MXPA00010277A - Neuropilin antisense oligonucleotide sequences and methods of using same to modulate cell growth - Google Patents

Abstract

This invention relates to oligonucleotides complementary to the neuropilin genes which modulate tumor cell growth and angiogenesis in mammals. This invention is also related to methods of using such compounds in inhibiting the growth of tumor cells and angiogenesis in mammals. This invention also relates to pharmaceutical compositions comprising a pharmaceutically acceptable excipient and an effective amount of a compound of this invention.

Description

NEUROPILINE ANTI-SUITENTIAL OLIGONUCLEOTIDE SEQUENCES AND METHOD OF USING THEM FOR MODULAR CELLULAR GROWTH Reference to Related Requests This application claims the priority of the Provisional Application of the United States of America Serial No. 60 / 082,791 filed on April 23, 1998, the application of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention This invention relates to oligonucleotides that are complementary to mammalian neuropilin (or VEGF165R) whose oligonucleotides modulate cell growth in mammals. This invention is also related to methods of using such compounds in inhibiting the growth of tumor cells in mammals and in inhibiting angiogenesis in mammals. This invention also relates to pharmaceutical compositions comprising a pharmaceutically acceptable excipient and an effective amount of a compound of this invention.

References The following publications, patent applications and patents are cited in this application as numbers of fÜillti ÍF 11 riiiii ilil ii li íittiliirpillili-liillif ilUTI i ll NII ilrHi MUI superscript:.. 1. Tischer, E., et al, "The human gene for vascular endothelial growth factor protein Multiple forms are encoded through alternative exon splicing , "J. Biol Chem. 266: 11947-54, (1991). 2. Poltorak, Z., et al., "VEGF1 5, a secreted vascular endothelial growth factor isoform that binds to extracellular matrix", J. Biol Chem. 272; 7151-8, 1997. 3. Terman, B. I., et al., "Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor", Biochem Biophys Res Commun. 187: 1579-86, 1992. 4. Millauer, B., et al., "High affinity VEIGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis", Cell. 72: 835-46, 1993. 5. Shibuya, M., et al., "Nucleotide sequence and expression of a novel human receptor-type tyrosine kinase gene (fit) closely related to the fms family", Oncogene. 5: 519-24, 1990. 6. de Vries, C, et al., "The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor" Science 225: 989-91, 1992. 7. Kawakami, A., et al., "Developmentally regulated expression of a cell surface protein, neuropilin, in the mouse nervous system", J Neurobiol. 29: 1-17, 1996. 8. Takagi, S., et al., "Expression of a cell adhesion molecule, neuropihn, in the developing chick nervous system", Dev Biol. 170: 207-22, 1995. 9. Soker, S., et al., "Neuropilin-1 is expressed by endothelial and tumor cells as a isoform-specific receptor for vascular endothehal growth factor", Cell. 92: 735-45, 1998. 10. Soker, S., et al, "I nh ibition of vascular endothelial growth factor (VEGF) -induced endothelial cell proliferation by a peptide Corresponding to the Exon 7-Encoded Domain of VEGF165". J. Biol Chem. 272: 31582-8, 1997. 11. He, Z. and Tessier-Lavigne, M. "Neuropilin is a receptor for the chemorepellent axonal Semaphorin III", Cell. 90: 739-51, 1997. 12 Mitsuhashi, M. "Strategy for designing specific antisense oligonucleotide sequences", J Gastroenterol. 32: 282-7, 1997. 13. Alama, A., et al., "Antisense oligonucleotides as therapeutic agents", Pharmacol Res. 36: 171-8, 1997. 14. Curcio, LD, et al., "Oligonucleotides as modulators of cancer gene expression", Pharmacol Ther . 74: 317-32, 1997. 15. Brem, S., et al., "Prolonged tumor dormancy by prevention of neovascularization in the vitreous", Cancer Res. 36: 2807-12, 1976. 16. Holmgren, L., et al, "Dormancy of micrometastases: balanced proliferation and apoptosis in the presence of angiogenesis suppression"., Nat Med. 1:. 149-53, 1995. 17. Parangi, S., et al, "Antiangiogenic therapy of transgenic mice impairs de novo tumor growth, Proc Nati Acad Sci USA 93: 2002-7, 1996. 18. Choy et al., "Molecular mechanisms of drug resistance involving ribonucleotide reducíase: hydroxyurea resistance in a series of . ^ ^^ - ^^^ ^ c I ITM on the mouse cell lines related ly selected in the presence of drug Increasing Concentrations ".. Cancer Res 48: 2029-2035 (1988) 19. Fan et al," Ribonucleotide reductase R2 component is a novel malignancy determinant that cooperates with activited oncogenes to determine transformation and malignant potential "Proc. Nati. Acad. Sci USA 93: 14036-40 (1996) 20. Huang and Wright" Fibroblast growth factor mediated alterations in drug resistance and evidence of gene amplification "Oncogene 9: 491-499 (1994) 21. International Patent Application Publication No. WO99 / 02556," Semaphopn Receptors "22. International Patent Application Publication No. WO99 / 04263," Semaphopn Receptors "23. Remington's Pharmaceutical Sciences , Mace Publishing Company, Philadelphia PA 17th ed. (1985) 24. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989, 1992) 25. Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore Maryland (1989). 26. Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988). 27. Hurta and Wright, "Malignant transformation by H-ras results n aberrant regulation of ribonucleotide reductase gene expression by transforming growth factor-beta" J. Cell Biochem 57: 543-556 (1995) 28. International Patent Application Publication No. WO97 / 21808, "Modlfied VEGF Antisense Oligonucleotides" 29. Nielsen et al .; Science (1991) 354: 1497 30. Good and Nielsen; "Inhibition of bacterial growth by translation and peptide nucleic acid targeted to ribosomal RNA" PNAS USA (1998) 95:. 2073-2076 31. Buchardt, deceased, et al, U.S. Patent No. 5,766,855 32. Buchardt, deceased, et al., U.S. Patent No. 5,719,262 33. U.S. Patent No. 5,034,506 34 Aitschui, et al. "Basic local alignment search tool" J. Mol. Biol. (1990) 215: 403-10; 35. Devereux J. et al., "A Comprehensive Being of Sequence Analysis Programs for the VAX", Nucleic Acids Res. (1984) 12: 387-395; 36. Chang et al .; Somatic Gene Therapy, CRC Press, Ann Arbor Ml (nineteen ninety five); 37. Vega et al., Gene Targeting, CRC Press, Ann Arbor Ml (1995) 38. Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Ma (1988) 39. Sulllvan U.S. Patent No. 5,225,347 40. U.S. Patent 5,023,252, issued June 11, 1991 41. Felgner et al., U.S. Patent No. 5,580,859 42. Dreeley et al., Science, 258: 1650-1564 (1992) 43. Uhlmann et al. Chem Rev. 90: 534-583 (1990) 44. Agrawal et al. Trends Biotechnol. 10: 152-158 (1992) ^^^ -. ^^ _ ^^^. ^ A.Í ^ ...-- ^^. ^^^^^^^ ... 45 Smith et al., (1994) Invest. Ophthaimol. Vis. Sci 35: 101-111 46 Plerce et al., (1995) Proc. Nati Acad Sci USA 92: 905-9 All publications, patent applications and patents above are incorporated herein by reference in their entirety to the same extent as if each publication, patent application or patent was specifically and individually indicated to be incorporated by reference in its entirety.

