihydrochloride, l-oxa-4,7,10-triazacyclododecane, 1,4,8,12-tetraaza cyclopentadecane or 1,4,7,10-tetraaza cyclododecane, preferably tetraethylpentamine.

Oligopeptides, either natural or synthetic, can bind Copper too. Glycyl-L- histidyl-L-lysine-Cu ²⁺ (GHL-Cu) is a tripeptide-Copper complex that was isolated from human plasma. It has been shown to have, in nanomolar concentrations, a variety of biological effects both in vitro and in vivo. It was first described as a growth factor for a

variety of differentiated cells. Subsequent data from various groups indicated that it exhibited several properties of a potent activator of the wound healing process. It was a potent chemotactic agent for monocytes/macrophages and mast cells. It stimulated nerve tissue regeneration and was reported to trigger the angiogenesis process in-vivo. It stimulated collagen synthesis in several fibroblast strains. It accelerated wound closure when injected into superficial wounds in animals and accumulation of collagen and dermatan sulfate proteoglycans. It also exerted metabolic effects, such as inhibition of lipid peroxidation by feritin. GHL-metal ions combinations were shown to promote monolayer formation and cellular adhesiveness in tumorigenic hepatoma (HTC ₄ ) cells in culture, resulting in marked enhancement of cell survival and growth under basal (growth limiting) conditions. The mode of action of GHL is unknown. It has been reported that GHL forms chelates with Copper and iron in human plasma and in buffered solution at physiological pH.

Inhibitors of lysyl oxidase may also be antibodies or antibody binding fragments against the active site of Lox. According to the invention, a LOX protein produced recombinantly or by chemical synthesis, and fragments or other derivatives or analogs thereof, including fusion proteins, may be used as an immunogen to generate antibodies that recognize the LOX polypeptide. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library. Such an antibody is preferably specific for human LOX and it may recognize either a mutant form of LOX or wild-type LOX, or both. One can use the hydropathic index of amino acids, as discussed by Kyte and Doolittle (1982), wherein it was determined that certain amino acids maybe substituted for other amino acids having similar hydropathic indices and still retain a similar biological activity; to determine epitope regions. In addition, polymorphisms and mutants of Lox have been known and described and can be used in the invention and/or the polymorphisms or mutations, one or more than one, can be incorporated into any of the sequences discussed or used in the invention {see for example, Chioza, et al., Amyotrophic Lateral Sclerosis and Other Motor-neuron Disorders, 2(2): 93-97 (2001), listing amino acids 158 and 159 as polymorphic positions).

Various procedures known in the art may be used for the production of polyclonal antibodies to a LOX polypeptide or derivative or analog thereof. For the production of antibody, various host animals can be immunized by injection with the LOX polypeptide, or a derivative (e.g., fragment or fusion protein) thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc. In one embodiment, the LOX polypeptide or fragment thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.

For preparation of monoclonal antibodies directed toward the LOX polypeptide, or fragment, analog, or derivative thereof, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein (Nature, 1975, 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 1983,4:72; Cote et al., Proc. Natl. Acad. Sci. 1983, 80:2026-2030), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985, pp.77-96). In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals (International Patent Publication No. WO89/ 12690). In fact, according to the invention, techniques developed for the production of "chimeric antibodies" (Morrison et al., J. Bacteriol., 1984, 159:870); Neuberger et al., Nature, 1984, 312:604-608; Takeda et al., Nature, 1985, 314:452-454) by splicing the genes from a mouse antibody molecule specific for a LOX polypeptide together with genes from a human antibody molecule of appropriate biological activity can be used; such antibodies are within the scope of this invention. Such human or humanized chimeric antibodies are preferred for use in therapy of human diseases or disorders (described infra), since the human or humanized antibodies are

much less likely than xenogenic antibodies to induce an immune response, in particular an allergic response, themselves.

Antibody fragments which contain the idiotype of the antibody molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab') ₂ fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') ₂ fragment, and the Fab fragments which can be generated by treating the antibody molecule w ith papain and a reducing agent.

According to the invention, techniques described for the production of single chain antibodies (U.S. Pat. Nos. 5,476,786, 5,132,405, and U.S. Pat. No. 4,946,778) can be adapted to produce LOX polypeptide-specific single chain antibodies. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al., Science, 1989, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for LOX polypeptide, or its derivatives, or analogs.

In the production and use of antibodies, screening for or testing with the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. For example, to select antibodies which recognize a specific epitope of LOX polypeptide, one may assay generated hybridomas for a product which binds to a LOX polypeptide fragment containing such epitope. For selection of an

antibody specific to a LOX polypeptide from a particular species of animal, one can select on the basis of positive binding with LOX polypeptide expressed by or isolated from cells of that species of animal.

According to this specific embodiment of the present invention, anti LOX antibodies antagonize the activity of LOX polypeptide and thus are useful for treatment of angiogenesis related diseases. Such antibodies can be tested using the assays described infra.

According another embodiment of the present invention, nucleotide sequences derived from the gene encoding LOX, and peptide sequences derived from LOX, are useful targets to identify drugs that are effective in treating disorders associated with angiogenesis malfunction processes. Drug targets include without limitation (i) isolated nucleic acids derived from the gene encoding LOX or the complement or RNA transcript thereof; (ii) isolated peptides and polypeptides derived from LOX polypeptides; and, most importantly, (iii) different compounds that selectively regulate LOX expression. Another mechanism of down regulating enzymes at the transcript level is RNA interference (RNAi), an approach which utilizes small interfering dsRNA (siRNA) molecules that are homologous to the target mRNA and lead to its degradation (Carthew, 2001). RNAi is an evolutionarily conserved surveillance mechanism that responds to double-stranded RNA by sequence-specific silencing of homologous genes (Fire et al., 1998; Zamore et al., 2000). RNAi is initiated by the dsRNA-specific endonuclease Dicer, which promotes cleavage of long dsRNA into double-stranded fragments between about 21 and 25 nucleotides long, termed small interfering RNA (siRNAs) (Zamore et al., 2000; Elbashir et al., 2001 ; Hammond et al., 2000; Bernstein et al., 2001). siRNA are incorporated into a protein complex that recognizes and cleaves target mRNAs (Nykanen et al., 2001). According this embodiment, the present invention concerns a medicament to treat angiogenesis related disorders, wherein the medicament contains siRNA or a double-stranded ribonucleic acid (dsRNA) that is suitable for inhibiting by RNA interference the expression of the LOX gene.

Preferably, a strand of dsRNA exhibits a region that is at least segmentally complementary to the LOX gene sequence, consisting, in particular, of fewer than 25

successive nucleotides. "Gene" is here understood to mean the DNA strand of the double- stranded DNA that codes for a protein or peptide, which is complementary to a DNA strand including all transcribed regions that serves as a matrix for transcription. With this gene we are generally dealing with the sense strand. The strand can be complementary to an RNA transcript or its processing product, such as an mRNA, that is formed during the expression of the gene. The complementary region of the dsRNA can exhibit, in order of ascending preference, 19 to 24, 20 to 24, 21 to 23, and particularly 22 or 23 nucleotides. A dsRNA having this structure is particularly efficient in inhibiting the LOX gene. The strand of the dsRNA can exhibit fewer than 30, fewer than 25, 21 to 24, and particularly 23 nucleotides. The number of these nucleotides is also the maximum possible number of base pairs in the dsRNA. It has been shown to be particularly advantageous when at least one end of the dsRNA exhibits a single-stranded overhang consisting of 1 to 4, in particular of 2 or 3, nucleotides. In comparison to dsRNA without single-stranded overhangs at least one end, such dsRNA demonstrates superior effectiveness in inhibiting expression of the gene. Here, one end is a dsRNA region in which a 5'- and a 3'-strand- end is present. DsRNA consisting only of a single strand (Sl) accordingly exhibits a loop structure and only one end. DsRNA consisting of the strand Sl and the second strand (S2) exhibits two ends. Here, one end is formed in each case by a strand end on the strand Sl and one on the strand S2. Many available and commercially available methods and reagents are known for introducing RNAi nucleic acids into cells and organisms, including poly-lysine and other polymers, a variety of lipids, liposomes, and physical projectiles, coated micro-beads, eletroporation and ultrasound poration methods and devices. Furthermore, direct injection of compositions containing the nucleic acids into a specific tissue site or near a tumor site can be used, together with the lipid-based or other administration methods or devices noted.

A medicament comprising the compositions or combinations of the invention may exhibit a preparation suitable for inhalation, oral ingestion, infusion or injection, in particular for intravenous or intraperitoneal infusion or injection, or for infusion or injection directly into a tissue affected by disease. A preparation suitable for inhalation, infusion, or injection can most simply consist, in particular exclusively, of the dsRNA

and a physiologically tolerated solvent, preferably a physiological saline solution or a physiologically tolerated buffer, in particular a phosphate-buffered saline solution. Surprisingly, it has been shown that dsRNA that has simply been dissolved and administered in such a buffer or solvent is taken up by the cells that express the gene. Expression of the gene is inhibited without a requirement that the dsRNA be packaged in a special vehicle, although specific vehicles are possible. The dsRNA can be present in a medicament in a solution, in particular a physiologically tolerated buffer or a physiological saline solution, surrounded by a micellar structure, preferably a liposome, a capsid, a capsoid, or polymeric nano- or microcapsule, or bound to a polymeric nano- or microcapsule. The physiologically tolerated buffer can be a phosphate-buffered saline solution. A micellar structure, a capsid, capsoid, or polymeric nano- or microcapsule can facilitate uptake of dsRNA in cells that express the gene. The polymeric nano- or microcapsule consists of at least one biologically degradable polymer such as polybutylcyanoacrylate. The polymeric nano- or microcapsule can transport and release in the body dsRNA that is contained in or bound to it.

Screening assays, particularly for high throughput screening of molecules that down-regulate the activity of LOX, e _^ g., by permitting expression of LOX in quantities greater than can be isolated from natural sources, or in indicator cells that are specially engineered to indicate the activity of LOX expressed after transfection or transformation of the cells. Accordingly, the present invention contemplates methods for identifying specific molecules that alter LOX expression, as well as molecules that act directly on LOX, using various screening assays known in the art. Any screening technique known in the art can be used to screen for LOX antagonists. The present invention contemplates screens for small molecule ligands or analogs and mimics, as well as screens for natural ligands that bind to and antagonize LOX expression activity in vivo. For example, natural products libraries can be screened using assays of the invention for molecules that antagonize LOX expression or activity.

Another approach uses recombinant bacteriophage to produce large libraries. Using the "phage method" (Scott and Smith, Science 1990, 249:386-390; Cwirla, et al., Proc. Natl. Acad. ScL, USA 1990, 87:6378-6382; Devlin et al., Science 1990, 49:404-

406), very large libraries can be constructed (10. sup.6-108 chemical entities). A second approach uses primarily chemical methods, of which the Geysen method (Geysen et al., Molecular Immunology 1986, 23:709-715; Geysen et al. J. Immunologic Method 1987 102:259-274; and the method of Fodor et al. (Science 1991 ,251 :767-773) are examples. Furka et al. (14th International Congress of Biochemistry, Volume #5 1988, Abstract FR:013; Furka, Int. J. Peptide Protein Res. 1991, 37:487-493), Houghton (U.S. Pat. No. 4,631 ,21 1) and Rutter (U.S. Pat. No. 5,010,175) describe methods to produce a mixture of peptides that can be tested as antagonists.

In another aspect, synthetic libraries (Needels et al., Proc. Natl. Acad. Sci. USA 1993, 90:10700-4; Ohlmeyer et al., Proc. Natl. Acad. Sci. USA 1993, 90: 10922-10926; Lam et al., PCT Publication No. WO 92/00252; Kocis et al., PCT Publication No. WO 94/28028) and the like can be used to screen for ligands that regulate LOX according to the present invention. Test compounds are screened from large libraries of synthetic or natural compounds. Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemical library is available from Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g. Pan Laboratories (Bothell, Wash.) or MycoSearch (NC), or are readily producible. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means (Blondelle et al., Tib Tech 1996, 14:60). Knowledge of the primary sequence of LOX, and the similarity of that sequence with proteins of known function, can provide options for additional inhibitors or antagonists of Lox enzyme activity or biological function. Identification and screening of antagonists is further facilitated by determining structural features of the protein, e.g., using X-ray crystallography, neutron diffraction, nuclear magnetic resonance spectrometry, and other techniques for structure determination. These techniques provide

for the rational design or identification of antagonists.

In vivo screening may be performed in intact cells or whole animals expressing a gene encoding LOX to identify candidate drugs or antagonists of LOX. A permanent cell line may be established. Alternatively, cells (including without limitation mammalian, insect, yeast, or bacterial cells) are transiently programmed to express LOX gene by introduction of appropriate DNA or mRNA. Identification of candidate compounds can be achieved using any suitable assay, including without limitation (i) assays that measure the ability of a test compound to inhibit a measurable activity or function of LOX and (ii) assays that measure the ability of a compound to inhibit the transcriptional activity of sequences derived from the promoter (i.e., regulatory) regions of the LOX gene.