State of the Art The proliferation of capillaries nodes, called angiogenesis or neovascularization is critical for the transition from a localized tumor is small to expand into more malignant growth. With the proper development of the blood supply, the growth of the tumor deteriorates dramatically. Neurovascular diseases of the retina such as diabetic retiniopathy, retiniopathy of premature and age-related macular degeneration are the main causes of blindness in the United States of America and in the world. During the course of diabetes mellitus, the retinal vessels undergo changes that result not only in vessels with effusion but also in vessel separation resulting in retinal hypoxia. One of the effects of this neovascularization of the retina results in bleeding and separation of the retina. Premature retiniopathy is a common cause of blindness in children. Cups The blood of the retina ceases to develop within the peripheral retina, resulting in ischemia and localized hypoxic conditions as the metabolic demands of the development of the retina are increased. The resulting hlpoxia stimulates the subsequent neovascularization of the retina which can lead to an irreversible loss of sight. Ocular neovascularization is also the underlying pathology in falsiform retiniopathy, neorovascular glaucoma, retinal vein occlusion and other hypoxic diseases. Recent experimental data show a high correlation between vascular endothelial growth factor expression and retinal neovascularization. Of the numerous angiogenic factors produced from tumor cells, vascular endothelial growth factor (VEGF) appears to be a major mediator of tumor angiogenesis and neovascularization. The monomers of human VEGF exist as five different isoforms between which VEGF121 and VEGF? 65 are the abundant ones (1,2). VEGF activities are exerted by their binding to high affinity tyrosine kinase receptors present in the tumoral tilt of endothelial cell lining. These two receivers have been isolated: KDR / Flk-1 (3, 4) which appears to be the main transducer of the VEGF and Flt-1 signals (5, 6). The neuropilin or VEGF165 of the vascular endothelial growth factor receptor that was originally isolated as a receptor for the collapsin / semaphorin that mediates cell guidance _i? ?? áü ?? u n iiiiiiiiEE.EMi.il Hip ?? il? l_? m_Éil_¡ü ?? i rifli «h i ¡i IIIIIIIIIII nerium 87, 8), has recently been cloned as a new specific isoform receptor expressed by endothelial cells for VEGF165 (9). The nucleic acid sequence for human neuropilin has been reported (9, 11, 21, 22). Neuropilin acts as a co-receptor for VEGF? 65 which binds to KDR / Flk-1 and modulates the subsequent bioactivity, ie tumor-induced angiogenesis. It is also highly expressed in tumor-derived cells such as breast carcinoma cells MDA-MB-231 and PC3 prostate carcinoma cells, among some tested (9, 10). VEGF has also demonstrated binding to Hela, melanoma and NIH 3T3 cells. Antisense technology has been widely adopted not only as a useful research tool (12), but also as a racial approach to acquiring new therapeutic compounds for the treatment of many human diseases including cancer (13, 14). Antisense oligonucleotides can specifically hybridize to mRNA sequences and inhibit the expression of proteins that are important in the initiation and / or progression of human cancer. Therefore, it would be desirable to identify antisense oligonucleotides directed against neuropilin which acts to inhibit the expression and production of neuropilin / VEGF ^ sR with greater specificity and less toxicity. lj ^ »^^» ^ mgr ^^ > This invention is directed to antisense oligonucleotides that modulate the expression of neuropilin genes and the production of neuropilin / VEGR165R in mammals and pharmaceutical compositions that comprise such antisense oligonucleotides This invention is also related methods of using such antisense oligonucleotides to inhibit the proliferation of new capillaries or angiogenesis or neovascularization Involved in tumor growth and metastasis in mammals.Accordingly, in one of its aspects of composition, this invention is directed to an antisense oligonucleotide of from about 3 to about 100 nucleotides, comprising nucleotides complementary to the neuropilin, mRNA of a mammal.The antisense oligonucleotide can be nuclease resistant and can have one or more antisense bonds. phosphorothioate internucleotide. antisense oligonucleotide may further comprise additional nucleotides that are not complementary to the neuroprone mRNA- In another aspect of composition, this invention is directed to an antisense oligonucleotide from about 20 to about 100 nucleotides, comprising a sequence selected from the group consisting of SEC. FROM IDENT. NOs: 1-30 established in Table 1 whose oligonucleotide inhibits the expression of neuropilin. In another aspect of composition, this invention ^ y | • Í..Í.Á ¡A? gA ^ £ ú¿ ^ t ^^^ ut ^^^^^^^^ is directed to a vector comprising an oligonucleotide sequence from about 20 to 100 nucleotides comprising a sequence selected from the group consisting of the SEC . FROM IDENT. NOs: 1-30 as set forth in Table 1 whose ohgonucleotide inhibits the expression of neuropilin. In yet another aspect of its composition, this invention is directed to a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an effective amount of an antisense oligonucleotide of from about 20 to about 100 nucleotides comprising a sequence selected from the group consisting of of SEC. FROM IDENT. NOs: 1-30 as set forth in Table 1 whose oligonucleotide inhibits the expression of neuropilin. In one of its aspects of the method, the invention is directed to a method for inhibiting the growth of a mammalian tumor comprising, administering to a mammal suspected of having the tumor an effective amount of an antisense oligonucleotide from about 3 hours per day. nucleotides up to about 100 nucleotides comprising a sequence complementary to the mammalian neuropilin mRNA under conditions such that tumor growth is inhibited. The antisense oligonucleotide can be administered with a chemotherapeutic agent. In another of its aspects of the method, this invention is directed to a method for inhibiting the metastasis of a tumor of ^ ¡¡^ M? "BMi máá" mammal comprising, administering to a mammal suspected of having the metastatic tumor, an effective amount of an antisense oligonucleotide from about 3 nucleotides to about 100 nucleotides comprising a sequence complementary to the mammalian neuropilin mRNA under conditions such that tumor metastasis is inhibited. The antisense oligonucleotide can be administered with a chemotherapeutic agent. In another of its aspects of the method, this invention is directed to a method for inhibiting angiogenesis or neovasculapzation in a mammal comprising, administering to a mammal an effective amount of an antisense oligonucleotide from about 3 nucleotides to about 100 nucleotides complementary to the mammalian neuropilin mRNA under conditions such that neovascularization is inhibited. In another aspect of the method, this invention is directed to a method of inhibiting neuropilin expression comprising contacting the neuropilin-specific nucleic acid with an antisense oligonucleotide from about 20 nucleotides to about 100 nucleotides comprising a sequence. selected from the group consisting of SEC. FROM IDENT. NOs: 1-30 as set forth in Table 1 whose oligonucleotide inhibits the expression of neuropilin.

^^ - ^ ^^ .. ^^ A, ^^ l ^^ ai ^ BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-F are graphs of percent inhibition of colony formation ability of different cell lines by administration of the indicated antisense oligonucleotides. Figure 1A shows the percentage of inhibition of the human melanoma cell line C8161; Figure 1B shows the percentage of inhibition of the human lung cancer cell line A549; Figure 1C shows the percentage of inhibition of the human melanoma cell line A2058; Figure 1D shows the percent inhibition of the human colon HT-29 cell line; Figure 1E shows the percentage of inhibition of the human prostate cancer cell line PC-3; and Figure 1F shows the inhibition percentage of the human pancreatic cancer cell line AsPC-1. Figures 2A and 2B are autoradiographies of stains Northern RNA from the human melanoma cancer cell line A2058 (Figure 2B) or the human breast cancer cells line MDA-MB-231 (Figure 1A) after administration with one of the following antisense oligonucleotides: GTI3601 [SEC. FROM IDENT. NO: 1] GTI3602 [SEC. FROM IDENT. NO: 2]; GTI3603 [SEC. FROM IDENT. NO: 3]; GTI3604 [SEC. FROM IDENT. NO: 4]; GTI3610 [SEC. FROM IDENT. NO: 10]; GTI3611 [SEC. FROM IDENT. NO: 11]; and GTI3612 [SEC. FROM IDENT. NO: 12]. Figure 3A is a graph of the volume of a tumor over time after injection of HT-29 human colon cancer cells into the right flank of mice with administration of antisense oligonucleotide GTI3602 [SEQ. FROM IDENT. NO: 2] or without that administration (saline). Figure 3B is a graph of the weight of a tumor 20 days after the injection of human colon cancer cells HT-29 on the right flank of mice with administration of antisense oligogonucleotide GTI3602 [SEC. FROM IDENT. NO: 2] or without such administration (saline). Figure 4 is a graph of the average number of lung metastases per mouse by the human melanoma cell line C8161 after treatment of the cell line with the antisense oligonucleotides GTI3611 [SEQ. FROM IDENT.

NO: 11] or GTI3602 [SEC. FROM IDENT. NO: 2] or without them (control). Figure 5 is the nucleotide sequence of the human neuropiiline cDNA [SEQ. FROM IDENT. NO: 33]. Figure 6 is the nucleotide sequence of rat neuropilin cDNA [SEQ. FROM IDENT. NO: 34 |; Figure 7 is the nucleotide sequence of the mouse neuropilin cDNA [SEQ. FROM IDENT. NO: 35].

DETAILED DESCRIPTION OF THE INVENTION This invention relates to oligonucleotides complementary to mammalian neuropllin mRNA whose oligonucleotide modulates cell growth. Neuropilin is a receptor for vascular endothelial growth factor or VEGF. VEGF has been found to modulate tumor-induced angiogenesis. Neuropilin is also highly expressed in tumor derived cells such as breast carcinoma cells MDA-MB-231 and in tissue culture cells such as Hela and NIH 3T3 cells. This suggests that, in addition to its role in angiogenic stimulation, neuropilin can act in an autocrine manner, as an individual signal transducer for VEGF activities on tumor cells improving survival, differentiation or motility. Another possibility may be that neuropilin has a storage or sequestration function.

Definitions As used herein, the following terms have the meanings indicated: The term "antisense oligonucleotide" as used herein means a nucleotide sequence that is complementary to the desired mRNA. Preferably, the antisense oligonucleotide is complementary to that portion of a mammalian neuropilin mRNA or VEGF165R mRNA that effectively acts to inhibit the expression of neuropilin. It is contemplated that the antisense oligonucleotide may be complementary to any 5'-untranslated region. of the mRNA, the coding region or the 3 'untranslated region of the mRNA. More preferably, the antisense oligonucleotide ^^ is complementary to the nucleotide sequence set forth in Figure 5. Without being limited to any theory or mechanism, it is generally considered that the activity of the antisense oligonucleotides depends on the binding of the oligonucleotide to the target nucleic acid (e.g. , at least a portion of a genomic region, gene or mRNA transcription thereof), thereby interrupting the function of the target, either by stopping the hybridization or by destroying the target RNA by means of Rnase H (the ability to activate Rnase H when it hybridizes to RNA) resulting in the inhibition of neuropilin expression. The term "oligonucleotide" refers to a nucleotide oligomer or polymer or nucleoside monomers that consist of naturally occuring bases, sugars, and inter-sugar bonds (base structure). The term also includes modified or substituted oligomers comprising monomers that are not naturally occurring or portions thereof, which function similarly. Such modified or substituted oligomers may be preferred over forms of natural occurrence due to properties such as improved cellular uptake, or increased stability in the presence of nucleases. The term also includes chimeric oligonucleotides which contain two or more chemically distinct regions. For example, the chimeric oligonucleotides may contain at least one region of modified nucleotides that confer beneficial properties (e.g. , ^ «- ^ - i. - - -. *. **** .. _..._. ^, ^ ... ^^. ^ - ^. ^ ^ ^ »Rfíjütür increased to nuclease, increased uptake within the cells) or two or more oligonucleotides of the invention can be joined to form a chimeric oligonucleotide. The antisense oligonucleotides of the present invention may be ribonucleic or deoxyribonucleic acid and may contain synthetic or naturally occurring monomer bases, including adenine, guanine, cytosine, tyrosine, and uracil. The oligonucleotides may also contain modified bases such as xanthine, hypoxanthine, 2-amino-adenine, 6-methyl, 2-propyl and other alkyladenines, 5-halouracil, 5-halocytosine, 6-azauracil, 6-azacytosine, 6-azathimine, pseudo uracil, 4-thououcil, 8-haloadenine, 8-aminoadenine, 8-thioladenine, 8-thiolalkyladenines, 8-hydroxyladenine and other 8-substituted adenines, 8-haloguanins, 8-amlnoguanine, 8-tlolguanine, 8-thioalkylguanines, 8 -hydroxylguanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines or guanines, 5-trifluoromethyluracil and 5-trifluorocytosine. The modifications may also include binding to other chemical groups such as methyl, ethyl or propyl to different parts of the oligonucleotides Including sugar, base or base structure components. The antisense oligonucleotides of the invention can also comprise phosphorus-oxygen heteroatoms modified in the phosphate base structure, short chain alkyl or cycloalkyl interazicar linkages or short chain heteroatom or heterocyclic linkers. For example, fr? it n luí i vmñn'í'iwí? 'Rmrr - ^ ^^ ^ M * - - - j ??? i ????? i i ii _aiÉi i_iiliiii antisense oligonucleotides may contain methylphosphonates, phosphorothioates, phosphorodithioates, phosphotriesters and morpholino oligomers???. Antisense oligonucleotides may comprise phosphorothioate linkages that bind between four to six nucleotides of 3 'terms. Phosphorothioate bonds can bind to all nucleotides. The phosphorothioate linkages can be mixed enantiomers RP and SP or can be stereoregular or substantially stereoregular in any RP and SP form. The antisense oligonucleotides can also have sugar mimetics. The oligonucleotide can have at least one nucleotide with a modified base and / or sugar, such as the 2'-O-substituted ribonucleotide. For purposes of the invention, the term 2'-0-substituted "means the substitution of the 2 'position of the pentose portion with a lower alkyl group -O- containing 1-6 saturated or unsaturated carbon atoms, or with an -O- aryl or allyl group having 2-6 carbon atoms, wherein the alkyl or aryl ayllu may be substituted or unsubstituted, for example, halo, hldroxi, trifluoromethyl, cyano, nitro, acyl group, acyloxy, alkoxy, carboxllo, carbalkoxy, or amino groups. oligonucleotides of the invention may include four or five ribonucleotides 2'-O-alqullados in terms 5 'and / or four or five ribonucleotides 2'-O-alllados in terms 3 'The antisense oligonucleotides of the invention may also comprise nucleotide analogs wherein the structure of nucleotides is fundamentally altered. ri? f? itr1ÉÍtlimr-? ffnpiiini¡ "my antinucleótido such analogue is a peptide nucleic acid (PNA) wherein the deoxyribose phosphate structure (or ribose) in DNA (or RNA) is replaced with a polyamide base structure which is similar to that found in the peptides. (Nielsen et al.29; Good and Nielsen30; Buchardt, deceased, et al31, United States Patent No. 5,766,855; Buchardt, deceased, et 32 Patent of the United States of America No. ,719,262). PNA analogs have been shown to be resistant to degradation by enzymes and to have prolonged lives in vivo and in vitro. The PNAs also bind more strongly to a complementary DNA sequence than to a nucleic acid molecule of natural occurrence due to the lack of charge repulsion between the PNA strand and the DNA strand. The oligonucleotides of the present invention may also include nucleotides comprising polymer base structures, cyclic base structures, or acyclic base structures. For example, the nucleotides may comprise morpholino base structures (U.S. Patent No. 5,034,506 33). The oligonucleotides of the present invention are "Resistant to nuclease" when they have been modified so that they are not susceptible to nuclease degradation of DNA and RNA or alternatively have been placed on a delivery vehicle which itself protects the oligonucleotide from nucleases of DNA or RNA. Oligonucleotides resistant to - - -1 and *** - * ^ - ** nuclease include, for example, methylphosphonates, phosphorothioates, phosphorodithioates, phosphotriesters and morpholino oligomers. Such delivery vehicles for conferring nuclease resistance include, for example, liposomes. The oligonucleotides of the present invention may also contain groups, such as groups, to improve the pharmacokinetic properties of an oligonucleotide or groups to improve the pharmacodynamic properties of an oligonucleotide. The antisense oligonucleotides are preferably selected from the sequence complementary to the neuropillin gene. Preferably, the sequence exhibits the lowest probability of demonstrating double formation, hairpin formation and homooligomer / sequence repeats although it has a high to moderate potential for binding to neuropillin gene sequences. These properties can be determined using the computer modeling program OLIGO Primer analysis Software, Version 5.0 (distributed by National Biosciences, Inc., Plymouth, MN). This computer program allows the determination of a qualitative estimate of these five parameters. Alternatively, the antisense oligonucleotides can also be selected on the basis that the sequence is highly conserved by the neuropilin gene between two or more mammalian species. These properties can be determined using the BLASTN program (Altschul, et al.34) of the University of Wisconsin Computer Group (GCG) software (Devereux J. et al., 35) with the National Center for Biotechnology Information (NCBI) databases. . Antisense oligonucleotides can include mutations, such as substitutions, insertions and deletions. Preferably less than 10% of the sequence will have mutations. Antisense oligonucleotides generally comprise from at least about 3 nucleotides or nucleotide analogues, more preferably there are at least about 5 nucleotides, more preferably there are at least about 7 nucleotides, still more preferably there are of at least about 9 nucleotides and more preferably there are at least about 20 nucleotides. The antisense oligonucleotides are preferably less than about 100 nucleotides or nucleotide analogues, more preferably less than about 50 nucleotides or nucleotide analogues, more preferably less than about 35 nucleotides or nucleotide analogs. Preferably, the antisense oligonucleotides comprise the sequences set forth in Table 1 (below).