Transgenic mammals can be prepared for evaluating the molecular mechanisms of LOX and the optimum inhibitors and combinations to use according to the invention. Such mammals provide excellent models for screening or testing drug candidates. Thus, human protein LOX "knock-in" mammals can be prepared for evaluating the molecular biology of this system in greater detail than is possible with human subjects. It is also possible to evaluate compounds or diseases on "knockout" animals, e.g., to identify a compound that can compensate for a defect in LOX activity. Both technologies permit manipulation of single units of genetic information in their natural position in a cell genome and to examine the results of that manipulation in the background of a terminally differentiated organism. Transgenic mammals can be prepared by any method, including but not limited to modification of embryonic stem (ES) cells and heteronuclear injection into blast cells. LOX knockout mammals can be prepared for evaluating the molecular pathology of this defect in greater detail than is possible with human subjects. Such animals also provide excellent models for screening drug candidates. A "knockout mammal" is a mammal (e.g., mouse, rabbit) that contains within its genome a specific gene that has been inactivated. Any method known in the art that may render the gene non-functional or not expressed may be used. A non-limiting example of such a method is gene targeting (see, e.g., U.S. Pat. Nos. 5,777,195 and 5,616,491). A knockout mammal includes both a heterozygote knockout (i.e., one defective allele and one wild- type allele) and a homozygous mutant (i.e., two defective alleles; a heterologous

construct for expression of LOX, such as a human LOX, could be inserted to permit the knockout mammal to live if the lack of LOX expression is lethal). Preparation of a knockout mammal requires first introducing a nucleic acid construct that will be used to suppress expression of a particular gene into an undifferentiated cell type termed an embryonic stem cell. This cell is then injected into a mammalian embryo. A mammalian embryo with an integrated cell is then implanted into a foster mother for the duration of gestation. Zhou, et al. (Genes and Development 1995, 9:2623-34) describes PPCA knock-out mice.

The term "knockout" refers to partial or complete suppression of the expression of at least a portion of a protein encoded by an endogenous DNA sequence in a cell. The term "knockout construct" refers to a nucleic acid sequence that is designed to decrease or suppress expression of a protein encoded by endogenous DNA sequences in a cell. The nucleic acid sequence used as the knockout construct is typically comprised of (1) DNA from some portion of the gene (exon sequence, intron sequence, and/or promoter sequence) to be suppressed and (2) a marker sequence used to detect the presence of the knockout construct in the cell. The knockout construct is inserted into a cell, and integrates with the genomic DNA of the cell in such a position so as to prevent or interrupt transcription of the native DNA sequence. Such insertion usually occurs by homologous recombination (i.e., regions of the knockout construct that are homologous to endogenous DNA sequences hybridize to each other when the knockout construct is inserted into the cell and recombine so that the knockout construct is incorporated into the corresponding position of the endogenous DNA). The knockout construct nucleic acid sequence may comprise (1) a full or partial sequence of one or more exons and/or introns of the gene to be suppressed, (2) a full or partial promoter sequence of the gene to be suppressed, or (3) combinations thereof. Typically, the knockout construct is inserted into an embryonic stem cell (ES cell) and is integrated into the ES cell genomic DNA, usually by the process of homologous recombination. This ES cell is then injected into, and integrates with, the developing embryo. Included within the scope of this invention is a mammal in which two or more genes have been knocked out. Such mammals can be generated by repeating the procedures set forth herein for generating each knockout

construct, or by breeding to mammals, each with a single gene knocked out, to each other, and screening for those with the double knockout genotype. Regulated knockout animals can be prepared using various systems, such as the tet-repressor system (see U.S. Pat. No. 5,654,168) or the Cre-Lox system (see U.S. Pat. Nos. 4,959,317 and 5,801 ,030). Transgenic animals may be created in which (i) a human LOX is stably inserted into the genome of the transgenic animal; and/or (ii) the endogenous LOX genes are inactivated and replaced with human LOX genes.

According to another embodiment, vectors comprising a sequence encoding a protein, including, but not limited to, full-length LOX, are provided to treat or prevent angiogenesis related disorder. In this embodiment of the invention, the therapeutic vector encodes a sequence that produces the protein of the invention.

Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are well known in the art such as recombinant DNA technology that can be used are described in Ausubel et al., (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al., (eds.), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY. Vectors suitable for gene therapy are also well known in the art.

In one aspect, the therapeutic vector comprises a nucleic acid that expresses a protein of the invention in a suitable host. In particular, such a vector has a promoter operationally linked to the coding sequence for the protein. The promoter can be inducible or constitutive and, optionally, tissue-specific. In another embodiment, a nucleic acid molecule is used in which the protein coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the protein (Koller and Smithies, Proc. Natl. Acad. Sci. U.S.A, 1989, 86:8932-8935; Zijlstra et al., Nature, 1989, 342:435-438).

Delivery of the vector into a patient may be either direct, in which case the patient is directly exposed to the vector or a delivery complex, or indirect, in which case, cells are first transformed with the vector in vitro then transplanted into the patient. These two

approaches are known, respectively, as in vivo and ex vivo gene therapy. Such vector may be directly provided in vivo, where it enters the cells of the organism and mediates expression of the protein. This can be accomplished by any of numerous methods known in the art, by constructing it as part of an appropriate expression vector and administering it so that it becomes intracellular, e.g., by infection using a defective or attenuated retroviral or other viral vector (see, U.S. Pat. No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont); or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in biopolymers (e.g., poly-S-l-64-N-acetylglucosamine polysaccharide; see, U.S. Pat. No. 5,635,493), encapsulation in liposomes, microparticles, or microcapsules; by administering it in linkage to a peptide or other ligand known to enter the nucleus; or by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem., 1987, 62:4429-4432), etc. In another embodiment, a nucleic acid ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publication Nos. WO 92/06180, WO 92/22635, WO 92/20316 and WO 93/14188). Alternatively, the nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA, 1989, 86:8932-8935; Zijlstra, et al., Nature, 1989, 342:435-438). These methods are in addition to those discussed above in conjunction with "Viral and Non-viral Vectors".

Alternatively, antibody molecules can also be administered, for example, by expressing nucleotide sequences encoding single-chain antibodies within the target cell population by utilizing, for example, techniques such as those described in Marasco et al. (Proc. Natl. Acad Sci. USA, 1993, 90:7889-7893). The form and amount of therapeutic nucleic acid envisioned for use depends on the type of disease and the severity of the desired effect, patient state, etc., and can be determined by one skilled in the art. In another most preferred embodiment, the synergistic combination of the present

invention uses at least one lysyl oxidase inhibitor with at least one chemotherapeutic agent.

In a further embodiment, the present invention provides for a method of screening for active compound capable of abrogating mature vasculature in patients affected with pathological angiogenesis.

The methods of screening according to the present invention comprise the step consisting of (i) introducing tumor cells to a collection of microvascular cells including mural cells and pericytes; (ii) regularly administering to the tumor cells lysyl oxidase inhibitor as described above; potentially (iii) administering to the tumor cells one or more anti-angiogenic chemotherapeutic; and (iv) measuring the mean vessel density, endothelial division, proliferation, migration, or endothelial cell apoptosis in the cells compared to a control, whereby a difference between the control and the cells administered the lysyl oxidase inhibitor can be detected.

Most preferably, methods of screening of the invention comprises introducing tumor cells to a collection of microvascular cells including mural cells and pericytes; (ii) regularly administering to the tumor cells a lysyl oxidase inhibitor as described above; eventually (iii) administering to the tumor cells one or more chemotherapeutic agent; (iv) and measuring the one or more of tumor volume, mean vessel density, endothelial cell division, proliferation, and migration, or endothelial cell apoptosis in the cells compared to a control, whereby a difference between the control and the cells administered the lysyl oxidase inhibitor can be detected.