Table 1. Antisense oligonucleotides having a complementary sequence for human neuropilin mRNA grt | The antisense oligonucleotides of Table I were selected from the complementary sequence for the human neuroplline / VEGF16sR mRNA so that the sequence exhibited the lowest probability of showing double formation, hairpin formation and repeats of homooligomers / sequence although it has a high potential to bind to the neuropilin / VEGF165R mRNA sequence. In addition, false priming for other sequences of frequent or repetitive occurrence in humans and mice was eliminated. These properties were determined using the computer modeling program OLIGO® Primer Analysis Software, Version 5.0 (distributed by National Biosciences, Inc., Plymouth, MN).

In Table 1 the "Tm" is the melting temperature of a double oligonucleotide calculated according to the thermodynamic values of the nearest neighbor. At this temperature 50% of the nucleic acid molecules are in the double state and 50% are denatured. The "? G" is the free energy of the oligonucleotide, which is a measurement of a double stability of the oligonucleotide. The term "alkyl" refers to monovalent alkyl groups preferably having from 1 to 20 carbon atoms and more preferably from 1 to 6 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, io-propyl, n-butyl, / so-butyl, / i-hexyl and the like. The term "aryl" refers to an unsaturated carbocyclic aromatic group of 6 to 14 carbon atoms having an individual ring (for example phenyl) or multiple (fused) condemned rings (for example naphthyl or anthryl). Preferred aryls include phenol, naphthyl and the like. The term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine and is preferably either fluorine or chlorine. For any of the above groups containing one or more substituents, it is of course understood that such groups do not contain any substitution or substitution patterns that are sterilically impractical and / or synthetically non-viable. In addition, the compounds of this invention include all the stereochemical isomers that arise from the substitution of those compounds. Í ??? -i-Ü,. - i | i i | M II iiiilÉllf MMÍ lli] _M¡ llllJMI l 11 lil i ?? ^^^ yja t ^ ijfc The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient (s). The material is compatible with a biological system such as a cell, cell culture, tissue or organism. The term "pharmaceutically acceptable salt" refers to salts that retain the effectiveness and biological properties of the antisense oligonucleotides of this invention and that are not biologically or otherwise undesirable. In many cases, the antisense oligonucleotides of this invention are capable of forming acid and / or base salts by virtue of the presence of the amino and / or carboxyl groups or groups similar thereto. The pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases include as an example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines such as alkylamines, dialkylamines, trialkylamines, substituted alkylamines, substituted dialkylamines, substituted trialkylamines, alkenylamines, dialkenylamines, trialkenylamines, substituted alkenylamines, substituted dialkenyl amines, substituted trialkenylamines, cycloalkylamines, dicycloalkylamines, tricycloalkylamines, substituted cycloalkylamines, cycloalkylamines disubstituted, trisubstituted cycloalkylamines, cycloalkenylamines, diclcloalkenylamines, tricycloalkenylamines, substituted cycloalkenylamines, disubstituted cycloalkenylamines, trisubstituted cycloalkenylamines, arylamines, diarylamines, triarylamines, heteroaryl amines, diheteroarylamines, triheteroarlamines, heterocyclic amines, diheterocyclic amines, triheterocyclic amines, combined di- and tri-amines where at least two of the substituents on the amine are different and are selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aplo, heteroaryl, heterocyclic, and the like. Also included are amines where two or three of the substituents, together with the amino nitrogen, form a heterocyclic or heteroaryl group. Examples of suitable amines include, by way of example only, / so-propylamine, trimethylamine, diethylamine, triisopropylamine, tri-propylamine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydraramine, choline , betaine, ethylenediamine, glucosamine, N-alkyl-glucamines, theobromine, purines, piperazine, piperidine, morpholine, N-ethylpiperidine, and the like. It should also be understood that other carboxylic acid derivatives would be useful in the practice of this invention, for example, carboxylic acid amides, including carboxamides, lower alkyl carboxamides, dialkylcarboxamides, and the like. The pharmaceutically acceptable addition salts tit * ^ * * - **., acceptable can be prepared from inorganic and organic acids. Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pluric acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, acid cinnamic, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, acid salicylic and the like. The term "neuropilin gene" refers to any gene that encodes a protein that is capable of acting as a receptor for semafopna or VEGF. Preferably, the neuropilin mRNA has a sequence substantially similar to that shown in the Figures , 5, 6 or 7. The term "complementary to" means that the antisense oligonucleotide sequence is capable of binding to the target sequence, i.e. the neuropilin (or mRNA) gene. Preferably, the antisense oligonucleotide binds to the nucleic acid sequence under physiological conditions, for example by Watson-Crick base pairs (interaction between the oligonucleotide and single-stranded nucleic acid) or by Hoogsteen base pairs (interaction between the oligonucleotide and double-stranded nucleic acid) or by any other means that are included in the case of an oligonucleotide binding to RNA, causing a pseudonudo formation. Binding by Watson-Crick or Hoogsteen base pairs under physiological conditions is measured as a practical matter by observing the interference with the function of the nucleic acid sequence. Preferably, the sequence of the antisense oligonucleotide has at least about 75% identity to the target sequence, preferably at least about 90% identity, and most preferably at least about 95% identity to the objective sequence that allows cavities or incompatibilities of different bases. Identity can be determined, for example, using the BLASTN program of the University of Wisconsin Computer Group (GCG) software. Preferably the antisense oligonucleotide sequence hybridizes to the neuropiline mRNA with a melting temperature of at least 45 ° C, more preferably of at least about 50 ° C and most preferably of at least about 55 ° C according to the invention. was determined through the OLIGO First Software Analysis program, version 5.0 described herein. The term "growth inhibition" means a reduction or inhibition in the growth of at least one type of tumor cell, of at least 10%, more preferably of at least 50% and more preferably of at least minus 75% The reduction in growth can be determined for tumor cells by measuring the size of the tumor in nude mice by the l ált i tptiÉÉ "*" '- •' - "• -" T iMlfifií'i • 1iMÍ_Í _._ lr1ÉlHtflÉÉM_ ^ l ^ lack of ability of tumor cells to form colonies in vitro. The term "inhibition of angiogenesis" means a reduction or inhibition to neobascularization. This can be determined by methods well known in the art. A murine model of retinal neovascularization induced by oxygen has been established that occurs in 100% of treated animals and is quantifiable (45, 46). Using this model, a correlation between neuropilin inhibition and inhibition of retinal neovascularization could be measured. This result can also be confirmed by changes in the level of neuropilin expression by Northern tinsion and in situ hybridization analysis. The term "inhibition of metastasis" means the reduction or inhibition of the number of metastatic tumors that develop, preferably of at least about % and more preferably of at least about 50%.

This can be determined by methods established in the examples and other methods known in the art. The term "inhibition of neuropilin expression" means that the antisense oligonucleotide reduces the level of Neuropilin mRNA or the level of the neuropilin protein produced by the cell when the oligogonucleotide is administered to the cell. The term "mammalian" or "mammalian" means that all mammals including humans, sheep, cattle, horses, sheep, canines, felines, and rodents, etc., preferably means i? l ii? l? EHÉrtlIpMÜ-T "" ^, ^ - * "****** human beings A" mammal suspected of having a tumor "means that the mammal may have a proliferative disorder or tumor that has been diagnosed with a proliferative disorder or tumor or has previously been diagnosed with a prollferative disorder or tumor, the tumor that has been removed surgically and the mammal is suspected to harbor some residual tumor cells.