According to the present invention, the chemotherapeutic agent used can be selected from docetaxel, which is commercially available as an injectable solution as TAXOTERE. Docetaxel is (2R, 3S)- N-carboxy-3-phenylisoserine, N-tert-butyl ester, 13- ester with 5(3-20-epoxy-l, 2a, 4, 7, 10, 130C-hexahydroxytax-l l-en-9-one 4-acetate 2benzoate, trihydrate, indicated for the treatment of breast cancer. Docetaxel is a semisynthetic derivative of paclitaxel q. v., prepared using a natural precursor, 10- deacetyl-baccatin III, extracted from the needle of the European Yew tree. Uses and process of preparation of docetaxel are described in US 4,814,470; US 5,438,072; US 5,698,582; US 6,714,512, incorporated herein by reference. Many other

chemotherapeutic agents or treatments can be used in combination, such as Gemcitabine, 2'-deoxy-2', 2'-difluorocytidine monohydrochloride (3-isomer), commercially available as GEMZAR. Gemcitabine which is a cytidine analog, exhibits cell phase specificity at S phase and by blocking progression of cells through the Gl/S boundary. Method of use and preparation are described inter alia in US 4,808,614 and US 5,464,826. Gemcitabine has been used in combination with cisplatin in the treatment of locally advanced non- small cell lung cancer and alone in the treatment of locally advanced pancreatic cancer.

Useful chemotherapeutic agents also include, but are not limited to, anti- microtubule agents such as diterpenoids and vinca alkaloids; platinum coordination complexes; alkylating agents such as nitrogen mustards, oxazaphosphorines, alkylsulfonates, nitrosoureas, and triazenes; antibiotic agents such as anthracyclins, actinomycins and bleomycins; topoisomerase II inhibitors such as epipodophyllotoxins; antimetabolites such as purine and pyrimidine analogues and antifolate compounds; topoisomerase I inhibitors such as camptothecins; hormones and hormonal analogues; signal transduction pathway inhibitors; non-receptor tyrosine kinase angiogenesis inhibitors; immunotherapeutic agents; proapoptotic agents; and cell cycle signaling inhibitors. In addition, anti-microtubule or anti-mitotic agents can be used and are phase specific agents active against the microtubules of tumor cells during M or the mitosis phase of the cell cycle. Examples of anti-microtubule agents include, but are not limited to, diterpenoids and vinca alkaloid. Diterpenoids, which are derived from natural sources, are phase specific anticancer agents that operate at the G2/M phases of the cell cycle. It is believed that the diterpenoids stabilize the b-tubulin subunit of the microtubules, by binding with this protein. Disassembly of the protein appears then to be inhibited with mitosis being arrested and cell death following. Examples of diterpenoids include, but are not limited to, paclitaxel. Paclitaxel, 5, 20-epoxy-l, 2O, 4,7 (3, 10a, 13a- hexa-hydroxytax-1 l-en-9-one 4,10diacetate 2-benzoate 13-ester with (2R, 3S)-N- benzoyl-3-phenylisoserine, is a natural diterpene product isolated from the Pacific yew tree Taxus brevifolia and is commercially available as an injectable solution TAXOL. It is a member of the taxane family of terpenes. It was first isolated in 1971 by Wani et al. J. Am. Chem, Soc, 93: 2325.1971), who characterized its structure by chemical and X-ray

crystal lographic methods. One mechanism for its activity relates to paclitaxel's capacity to bind tubulin, thereby inhibiting cancer cell growth. Schiff et al., Proc. Natl, Acad, Sci. USA, 77: 1561-1565 (1980); Schiff et al., Nature, 277: 665-667 (1979); Kumar, J. Biol, Chem, 256: 10435-10441 (1981). For a review of synthesis and anticancer activity of some paclitaxel derivatives see: D. G. I. Kingston et al., Studies in Organic Chemistry vol. 26, entitled "New trends in Natural Products Chemistry 1986", Attaur-Rahman, P. W. Le Quesne, Eds.(Elsevier, Amsterdam, 1986) pp 219-235.

Paclitaxel has been approved for clinical use in the treatment of refractory ovarian cancer in the United States (Markman et al., Yale Journal of Biology and Medicine, 64: 583,1991 ; McGuire et al., Ann. Intern, Med., I l l : 273, 1989) and for the treatment of breast cancer (Holmes et al., J. Nat. Cancer Inst., 83 1797, 1991.) It is a potential candidate for treatment of neoplasms in the skin (Einzig et. al., Proc. Am. Soc. Clin. Oncol., 20: 46) and head and neck carcinomas (Forastire et. al., Sem. Oncol., 20: 56, 1990). The compound also shows potential for the treatment of polycystic kidney disease (Woo et. al., Nature, 368:750, 1994), lung cancer and malaria. Treatment of patients with paclitaxel results in bone marrow suppression (multiple cell lineages, Ignoff, R. J. et. al, Cancer Chemotherapy Pocket Guide, 1998) related to the duration of dosing above a threshold concentration (5OnM) (Kearns, C. M. et. al., Seminars in Oncology, 3 (6) p. 16-23, 1995). Vinca alkaloid are phase specific anti-neoplastic agents derived from the periwinkle plant. Vinca alkaloids act at the M phase (mitosis) of the cell cycle by binding specifically to tubulin. Consequently, the bound tubulin molecule is unable to polymerize into microtubules. Mitosis is believed to be arrested in metaphase with cell death following. Examples of vinca alkaloids include, but are not limited to, vinblastine, vincristine, and vinorelbine. Vinblastine, vincaleukoblastine sulfate, is commercially available as VELBANO as an injectable solution. Although it has possible indications for a second line therapy of various solid tumors, it is primarily indicated in the treatment of testicular cancer and various lymphomas including Hodgkin's Disease; and lymphocytic and histiocytic lymphomas. Vincristine, vincaleukoblastine, 22-oxo-, sulfate, is commercially available as ONCOVINO as an injectable solution. Vincristine is indicated

for the treatment of acute leukemias and has also found use in treatment regimens for Hodgkin's and non Hodgkin's malignant lymphomas. Alopecia and neurologic effects are the most common side effect of vincristine and to a lesser extent myelosupression and gastrointestinal mucositis effects occur. Vinorelbine, 3', 4'-didehydro-4'-deoxy-C- norvincaleukoblastine [R-(R*, R*)-2, 3dihydroxybutanedioate (1 :2) (salt)], commercially available as an injectable solution of vinorelbine tartrate (NA VELBINEO), is a semisynthetic vinca alkaloid. Vinorelbine is indicated as a single agent or in combination with other chemotherapeutic agents, such as cisplatin, in the treatment of various solid tumors, particularly non-small cell lung, advanced breast, and hormone refractory prostate cancers. Myelosuppression is the most common dose limiting side effect of vinorelbine.

Platinum coordination complexes can also be used and are non-phase specific anti-cancer agents, which are interactive with DNA. The platinum complexes enter tumor cells, undergo, aquation and form intra-and interstrand crosslinks with DNA causing adverse biological effects to the tumor. Examples of platinum coordination complexes include, but are not limited to, cisplatin and carboplatin. Cisplatin, cis- diamminedichloroplatinum, is commercially available as PLATINOL; as an injectable solution. Cisplatin is primarily indicated in the treatment of metastatic testicular and ovarian cancer and advanced bladder cancer. Carboplatin, platinum, diammine [1,1- cyclobutane-dicarboxylate (2-)-O, O'], is commercially available as PARAPLATINO as an injectable solution. Carboplatin is primarily indicated in the first and second line treatment of advanced ovarian carcinoma.

Alkylating agents are non-phase anti-cancer specific agents and strong electrophiles. Typically, alkylating agents form covalent linkages, by alkylation, to DNA through nucleophilic moieties of the DNA molecule such as phosphate, amino, sulfhydryl, hydroxyl, carboxyl, and imidazole groups. Such alkylation disrupts nucleic acid function leading to cell death. Examples of alkylating agents include, but are not limited to, nitrogen mustards such as cyclophosphamide, melphalan, and chlorambucil; alkyl suffonates such as busulfan; nitrosoureas such as carmustine; and triazenes such as dacarbazine. Cyclophosphamide, 2-[bis (2-chloroethyl) amino] tetrahydro-2H-l, 3,

2oxazaphosphorine 2-oxide monohydrate, is commercially available as an injectable solution or tablets as CYTOXAN. Cyclophosphamide is indicated as a single agent or in combination with other chemotherapeutic agents, in the treatment of malignant lymphomas, multiple myeloma, and leukemias. Melphalan, 4- [bis (2-chloroethyl) amino]-L-phenylalanine, is commercially available as an injectable solution or tablets as ALKERANO. Melphalan is indicated for the palliative treatment of multiple myeloma and non-resectable epithelial carcinoma of the ovary.

Chlorambucil, 4-[bis (2-chloroethyl) amino] benzenebutanoic acid, is commercially available as LEUKERAN tablets. Chlorambucil is indicated for the palliative treatment of chronic lymphatic leukemia, and malignant lymphomas such as lymphosarcoma, giant follicular lymphom, and Hodgkin's disease. Bone marrow suppression is the most common dose limiting side effect of chlorambucil.

Busulfan, 1, 4-butanediol dimethanesulfonate, is commercially available as MYLERAN. Busulfan is indicated for the palliative treatment of chronic myelogenous leukemia. Bone marrow suppression is the most common dose limiting side effects of busulfan.

Carmustine, 1, 3- [bis (2-chloroethyl)-l-nitrosourea, is commercially available as single vials of lyophilized material as BiCNU;. Carmustine is indicated for the palliative treatment as a single agent or in combination with other agents for brain tumors, multiple myeloma, Hodgkin's disease, and non-Hodgkin's lymphomas. Delayed myelosuppression is the most common dose limiting side effects of carmustine.

Dacarbazine, 5-(3, 3-dimethyl-l-triazeno)-imidazole < 4-carboxamide, is commercially available as single vials of material as DTIC. Dacarbazine is indicated for the treatment of metastatic malignant melanoma and in combination with other agents for the second line treatment of Hodgkin's Disease.

Antibiotic anti-neoplasties are non-phase specific agents, which bind or intercalate with DNA. Typically, such action results in stable DNA complexes or strand breakage, which disrupts ordinary function of the nucleic acids leading to cell death. Examples of antibiotic anti-neoplastic agents include, but are not limited to, actinomycins

such as dactinomycin, anthrocyclins such as daunorubicin and doxorubicin; and bleomycins. Dactinomycin, also know as Actinomycin D, is commercially available in injectable form as COSMEGENO. Dactinomycin is indicated for the treatment of Wilrn's tumor and rhabdomyosarcoma. Nausea, vomiting, and anorexia are the most common dose limiting side effects of dactinomycin. Daunorubicin, (8S-cis-)-8-acetyl-10- [(3- amino-2, 3,6-trideoxy-a-L-lyxohexopyranosyl) oxy]-7, 8,9,10-tetra hyd ro-6,8,1 1-trihyd roxy-l-methoxy-5,12 naphthacenedione hydrochloride, is commercially available as a liposomal injectable form or as an injectable form. Daunorubicin is indicated for remission induction in the treatment of acute nonlymphocytic leukemia and advanced HIV associated Kaposi's sarcoma. Doxorubicin, (8S, 10S)-IO- [(3-amino-2, 3, 6-trideoxy- a-L-lyxohexopyranosyl) oxy]-8-glycoloyl, 7,8,9, 10-tetrahydro-6,8,l 1-tri hydroxy- 1- methoxy-5,12 naphthacenedione hydrochloride, is commercially available as an injectable form as RUMEX; or ADRIAMYCIN RDFO. Doxorubicin is primarily indicated for the treatment of acute lymphoblastic leukemia and acute myeloblastic leukemia, but is also a useful component in the treatment of some solid tumors and lymphomas. Bleomycin, a mixture of cytotoxic glycopeptide antibiotics isolated from a strain of Streptomyces verticillus, is commercially available as BLENOXANE. Bleomycin is indicated as a palliative treatment, as a single agent or in combination with other agents, of squamous cell carcinoma, lymphomas, and testicular carcinomas. Topoisomerase II inhibitors include, but are not limited to, epipodophyllotoxins.

Epipodophyllotoxins are phase specific anti-neoplastic agents derived from the mandrake plant. Epipodophyllotoxins typically affect cells in the S and G2 phases of the cell cycle by forming a ternary complex with topoisomerase II and DNA causing DNA strand breaks. The strand breaks accumulate and cell death follows. Examples of epipodophyllotoxins include, but are not limited to, etoposide and teniposide. Etoposide, 4'-demethyl-epipodophyllotoxin 9 [4,6-0- (R)-ethylidene-p-D glucopyranoside], is commercially available as an injectable solution or capsules and is commonly known as VP-16. Etoposide is indicated as a single agent or in combination with other chemotherapy agents in the treatment of testicular and nonsmall cell lung cancers.