Preparation of the Antisense Oligonucleotides The antisense oligonucleotides of the present invention can be prepared by conventional and well-known techniques. For example, ollgonucleotides can be prepared using solid phase synthesis and in particular using commercially available equipment such as the equipment available from Applied Biosystems Canada Inc., Mississauga, Canada. Oligonucleotides can also be prepared by enzymatic digestion of the naturally occurring neuropilin gene through methods known in the art. These oligonucleotides can be prepared by methods recognized in the art such as phosphoroamidate or H-phosphoate chemistry that can be carried out manually or through the automated synthesizer as described by Uhlmann et al. (43) and Agrawal et al (44). ._ ".. _« * _ A -.... < -, * - »-. -g .t U? É ???? ^? ^^^^^^ ^ Isolation and Purification of Antisense Oliqnucleotides The isolation and purification of the antisense oligonucleotides described herein can be effected, if desired, through proper separation or purification such as, for example, filtration, extraction, crystallization, column chromatography, thick layer chromatography, thin layer chromatography, liquid chromatography of high or low pressure preparation or a combination of those procedures. However, other equivalent separation or isolation procedures could, of course, be used. An expression vector comprising the antisense ollgonucleotide sequence can be constructed by considering the ollgonucleotide sequence and using methods known in the art. The vectors can be constructed by those skilled in the art to contain all the expression elements required to achieve the desired transcription of the antisense oligonucleotide sequences. Therefore, the invention provides vectors comprising a transcription control sequence operatively associated with a sequence encoding an antisense oligonucleotide. Suitable transcription and translation elements can be derived from a variety of sources, including bacterial, fungal, viral, mammalian or insect genes. The selection of appropriate elements depends on the selected host cell.

Reporter genes can be included in the vector. Suitable reporter genes include β-galactosidase (e.g., lacZ), chloramphenicol, acetyltransferase, firefly luciferae, or an immunoglobulin or portion thereof. Transcription of the antisense olinucleotide can be monitored through monitoring for the expression of the reporter gene. Vectors can be introduced into cells or tissues through a variety of methods known in the art. Such methods can be described generically in Sambrook et al.24; Ausubel et al.25; Chang et al.36; Vega et al.37; and Vectors: A Survey of Molecular Cloning Vectors and Their Uses38 and include, for example, transfection, lipofection, electroporation and stable or transient infection with recombinant viral vectors. The introduction of nucleic acids by infection offers several advantages. The highest efficiency and specificity for the type of fabric that can be obtained. Viruses are typically infected and spread in specific cell types. Thus, the specificity of the virus can be used to target the vector for specific cell types in vivo or within a tissue or mixed cell culture. Viral vectors can also be modified with specific receptors or ligands to alter target specificity through events mediated by the receptor. It is contemplated that the oligonucleotide of the invention may be a ribozyme that cleaves the mRNA. The ribozyme A ????? ^ - r? 1T rmmüif .i, ti w? The reaction preferably has a sequence homologous to a sequence of an oligonucleotide of the invention and the catalytic center necessary to divide the mRNA. For example, a homologous ribozyme sequence can be selected that destroys the neuropilin mRNA. The type of ribozyme used in the present invention can be selected from types known in the art. Several ribozyme structural families have been identified including Group I introns, RNase P, hepatitis delta virus ribozyme, hammerhead-shaped ribozymes and fork-shaped ribozyme originally derived from the negative strand of satellite RNA of the ribozyme. tobacco ring-shaped spots virus (sTRSV) (Sullivan 1994, U.S. Patent No. 5,22534739). Ribozymes in the form of a hammerhead and fork are more commonly adapted for transshipment of mRNAs for gene therapy (Sullivan 1994). The hair ribozymes are preferably used in the present invention. In general, the ribozyme is 30 to 100 nucleotides in length. The oligonucleotides of the invention can be insolubilized. For example, the oligonucleotide may be linked to a suitable carrier. Examples of suitable carriers are agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethylcellulose polystyrene, filter paper, ion exchange resin, plastic film, plastic tube, glass beads, polyamine-methylvinyl ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The Porter Httif Ttíif iiritriil TlÉHlT • -riifÉiiri it OtlilUlttír * '** "" - * - * - ^ ~ * - *** - **** may be in the form of, for example, a tube, test plate, disk of beads, sphere, etc. The insolubilized oligonucleotide can be prepared by reacting the material with the suitable insoluble carrier using known chemical or physical methods, for example, coupling cyanogen bromide.

Pharmaceutical Formulations When used as pharmaceuticals, antisense oligonucleotides are usually administered in the forms of pharmaceutical compositions. These compounds can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. These compounds are effective in injectable and oral compositions. Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound. The pharmaceutical composition is for example, administered intravenously, it is contemplated that the pharmaceutical composition can be administered directly into the tumor to be treated. This invention also includes pharmaceutical compositions containing, as the active ingredient, one or more of the antisense oligonucleotides associated with pharmaceutically acceptable carriers or carriers. In making the compositions of this invention, the active ingredient is mixed usually with an excipient, diluted mediant to an excipient or enclosed within such a carrier so that it may be in the form of a capsule, sack, paper or other container. When the excipient serves as a diluent, it can be a solid, semi-solid or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions may be in the form of tablets, pills, powders, lozenges, sachets, capsules, elixirs, suspensions, emulsions, solutions, syrups, sprays (as in a solid medium or in a liquid medium) containing ointments, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions and powders packed in sterile form. In the preparation of a formulation, it may be necessary to grind the active compound to provide the proper particle size before combining it with other ingredients. If the active compound is substantially insoluble, ordinarily, it is ground to a particle size of less than 200 mesh. If the active compound is substantially soluble in water, the particle size is usually adjusted by grinding to provide a substantially uniform distribution in the formulation, for example about 40 meshes. Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia gum, calcium phosphate, alginates, tragacanth, gelatin, silicate '"• iJ • * - * - * - * -' - *" • - * - m - * t * < l * < t, t ^ "-, - j -" - • - ** - * *] TiliTtliiltlIlriMÉí calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup and methylcellulose. The formulations may additionally include: lubricating agents such as talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl and propylhydroxybenzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated to provide rapid, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art. The compositions are preferably formulated in a unit dosage form, each dose containing from about 1% to about 95%, most typically from about 5% to about 90% of the active ingredient. The term "unit dosage forms" refers to physically discrete units suitable as unit doses for humans and for other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. . The antisense oligonucleotide is effective over a wide range of doses and is generally administered in a pharmaceutically effective amount. An effective amount is that amount that when administered relieves symptoms. l i l i ili • utrfcl "» * -'- * 1 '' * »- * - '-" - ^ * «^" ^ "' ^^ - • •« -. ^ _-_ * - > i -.? Preferably, the effective amount is that amount capable of inhibiting the growth of the tumor cell. Preferably the effective amount is from about 0.1 mg / kg of body weight to about 20 mg / kg of body weight. However, it is to be understood that the amount of antisense oligonucleotide administered will actually be determined by a physician, in light of relevant circumstances, including the condition to be treated, the route of administration selected, the actual compound administered, the age, weight and response of the patient, the severity of the patient's symptoms and the like. The course of therapy may last several days or up to several months or until the reduction of the disease is achieved. The antisense oligonucleotide can be administered in combination with other known therapies. When co-administered with one or more other therapies, the oligonucleotide can be administered either simultaneously with another or other treatments, or sequentially. If administered in sequence, the attending physician will decide on the appropriate sequence of administration of the oligonucleotide in combination with the other therapy. To prepare solid compositions such as tablets, the main active ingredient / antisense oligonucleotide is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When those preformulation compositions are referred to as homogeneous, it is given to It is also understood that the active ingredient is dispersed uniformly throughout the composition so that it can be easily subdivided into equally effective unit dose forms such as tablets, pills and capsules. The tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form that achieves the advantage of prolonged action. For example, the tablet or pill may comprise an internal dose and an external dose component, the latter being in the form of a cover over the first. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and allows the internal component to pass intact to the duodenum or be delayed in its release. A variety of materials can be used for such enteric coatings or coatings, such materials include a number of polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol and cellulose acetate. Liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, flavored syrups with suitable flavors, aqueous or oil suspensions, and flavored emulsions with edible oils such as corn oil. , cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