Antimetabolite neoplastic agents are phase specific anti-neoplastic agents that act at S phase (DNA synthesis) of the cell cycle by inhibiting DNA synthesis or by inhibiting purine or pyrimidine base synthesis and thereby limiting DNA synthesis. Consequently, S phase does not proceed and cell death follows. Examples of antimetabolite anti-neoplastic agents include, but are not limited to 5-fluorouracil, methotrexate, cytarabine, mecaptopurine, and thioguanine. 5-fluorouracil, 5-fluoro-2, 4- (IH, 3H) pyrimidinedione, is commercially available as fluorouracil. Administration of 5-fluorouracil leads to inhibition of thymidylate synthesis and is also incorporated into both RNA and DNA. The result typically is cell death. 5-fluorouracil is indicated as a single agent or in combination with other chemotherapy agents in the'treatment of carcinomas of the breast, colon, rectum, stomach and pancreas. Other fluoropyrimidine analogs include 5-fluoro deoxyuridine (floxuridine) and 5-fluorodeoxyuridine monophosphate. Cytarabine, 4-amino-l-p-D-arabinofuranosyl-2 (H)-pyrimidinone, is commercially available as CYTOSAR-U, and is commonly known as Ara-C. It is believed that cytarabine exhibits cell phase specificity at S-phase by inhibiting DNA chain elongation by terminal incorporation of cytarabine into the growing DNA chain. Cytarabine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia. Another cytidine analog includes 5- azacytidine. Cytarabine induces leucopenia, thrombocytopenia, and mucositis. Mercaptopurine, l,7-dihydro-6H-purine-6-thione monohydrate, is commercially available as PURINETHOLO. Mercaptopurine exhibits cell phase specificity at S-phase by inhibiting DNA synthesis by an as of yet unspecified mechanism. Mercaptopurine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia. A useful mercaptopurine analog is azathioprine. Thioguanine, 2-amino-l , 7-dihydro-6H-purine-6-thione, is commercially available as TABLOID. Thioguanine exhibits cell phase specificity at S-phase by inhibiting DNA synthesis by an as of yet unspecified mechanism. Thioguanine is indicated as a single agent or in combination with other chemotherapy agents in the treatment of acute leukemia. Other purine analogs include pentostatin, erythrohydroxynonyladenine, fludarabine phosphate, and cladribine. Methotrexate, N- [4 [[ (2, 4-diamino-6-pteridinyl)

methyl] methylamino] benzoyl]-L-glutamic acid, is commercially available as methotrexate sodium. Methotrexate exhibits cell phase effects specifically at S-phase by inhibiting DNA synthesis, repair and/or replication through the inhibition of dyhydrofolic acid reductase which is required for synthesis of purine nucleotides and thymidylate. Methotrexate is indicated as a single agent or in combination with other chemotherapy agents in the treatment of choriocarcinoma, meningeal leukemia, non-Hodgkin's lymphom, and carcinomas of the breast, head, neck, ovary and bladder.

Camptothecins, including, camptothecin and camptothecin derivatives are available or under development as Topoisomerase I inhibitors. Camptothecins cytotoxic activity is believed to be related to its Topoisomerase I inhibitory activity. Examples of camptothecins include, but are not limited to irinotecan, topotecan, and the various optical forms of 7- (4-methylpiperazino-methylene)-10, 1 l-ethylenedioxy-20- camptothecin described below. Irinotecan HCI, (4S)-4, 1 1 -diethyl-4-hydroxy-9- [ (4- piperidinopiperidino) carbonyloxy]-l H-pyrano [3 ¹ , 4', 6,7] indolizino [1, 2-b] quinoline- 3, 14 (4H, 12H)-dione hydrochloride, is commercially available as the injectable solution CAMPTOSARO. Irinotecan is a derivative of camptothecin which binds, along with its active metabolite SN-38, to the topoisomerase I-DNA complex. It is believed that cytotoxicity occurs as a result of irreparable double strand breaks caused by interaction of the topoisomerase I : DNA: irintecan or SN-38 ternary complex with replication enzymes, irinotecan is indicated for treatment of metastatic cancer of the colon or rectum. Topotecan HCI, (S)-IO- [(dimethylamino) methyl]-4-ethyl-4, 9-dihydroxy-l H- pyrano [3', 4', 6,7] indolizino [1, 2-b] quinoline-3, 14- (4H, 12H)-dione monohydrochloride, is commercially available as the injectable solution HYCAMTINO. Topotecan is a derivative of camptothecin which binds to the topoisomerase I-DNA complex and prevents religation of singles strand breaks caused by Topoisomerase I in response to torsional strain of the DNA molecule. Topotecan is indicated for second line treatment of metastatic carcinoma of the ovary and small cell lung cancer.

The present invention also relates to methods of treating or preventing a variety of angiogenesis related diseases or conditions, including, but not limited to hemangioma, solid tumors, blood borne tumors, leukemia, metastasis, telangiectasia, psoriasis,

scleroderma, pyogenic granuloma, myocardial angiogenesis, Crohn's disease, plaque neovascularization, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, corneal diseases, rubeosis, neovascular glaucoma, diabetic retinopathy, retrolental fibroplasia, arthritis, rheumatoid arthritis, diabetic neovascularization, diabetic retinopathy, macular degeneration, wound healing, obesity, peptic ulcer, Helicobacter related diseases, fractures, keloids, vasculogenesis, hematopoiesis, psiorasis, ovulation-related disorders, menstruation-related disorders, placentation, and cat scratch fever.

According to another aspect of the invention, the lysyl oxidase inhibitor is administered as dual therapy in parallel of the second biologically active compound and can comprise treatment regimens where one is administered first, followed by the other, where both are administered at the same time, where one is administered for a period of time and the other for another period of time, or combinations of any of these regimens. A preferred mode of administration is one or more of intramuscular, intratumoral, intraperitoneal, intracranial, intratumoral, or intravenous. Most preferred, is the administration at low dose of chemotherapeutic agent, in association with the lysyl oxidase inhibitor as described above. This type of regimen can provide a particularly satisfying result on tumors that are refractory to standard care chemotherapy.

The combination according to the present invention can be administered especially for tumor therapy in combination with chemotherapy, radiotherapy, immunotherapy, surgical intervention, or a combination of these. Long-term therapy is equally possible as is adjuvant therapy in the context of other treatment strategies, as described above.

As used herein, the term "effective amount" means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician. Furthermore, the term "effective amount" means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope

amounts effective to enhance normal physiological function. In particular, an effective amount would be that amount producing a detectable change in lysyl oxidase activity and/or microtubule and/or microvascular formation in a tissue.

The present invention also provides a pharmaceutical composition including therapeutically effective amounts of at least a lysyl oxidase inhibitor alone or in combination with at least an effective amount of a chemotherapeutic as described above. The pharmaceutical combinations according to the present invention for use in a method for the prophylactic or especially therapeutic treatment of angiogenesis related disease, especially those mentioned hereinabove, as well as tumor diseases. For pharmaceutical compositions, the anti-mature vasculature compounds and synergistic combinations of the invention are administered to an individual in need of a cancer treatment, such as for example prostate cancer, ovarian cancer and pancreatic cancer. In therapeutic applications, compositions are administered to a patient in an amount sufficient to effectively abrogate mature vasculature within the tumor, and thereby cure or at least partially arrest the cellular proliferation and tumor growth. An amount adequate to accomplish this is defined as "effective amount" or "therapeutically effective dose." Amounts effective for this use will depend on the stage and severity of the pathology disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician. It should be kept in mind that the combination according to the present invention may be employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these compositions.

Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician. In any event, the pharmaceutical formulations should provide a quantity of composition of lysyl oxidase inhibitor alone or in combination with a chemotherapeutic agent of the invention sufficient to effectively treat the patient.

Administration should begin at the first indication of tumor being resistant to regular cancer treatment regimen or shortly after diagnosis, and continue until symptoms

are substantially abated and for a period thereafter. In well established cases of disease, loading doses followed by maintenance doses will be required.