• IIIh iiiilriÉiii i i iiini Má ititlá EII tiii aA x iiiitiriiiiiÉiiilÉi Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described herein. Preferably, the compositions are administered by the oral or respiratory nasal route for local or systemic effect. Preferred pharmaceutically acceptable solvent compositions can be nebulized by the use of inert gases. The nebulized solutions can be inhaled directly from the nebulizer device or the nebulizer device can be attached to a mask holder, or intermittent positive pressure breathing machine. The solution, suspension or powder compositions can be preferably administered orally or nasally, from devices that deliver the formulation in an appropriate manner. The pharmaceutical composition of the invention may be in the form of a liposome, in which the oligonucleotide, in addition to other pharmaceutically acceptable carriers, is combined with antipathetic agents such as lipids that exist in aggregate form such as micelles, insoluble monolayers, liquid crystals or Lamellar layers that are in aqueous solution. Suitable lipids for the liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. A lipid carrier llU-ÉIH. '?? HiHülí II mi ilÉ? Illl I1Í. I particularly useful is lipofectin. The preparation of liposomal formulations is within the skill of the art, for example, in International Patent No. WO97 / 21808 (28). The pharmaceutical composition may further include compounds such as cyclodextrins and the like that improve the delivery of oligonucleotides within cells or slow release polymers. Another preferred formulation that is employed in the methods of the present invention utilizes transdermal delivery devices ("patches"). Such transdermal patches can be used to provide a continuous or discontinuous infusion of the antisense oligonucleotides of the present invention in controlled amounts. The construction and use of transdermal patches for delivery of pharmaceutical agents is well known in the art. See, for example, U.S. Patent 5,023,25240, incorporated herein by reference. Such patches can be constructed for continuous, pulsatile delivery or upon request of pharmaceutical agents. Another preferred delivery method involves the "firing" delivery of the pure antisense oligonucleotides through the dermal layer. The delivery of "pure" antisense oligonucleotides is well known in the art. See, for example, Felgner et al., U.S. Patent No. 5,580,85941. It is contemplated that oligonucleotides ^^ .j ^ mu ^ ** ^^. ^^. ^ antisentldo can be packaged in a lipid vesicle before the "firing" delivery of the antisense oligonucleotide. The following formulation examples illustrate representative pharmaceutical compositions of the present invention. Formulation Example I Hard gelatin capsules were prepared containing the following ingredients: Amount Ingredient (mg / capsule) Active ingredient 30.0 Starch 305.0 Magnesium stearate 5.0 The above ingredients were mixed and filled into hard gelatin capsules in amounts of 340 mg.

Formulation Example 2 A tablet formula is prepared using the following ingredients: Amount Ingredient (mg / tablet) Active ingredient 25.0 Cellulose, microcrystalline 200.0 Colloidal silicon dioxide 10.0 Stearic acid 5.0 HÜ i ^ nm - * ^^ ** - - - The components were mixed and compressed to form tablets, each weighing 240 mg.

Formulation Example 3 A formulation for dry powder inhaler is prepared which contains the following components: Ingredient% by weight Active ingredient 5 Lactose 95 The active ingredient was mixed with the lactose and the mixture was added to a dry powder inhalation device .

Formulation Example 4 Tablets each containing 30 mg of active Ingredient were prepared as follows: Amount Ingredient (mg / tablet) Active ingredient 30.0 mg Starch 45.0 mg Microcrystalline cellulose 35.0 mg Polyvinylpyrrolidone (as a 10% solution in sterile water) 4.0 mg Starch of sodium carboxymethyl 4.5 mg Magnesium stearate 0.5 mg ItMlIl '^ llMtr * "^ .M- ^ 1 | ^^ ^ -b ^ .-- t - ^ - ^ - ^. >.-.--» - * ..., .., a ^ - * a. «_ teiA.

Talc 1.0 mg Total 120 mg The active ingredient, starch and cellulose were passed through a No. 20 mesh American screen and mixed thoroughly. The solution of polyvinylpyrrolidone was mixed with the resultant powders, which are passed through a North American mesh screen of No. 20. The granules thus produced were dried at 50 to 60 ° C and passed through a North American mesh screen. No. 16. Sodium carboxymethyl starch, magnesium stearate and talc were previously passed through a No. 30 mesh American sieve and added to the granules which, after mixing, were compressed in a tabletting machine to produce tablets each weighing 120 mg.

Formulation Example 5 The capsules, each containing 40 mg of medicament are made as follows: Quantity Ingredient (mg / capsule) Active ingredient 40.0 mg Starch 109.0 mg Magnesium stearate 1.0 mq Total 150.0 mg ^^^ M im t The active ingredient, starch and magnesium stearate are mixed, passed through a No. 20 mesh American sieve, and filled into a hard gelatin capsule in amounts of 150 mg.

Formulation Example 6 Suppositories are prepared each containing 25 mg of active ingredient as follows: Ingredient Quantity Active ingredient 25. mg Saturated fatty acid glycerides up to 2,000 mg The active ingredient is passed through a No. 60 mesh American sieve and suspended in saturated fatty acid glycerides previously melted using the minimum necessary heat. The mixture is then poured into a suppository mold of a nominal capacity of 2.0 g and allowed to cool.

Formulation Example 7 The suspensions, each containing 50 mg of drug per 5.0 mL of dose are made as follows: Ingredient Quantity Active ingredient 50.0 mg Xanthan gum 4.0 mg Sodium carboxymethyl cellulose (11%) Microcrystalline cellulose (89%) 50.0 mg . ^. ^ .. ^ > .. _ ^ ,. .._-_. , ..- ^^ ... ^^ h. ^ »^^^^ -. ^ Sucrose 1.75 g Sodium benzoate 10.0 mg Sabopzante and dye c.v. Purified water up to 5.0 mL The active ingredient, sucrose and xanthan gum are mixed, passed through a No. 10 mesh American sieve, and then mixed with a previously made solution of microcrystalline cellulose and carboxymethylcellulose sodium in water. The sodium benzoate, the flavoring and the dye are diluted with part of the water and added with agitation. Sufficient water is then added to produce the required volume.

Formulation Example 8 Amount Ingredient (mg / capsule) Active ingredient 15.0 mg Starch 407.0 mg Magnesium stearate 3.0 mq Total 425.0 mg The active ingredient, starch and magnesium stearate are mixed, passed through a North American mesh screen No. 20, and filled into hard gelatin capsules in amounts of 425.0 mg. ^ - - - - - »- > - ~, ^^ - ^? ^ á ^ lká ^^ a? ^^ ^.