The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral or local administration. Preferably, the pharmaceutical compositions are administered parenterally, e _^ g., intravenously, subcutaneously, intradermal Iy, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a solution of the lysyl oxidase inhibitor alone or in combination with a chemotherapeutic agent in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

The concentration of the combination of the invention in the pharmaceutical formulations can vary widely, Le _í , from less than about 1%, usually at or at least about 10-15% to as much as 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.

Thus, a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 100 mg of the combination. Actual methods for preparing parenterally administrable compounds will be known or apparent to those skilled in the art and are described in more detail in for example, Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), and the 18 ^th and 19 ^th editions thereof, which are incorporated herein by reference.

For solid compositions conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more lysyl oxidase inhibitor, and more preferably at a concentration of 25%-75%.

It is another aspect or object of the present invention to provide a method of treating diseases and processes that are mediated by angiogenesis. It is yet another aspect of the present invention to provide a method and composition for treating diseases and processes that are mediated by angiogenesis including, but not limited to, hemangioma, solid tumors, blood borne tumors, leukemia, metastasis, telangiectasia, psoriasis, scleroderma, pyogenic granuloma, myocardial angiogenesis, Crohn's disease, plaque neovascularization, coronary collaterals, cerebral collaterals, arteriovenous malformations, ischemic limb angiogenesis, corneal diseases, rubeosis, neovascular glaucoma, diabetic retinopathy, retrolental fibroplasia, arthritis, rheumatoid arthritis, diabetic neovascularization, diabetic retinopathy, macular degeneration, wound healing, peptic ulcer, Helicobacter related diseases, fractures, keloids, vasculogenesis, hematopoiesis, ovulation, menstruation, placentation, psiorasis, obesity and cat scratch fever.

It is another aspect of the present invention to provide a composition for treating or repressing the growth of a cancer. It is still another aspect of the present invention to provide a method for treating ocular angiogenesis related diseases such as macular degeneration or diabetic retinopathy by direct ophthalmic injections of the eye. Another aspect of the present invention is to provide a method for targeted delivery of compositions to specific locations. Yet another aspect of the invention is to provide compositions and methods useful for gene therapy for the modulation of angiogenic processes. Vectors and methods for gene therapy are available in the art.

Throughout this disclosure, applicants refer to journal articles, patent documents, published references, web pages, sequence information available in databases, and other

sources of information. One skilled in the art can use the entire contents of any of the cited sources of information to make and use aspects of this invention. Each and every cited source of information is specifically incorporated herein by reference in its entirety. Portions of these sources may be included in this document as allowed or required. However, the meaning of any term or phrase specifically defined or explained in this disclosure shall not be modified by the content of any of the sources.

The examples that follow are merely exemplary of the scope of this invention and content of this disclosure. One skilled in the art can devise and construct numerous modifications to the examples listed below without departing from the scope of this invention.

EXAMPLES

Example 1: Inhibition of tubules formation in HUVEC spheroid assays

1. 1 Preparation of the spheroid assays Pure powder of Methylcellulose (Sigma #M-0512, 4000 centipoises) (6g) is autoclaved with a magnetic stirrer in a 500ml capped bottle. The powder is dissolved in preheated 250ml EBM-2 (60 ⁰ C) and stirred for 20min. Another 250ml EBM-2, at room temperature, is added to a final volume of 500ml and this stock solution is stirred for 1- 2h at 4°C. Then the solution is cleared by centrifugation (5000g x 2hr, room temperature). The resulting solution, called methocel stock, is collected. All processes are under sterile conditions.

The HUVEC cells are grown in EGM-2 and trypsinized and counted as indicated above. A defined number of cells (750 cells in 150ul medium/ well of 96 well plate) are mixed with methocel containing medium which is 20% methocel stock solution and 80% EGM-2. For example, for one 96-well-plate, 7.5 x 104 cells are suspended in 15ml of well mixed medium, which contains 12ml of EGM-2 and 3ml of methocel stock. 150ul of the cells are distributed to each well of the 96-well-plate (Cat, # 650185 from Greiner, suspension culture, U-form). After 24h at 37°C in the incubator, all suspended cells in each well contribute to the formation of a single endothelial cell spheroid, called standard sized spheroid.

The following reagents are prepared:

Fibrinogen stock: 4 mg/ml Fibrinogen (Sigma #F-4753) is prepared in EBM-2, filtered with 0-2um filter (VWR#28144-040) under sterile conditions. The fibrinogen stock is prepared fresh for each assay. Thrombin stock: 100U/ml thrombin (Sigma #T-4648) is prepared in EBM-2, filtered and aliquoted to small volume. The thrombin stock is stored at -20°C, and thawed before use. Plasma stock: lyophilized plasma (Sigma #P4639) is dissolved as the indicated volume with ddH2O, filtered and aliquoted to small volumes. The plasma stock is stored at - 2O ⁰ C, and thawed before use. 2x assay control medium: 2 sets of BulletKits are added into one EBM-2, but the FBS, bFGF and VEGF Bullets from the kits are not added to the EBM-2 medium. So the 2x assay control medium is the 2x EGM-2 without any FBS, bFGF and VEGF. 2x assay medium: 2x assay control medium is combined with l-2ug/ml bFGF CR&D#234-FSE) and l-2ug/ml VEGF (R&D #293-VE). 0.25ml of 2x assay or control medium is mixed with 0.25ml of Fibrinogen stock, lOul of Thrombin stock, and lOul of Plasma stock. The mixture is quickly spread on the bottom of one well of a 24-well tissue culture plate (VWR # 29442-044). The culture plate is kept at 37°C in an incubator until the fibrin gels solidify. The spheroids are harvested within 24h, using standard pipettes (VWR # 14670-1 14). 12 spheroids are transferred into one 15ml centrifuge tube with 10ml of PBS, and centrifuged at 300-500 xg, for 3 minutes. The supernatant is removed and the tube briefly scratched over a rough surface to loosen the pellet. The loosened pellet is then not allowed to stay in the tube for more than 15-30 minutes, otherwise the spheroids comprising the pellet sticks together. Each pellet is overlaid (not mixed) with 0.25 ml of 2x assay or control medium, lOul of Plasma stock, lOul of Thrombin stock, and 0.25ml of Fibrinogen stock in each 15ml spheroid-containing tube. The contents of the tube are mixed well and quickly spread onto the corresponding fibrin gel of each well of a 24-well-plate. The plates are incubated at 37 ⁰ C until the gels solidified. 500 ul of IX corresponding medium is added on top of the gel. The plates are placed back in incubator.

1.2 Identification of tubule formation activity - Lysyl Oxidase The spheroid assay noted above is used to identify genes differentially expressed during tubule formation. A comparison of the mRNA expressed at different days, for example before and then during tubule development, can reveal active genes and proteins to be targeted to control or modulate angiogenesis in mammals. Similarly, these genes and proteins can be used to identify therapeutic modulators for angiogenesis and anti- angiogenesis related conditions and diseases. In particular, modulating the up-regulated genes during tubule formation or cell invasion activities during the stages of microvasculature development can be used to prevent the angiogenesis commonly associated with tumor cells, many cancers, and metastasis, for example.

The spheroids are plated at day 0 (TO) and cultured until day 9 (T2) or longer. Serial cultures are used so that mRNA from each time period can be collected and differentially compared. The lysyl oxidase (lox) mRNA is detected at TO. At T2, an approximately seven-fold increase in lox mRNA can be detected. As lox is known to function in the development of extracellular matrices, and extracellular matrix development is critical to new microvasculature development, lox is targeted as a protein and mRNA to be modulated in order to effect angiogenesis.

1. 3 Test of L OX protein inhibition

Spheroids can also be used to identify inhibitors of Lox function, enzymatic activity, microvasculature generation, and RNAi and anti-sense inhibitors. For example, spheroids are treated with various concentrations of Lox inhibitor or combinations of compounds including a Lox inhibitor, i.e., 0.1 μM, lμM, 5μM, and lOμM administered each day. Spheroids are examined and the results measured at around 1 1 days after initial treatment begins. Spheroids treated with an optimum inhibitor concentration or combination will demonstrate an inhibition of tubule formation when compared to controlled, untreated spheroids.

Example 2: Inhibition of tubule formation in HUVEC spheroid assays

2.1 Preparation of inhibiting nucleic acid

Inhibiting nucleic acids that interfere with Lox expression can be used according to methods known in the art. For example, an expression cassette comprising an isolated nucleic acid sequence encoding a small interfering RNA molecule (siRNA) targeted against the Lox gene (SEQ ID NO: 3). The siRNA may form a hairpin structure comprising a duplex structure and a loop structure. The loop structure may contain from 4 to 10 nucleotides, such as 4, 5 or 6 nucleotides. The duplex is less than 30 nucleotides in length, such as from 19 to 25 nucleotides. The siRNA may further comprise an overhang region. Such an overhang may be a 3 Overhang, a 5' overhang, or both 3' and 5' overhangs. The overhang region may be from 1 to 6 nucleotides in length. The expression cassette may further comprise a promoter. Examples of promoters include regulatable promoters and constitutive promoters. For example, the promoter may be a CMV, RSV, or polIII promoter. Similarly, an anti-sense sequence or siRNA short nucleotide can be used as a agent directly. One of skill in the art is familiar with methods of producing short RNA and DNA molecules for such purposes.

The selection of the region of the Lox gene to use in the inhibition should take into account the related genes and isozymes. Accordingly, an optimum inhibiting sequence would be from the N-terminal end of the Lox sequence and avoid the copper coordination domain (SEQ ID NO: 2) and sequence C-terminal to it. A comparison of available sequences can identify several 19 to 25 nucleotide regions suitable for inhibiting Lox expression. The design of siRNAs and hairpins in particular can be guided by structural and thermodynamic rules identified in the literature. See Schwarz, et. al., Asymmetry in the assembly of the RNAi enzyme complex, Cell, 1 15(2): 199-208 (2003); Khvorova, et. al., Functional siRNAs and miRNAs exhibit strand bias, Cell, 1 15(4):505 (2003); Reynolds, et. al., Rational siRNA design for RNA interference, Nature Biotechnology, 22(3):326-30 (2004). A number of commercial and open source algorithms for designing siRNAs and their target sequences are also available to one of skill in the art. Some of the software available or the companies offering such software

include (but are not limited to): EMBOSS, Promega siRNA Target Designer, GeneScript, Quiagen, Ambion, OligoEngine, Whitehead, Sfold, HannonLab and Sonnhammer Bioinformatics Group. These programs offer the user a wide range of selections and rule sets to choose from when designing siRNAs and their targets. Representative nucleotide regions suitable for antisense or RNAi inhibition within the human Lox gene would include those that specifically bind to any one of the following regions of SEQ ID NO: 3 : 1028-1046 or 406-424 or 810-828 or 1033- 1051 or 828-846 or 1 1 17-1 135 or 1 131-1 149 or 1227-1245 or 1251-1269 or 1334-1352 or 1361- 1379 or 191 1-1929 or 1033-1051 or 1 167-1 185 or 1249-1267 or 1356-1374 or 1432-1450 or 1761-1779 or 1704-1722. Many other possible regions can be selected, and specifically regions that differ from those listed above by moving 1, 2, 3, 4, 5, or 6 nucleotides in one direction (3' or 5') of the sequence or in both directions. Also, as noted above, RNAi design algorithms are available {see http://sonnhammer.cgb.ki.se/siSearch/siSearch 1.4.html using the default rules) and can be used to identify candidate RNAi target sites with the full length lox sequence or a fragment of it, particularly N-terminal fragments or fragments encompassing exon 1 and/or fragments excluding the encoded copper coordination site. Additional rules or suggestions for RNAi design can be found at http://www.protocol- online.org/prot/Detailed/3210.html and other web sites.

2.2 Combination Treatments

In combination with the lysyl oxidase inhibitors and nucleic acid inhibitors, one can select one or more of a variety of available chemotherapeutics, such as Gemcytabine and cyclophosphamide, non-limiting examples of chemotherapeutic agents that can be combined. Those skilled in the art will recognize that other anti-cancer compounds and therapies can similarly be readily combined with the lysyl oxidase and nucleic acid inhibitors of the invention and are within the scope of the invention. Such compounds and therapies are well known in the art (see for example Cancer: Principles and Practice of Oncology, Volumes 1 and 2, eds Devita, V. T., Hellman, S., and Rosenberg, S. A., J. B. Lippincott Company, Philadelphia, USA; incorporated herein by reference) and

include, without limitation, folates, antifolates, pyrimidine analogs, fluoropyrimidines, purine analogs, adenosine analogs, topoisomerase I inhibitors, anthrapyrazoles, retinoids, antibiotics, anthacyclins, platinum analogs, alkylating agents, nitrosoureas, plant derived compounds such as vinca alkaloids, epipodophyllotoxins, tyrosine kinase inhibitors, taxols, radiation therapy, surgery, nutritional supplements, gene therapy, radiotherapy, for example 3D-CRT, immunotoxin therapy, for example ricin, and monoclonal antibodies. Specific examples of chemotherapeutic compounds that can be combined with or used in conjunction with the nucleic acid molecules of the invention include, but are not limited to, Paclitaxel; Docetaxel; Methotrexate; Doxorubin; Edatrexate; Vinorelbine; Tomaxifen; Leucovorin; 5-fluoro uridine (5-FU); lonotecan; Cisplatin; Carboplatin; Amsacrine; Cytarabine; Bleomycin; Mitomycin C; Dactinomycin; Mithramycin; Hexamethylmelamine; Dacarbazine; L-asperginase; Nitrogen mustard; Melphalan, Chlorambucil; Busulfan; Ifosfamide; 4-hydroperoxycyclophospham- ide; Thiotepa; Irinotecan (CAMPTOSAR.RTM., CPT-1 1, Camptothecin-1, Campto) Tamoxifen; Herceptin; IMC C225; ABX-EGF; and combinations thereof. The above list of compounds are non-limiting examples of compounds and/or methods that can be combined with or used in conjunction with the inhibitors of the invention. Those skilled in the art will recognize that other drug compounds and therapies can similarly be readily.