Formulation Example 9 A formulation was prepared as follows: Ingredient Quantity Active ingredient 5.0 mg Corn oil 1.0 mL Formulation Example 10 A topical formulation was prepared as follows: Ingredient Amount Active ingredient 1-10 g Emulsification wax 30 g Liquid paraffin 20 g White soft paraffin to 100 g 15 The white soft paraffin is heated to melt. The liquid paraffin and the emulsification wax are incorporated and stirred until dissolved. The active ingredient is added and stirring is continued until it disperses. The mixture is then cooled until solidified. Other formulations suitable for use in the present invention can be found in Remington's Pharmaceutical Sciences 23 Antisense oligonucleotides or pharmaceutical composition comprising the antisense oligonucleotides can be packaged in convenient cases that provide the -MMH ^ MYMMIIII 11 ii ?? Mtii u finim im ^^ ~ ^^^ * ^^^ * - ^^ ~ - »--- --nmüli necessary materials packed in suitable containers. The antisense oligonucleotides of the invention in the form of a therapeutic formulation are useful in the treatment of diseases and disorders and conditions associated with anglogenesis and neovascularization including, but not limited to, retinal neovascularization and tumor growth. In such methods a therapeutic amount of an oligonucleotide effective in inhibiting the expression of neuropilin is administered to the cell. This cell can be part of a cell culture, a tissue culture, or the whole body of a mammal such as a human being. The oligonucleotides and ribosomes of the invention modulate the growth of the tumor cell. Therefore methods are provided for interfering with or inhibiting the growth of the tumor cell in mammals comprising contacting the tumor or tumor cells with an antisense oligonucleotide of the present invention. Without being limited to theory or mechanisms, it is considered that antisense oligonucleotides can inhibit tumor growth in two ways. They can inhibit growth in an autocrine way by acting directly on the tumor cells. Alternatively or optionally, antisense oligonucleotides can act by inhibiting neovascularization associated with tumor growth, thereby reducing the supply of available blood to the tumor. The term "contact" refers to the addition of an oligonucleotide, ribozyme, etc., to a cell suspension or invention can be administered in conjunction with in addition to known anticancer compounds or chemotherapeutic agents. Chemotherapeutic agents are compounds that can inhibit the growth of tumors. Such agents include, but are not limited to 5-fluorouracil, mitomycin C, methotrexate and hydroxyurea. It is contemplated that the amount of the chemotherapeutic agent administered may be any effective amount, i.e., an amount sufficient to inhibit tumor growth or a less than effective amount. The oligonucleotides of the present invention have been found to reduce the growth of tumors that are metastatic such as the breast cancer adenocarcinoma MDA-MB-231, HT-29 colon adenocarcinoma, A549 lung carcinoma, and A2058 melanoma cancer cells. In a modality of In another embodiment, a method for reducing the growth of metastatic tumors is provided in a mammal comprising administering an amount of an oligonucleotide complementary to the neuropilin mRNA, or an oligonucleotide shown in Table 1. The oligonucleotides of the present invention can reduce angiogenesis. In one embodiment of the invention, a method for the treatment of neurovascular disorders is provided. Oligonucleotides can be delivered using viral or non-viral vectors. The sequences can be incorporated in cartridges or constructs so that an oligonucleotide from The invention is expressed in a cell. Preferably, the construct contains the appropriate transcription control region to allow the ollgonucleotide to be transcribed in the cell. Therefore, the invention provides vectors that comprise a transcription control sequence operably linked to a sequence encoding an oligonucleotide of the invention. The present invention further provides host cells, selected from suitable eukaryotic and prokaryotic cells, which are transformed with those vectors. Suitable vectors are known and preferably contain all the expression elements necessary to achieve the desired transcription of the sequences. Phagemids are a specific example of such beneficial vectors because they can used either as a plasmid or as bacteriophage vectors. Examples of the vectors include viruses such as bacteriophages, vaculoviruses, retroviruses, DNA viruses, liposomes and other recombinant vectors. The vectors may also contain elements for use either in prokaryotic or eukaryotic host systems.

Someone with ordinary skill in the art will know that host systems are compatible with a particular vector. The vectors can be introduced into the cells by transient or stable transfection, lipofection, electrophoresis and infection with recombinant viral vectors. 25 Additional features can be added to the vector i É mn M t T i • - - ^ .....-_ _._.._ .. f | n¡, flg i ^ ggg ^ y ^^ to ensure its safety and / or improve its therapeutic efficacy . Such characteristics include, for example, markers that can be used to select negatively against cells infected by recombinant viruses. An example of such a negative selection marker is the TK gene that confers sensitivity to antiviral ganciclovir. Characteristics that limit expression to particular cell types can also be included. Such features include, for example, promoter and regulatory elements that are specific to the type of cell desired. Retroviral vectors are another example of vectors useful for the in vivo introduction of a desired nucleic acid since they offer advantages such as lateral infection and target specificity. Lateral infection is the process by which a single infected cell produces many progeny virions that infect neighboring cells. The result is that it quickly infects a large area. A vector for use in the methods of the invention may be selected depending on the type of cell desired to be targeted. For example, if cancer is going to be treated of the breast, then a specific vector for epithelial cells can be used. Similary, if the cells of the hematopoietic system are going to be treated, then a viral vector that is specific for blood cells is preferred. Utility 25 Antisense Oligonucleotides of the Present invention can be used for a variety of purposes. They can be used to inhibit the expression of the neuropilin gene in a mammalian cell, resulting in the inhibition of that cell's growth. They can be used to inhibit tumor cell growth and / or neovascularization. Oligonucleotides can be used as hybridization probes to detect the presence of neuropilin mRNA in mammalian cells. When used in this manner the oligonucleotides can be labeled with a suitable detectable group (such as a radioisotope, a ligand, or another member of a pair of specific bonds, for example biotin). Finally, the oligonucleotides can be used as molecular weight markers. In order to further illustrate the present invention and the advantages thereof, the following specific examples are given which do not represent a limitation of the scope of the claims in any way.

EXAMPLES In the following examples, all temperatures are in degrees Celsius (unless otherwise indicated) and all percentages are percentages by weight (also unless indicated otherwise). In the following examples, the following abbreviations have the following meanings. IF an abbreviation is not defined, it has its generally accepted meaning: AS = antisense cDNA complementary deoxyribonucleic acid ODN = oligodeoxynucleotide μM = micromolar mM = millimolar M = molar ml = milliliters μl microliters mg = milligram μg = microgram PAGE = polyacrylamide gel electrophoresis rpm = revolutions per minute? G = free energy, one measurement of the double stability of the oligonucleotide kcal = kilocalories FBS = fetal bovine serum DTT = dithiothriethol SDS = sodium dodecyl sulphate PBS = phosphate-buffered saline solution PMSF = phenylmethyl sulfonyl fluoride GAPDH = glyceraldehyde-3-phosphate dehydrogenase IgG = immunoglobulin G KDa = kilodalton PCR = polymerase chain reaction Tros-HCL = Tris (hydroxymethyl) a? Minoethane hydrochloric acid JéiiiiÉíiilli- ^ - '- * - .._.... > * .- i- > M - t-ái ^^ TRlzol = total RNA isolation reagent VEGF = vascular endothelial growth factor General Methods in Molecular Biology Standard molecular biology techniques known in the art and not specifically described were generally followed as in Sambrook et al24; Ausubel et al.25; and Perbal26.

Oligonucleotides The antisense oligonucleotides were selected from the complementary sequence for the neuropilin mRNA so that the sequence exhibited the lowest probability of showing double formation, hairpin formation, and repeats of homooligomers / sequence although they have a high potential to bind to the Neuropilin mRNA sequence. In addition, a false primer for other sequences of frequent or repetitive occurrence in humans and mice was eliminated. These properties were determined using the computer modeling program OLIGO® Primer Analysis Software, Version 5.0 International Biosciences, Inc. PIymouth MN). Based on this analysis, the phosphorothioate antisense oligonucleotides were designated and then made by methods well known in the art.

Cell Lines Seven different human cancer cell lines LA ^ -.....-- .. .. ^ ^ ^^ ^^^. S *** »,.! * ^^ including lung carcinoma (A549), melanoma (C8161), cell adenocarcinoma of the Ceno (MDA-MB-231), metastatic pancreatic adenocarcinoma (AsPC-1), colon adenocarcinoma (HT-29), human melanoma cell line A2058, human pancreatic cancer 5 PC3, were obtained from American Type Culture Collectlon ( ATCC). The cell lines were maintained in a-MEM medium (Gibco BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS).

Example 1 Inhibition of growth of cancer cell lines by antisense oligonucleotides complementary to neuropilin The ability of colony formation of cancer cell lines treated with different oligonucleotides of Antisense was estimated using a previously described method (Choy et al.18). Specifically, aliquots of a tumor cell suspension were seeded in 60 mm tissue culture dishes at a density of approximately 1X104 and incubated overnight at 37 ° C in a-MEM medium supplemented with 10% FBS. The cells were washed once in 5 ml of PBS and treated with 0.2 μM of the indicated antisense oligonucleotides in the presence of cationic lipid (lipofectin reagent, final concentration, 5 μg / ml, Gibco-BRL, Gaithersburg, MD) for 4 hours. hours. The antisense oligonucleotides were removed by washing the cells once with PBS and the cells were cultured in the growth medium (a-MEM medium supplemented with 10% FBS) for 7 to 10 days at 37 ° C. The colonies were stained with methylene blue and marked by direct counting as described (Choy et al.18 and Huang and Wright20). Percent inhibition was calculated by comparison with the number of colonies present in the growth cultures in the absence of the antisense oligonucleotides. All the experiments were executed in quadruplicate. The antisense oligonucleotides exerted inhibitory effects on the ability of colony formation of human tumor cell lines. Percent inhibition of each antisense oligonucleotide is shown in Figure 1A for the human melanoma cell line C8161; Figure 1B for the human lung cancer cell line A549; Figure 1C for the human melanoma cell line A2058; Figure 1D for human colon cancer cell line HT-29; Figure 1E for the human prostate cancer cell line PC-3; and Figure 1F for the human pancreatic cancer cell line AsPC-1.

Example 2 Reduced levels of mRNA after treatment with antisense oligonucleotides complementary to neuropilin Human melanoma cancer cells (A2058) or cancer cells from the breast (MDA-MB-231) were cultured to subconfluence (70-80%) and were treated with 0.2 μM of phosphorothioate antisense oligonucleotides complementary to neuropilin for 4 hours in the presence of cationic lipid (lipofectin reagent, final concentration, 5 μl / ml, Gibco-BRL) and Opti-MEM (Gibco-BRL). The cells were washed once with PBS and 1 ° * ^ * '• - - "" - ^ - ^ "M - ^ - Mai-i-i-iu--' incubated for 16 hours in a-MEM medium (Gibco-BRL) containing 10% FBS. Total RNA was prepared in TRIzol reagent (Glbco-BRL) and Northern staining analysis was run as described in Hurta and Wright (27) with some modifications. The stains were hybridized with fragments of 598 bp PCR labeled with 32P synthesized using the previous primer (5'-CGC TCC CGC CTG AAC TAC CC-3") [SEQ ID NO: 31], subsequent primer (5'- TCC CAC CCT GAA TGA TGA TG-3 ') [SEQ ID NO: 32] and 5'-human human colorectal adenocarcinoma plus the cDNA library (Clonetech, Palo Alto CA) as a template Neuropilin / VEGF165R Human mRNA was expressed as a nucleotide transcriptor ~ 7 kb (Soker et al.) The equal loading of RNA was demonstrated by methylene blue staining of the pre-hybridization stain, Figure 2A and 2B show the antisense oligonucleotides that reduce levels of neuropilin mRNA to at least 50% of the control cells.

Example 3 Inhibition of human tumor cell growth in mice by intravenous treatment with antisense oligonucleotides complementary to neuropilin CD-1 nude hairless mice were purchased from Charles River Laboratories (Montreal, Canada). Ht-29 human colon cancer cells (commonly 3X106 cells in 100 μl of PBS) were injected subcutaneously into the right flank of 6-7 week old nude female CD-1 nude mice. Each group experimental included 5 mice. After the tumor size reached a volume of approximately 100 mm3, typically 5 days after injection of the tumor cell, the antisense oligonucleotide was administered by infusion of the bolus into the tail vein every third day at 10 mg / kg. The control animals received only saline during the same period. The treatments lasted in a common way for 14 days. Figure 3A shows the effects of antisense oligonucleotide GTI3602 on tumor growth of HT-29 in hairless CD-1 mice. Antitumor activities were estimated by inhibiting tumor volume, which was measured with an average caliber of two-day intervals over the 14-day period. Each point in the figure represents the calculated mean tumor volume of 5 animals per experimental group. The covariance analyzes were used to compare the regression curves of the mice over time within each treatment group. The specific hypothesis of equality of inclinations or equality of intersections when the inclinations are equal are derived from the analysis. The entire analysis used the SAS (Statistical Analysis System) version 6.12. When compared to the saline control, administration of the antisense oligonucleotide inhibited tumor growth with a p-value of 0.001. At the end of the treatment (usually 24 hours after the last treatment) the animals were sacrificed and the tumor weights were measured. Figure 3B shows the average weight of the tumors. The antisense oligonucleotide showed inhibitory effects useful. A significant analysis of variance in one direction was used to compare the averages of the treatment groups. Where the overall group effect was significant, the a priori multiple comparisons using the minimum quadratic means were used to find the pairs of treatment groups that were significantly different.When the tumor weight was compared to the antisense oligonucleotide it also showed a statistically significant inhibition when compared to saline control.

Example 4 Inhibition of Experimental Metastasis by Antisense Oligonucleotides. The experimental metastasis of C8161 human melanoma cells treated with different antisense oligonucleotides was estimated as previously described (Fan et al, 199619). Aliquots of suspension were seeded in 100 mm tissue culture dishes at a density of 2 X 106 and incubated overnight at 37 ° C in a-MEM medium supplemented with 10% FBS. The cells were washed once in 10 ml of PBS and treated with 0.2 μM of oligonucleotides in the presence of cationic lipid (lipofectin reagent, final concentration, 5 μg / ml, Gibco-BRL) for 4 hours. The antisense oligonucleotides were removed by washing the cells once with PBS and the cells were trypsinized. The cells were then harvested by centrifugation and approximately 1X105 cells suspended in 0.1 ml of PBS were injected into the tail vein in atypical CD-1 female hairless mice from 6 to 8 weeks of age. Estimates of the number of lung tumors were made 5 weeks later, then the lungs of the mice were excised and stained with a solution of dye of pícpco acid (75% picric acid, 20% formaldehyde, 5% glacial acetic acid). Figure 4 shows the reduced number of lung tumors in mice without female hair after treatment of the tumor cells with the different antisense oligonucleotides.

Claims (16)

1. An antisense oligonucleotide of about 20 to about 100 nucleotides, comprising a sequence selected from the group consisting of SEQ ID NO: 1-30 as set forth in Table 1 whose oligonucleotide inhibits the expression of neuropilin.

2. The antisense oligonucleotide according to claim 1, further comprising one or more phosphorothioate internucleotide linkages.

3. The antisense oligonucleotide according to claim 1, further comprising additional nucleotides non-complementary to neuropilin mRNA.

4. A vector comprising an ollgonucleotide sequence of about 20 to 100 nucleotides comprising a sequence selected from the group consisting of SEQ. DE IDENT: NOs: 1-30 as established in Four 1 whose oligonucleotide inhibits the expression of neuropilin.

5. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and an effective amount of the antisense oligonucleotide from about 20 to 100 nucleotides comprising a sequence selected from the group consisting of SEQ. OF IDEN. NOs: 1-30 as established in Table 1 which inhibits the expression of neuropilin.

6. A method for inhibiting the growth of a mammalian tumor comprising, administering to a mammal that is What is the suspicion that the tumor has an effective amount of an antisense oligonucleotide from about 3 to about 100? nucleotides comprising a sequence complementary to a mammalian neuropilin mRNA under conditions such that tumor growth is inhibited

7. The method according to claim 6, further comprising the step of administering to the mammal a chemotherapeutic agent. The method according to claim 6, characterized in that the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NO: 1-30 as set forth in Table 1. 9. The method according to claim 1. 6, characterized in that the oligonucleotide is nuclease resistant 10. A method for inhibiting the metastasis of a mammalian tumor comprising, administering to a mammal suspected of having a tumor etastatic, an effective amount of an antisense oligonucleotide from about 3 nucleotides to about 100 nucleotides, comprising a sequence complementary to a mammalian neuropilin gene under conditions such that tumor metastasis is inhibited. The method according to claim 10, further comprising the step of administering to the mammal a chemotherapeutic agent. 12. The method according to claim 10, characterized in that the oligonucleotide is nuclease resistant. The method according to claim 10, characterized in that the oligonucleotide comprises a sequence selected from the group consisting of SEC. DE IDENT NOs: 1-30 as set forth in Table 1. 14. A method for inhibiting neovascularization comprising, administering to a mammal an effective amount of an antisense oligonucleotide from about 3 nucleotides to about 100 nucleotides comprising a sequence complementary to the mammalian neuropilin gene under conditions such that neovascularization is inhibited. 15. The method according to claim 14, characterized in that the oligonucleotide is nuclease resistant. 16. The method according to claim 14, characterized in that the oligonucleotide comprises a sequence selected from the group consisting of SEC. FROM IDENT. NOs: 1-30 as established in Table 1.