MX2008000993A - Compositions and methods to control angiogenesis with cupredoxins. - Google Patents

Abstract

The present invention relates to compositions comprising cupredoxins, and their use to inhibit angiogenesis in mammalian cells, tissues, and animals, and particularly the angiogenesis that accompanies tumor development and particularly in humans. Specifically, the present invention relates to compositions comprising the cupredoxin(s), and or peptides that are variants, derivatives or structural equivalents of cupredoxins, which retain the ability to inhibit angiogenesis in mammalian cells, tissues or animals. These compositions may be peptides or pharmaceutical compositions, among others. The compositions of the invention may be used to treat any pathological condition that has as a symptom or cause, inappropriate angiogenesis, and particularly inappropriate angiogenesis related to tumor development.

Description

COMPOSITIONS AND METHODS TO CONTROL ANGIOGENESIS WITH CUPREDOXINS RELATED APPLICATIONS This application claims the priority under the 35 U.S.C. §§ 119 and 120 for the North American Provisional Patent Application 60 / 700,297, filed on July 19, 2005, and the North American Patent Application 11 / 436,592 filed on May 19, 2006, which claims the priority for the Application for Provisional North American Patent 60 / 764,749, filed on February 3, 2006; and U.S. Patent Application No. 11 / 244,105, filed October 6, 2005, which claims priority for the US Provisional Patent Application Serial Number 60 / 616,782, filed October 7, 2004, and the Application US Provisional Patent Serial Number 60 / 680,500, filed May 13, 2005, and is a continuation in part of the US Patent Application Serial Number 10 / 720,603, filed on November 11, 2003, which claims the Priority for the US Provisional Patent Application Serial Number 60 / 414,550, filed on August 15, 2003, and which is a continuation in part of the US Patent Application Serial No. 10 / 047,710, filed on 15 January 2002, which claims priority for the US Provisional Patent Application Serial Number 60 / 269,133, filed on February 15, 2001. The full contents of these prior applications are fully incorporated herein by reference.

DECLARATION OF GOVERNMENT INTEREST The subject of this application has been supported by the research grants of the National Institute of Health (NIH), Bethesda, Maryland, USA, (Concession Numbers AI 16790-21, ES 04050-16, AI 45541, CA09432 and N01-CM97567). The government may have certain rights in this invention.

FIELD OF THE INVENTION The present invention relates to cupredoxins and variants, derivatives and structural equivalents of cupredoxins, specifically the azure of Pseudomonas aeruginosa, and their use in the inhibition of angiogenesis in mammals, to treat conditions related to inappropriate angiogenesis in mammals, and in particular in the inhibition of angiogenesis associated with tumor development. The invention also relates to pharmaceutical compositions comprising cupredoxins and variants, derivatives and structural equivalents of the cupredoxins that can be administered to a mammalian patient, and specifically can be administered to inhibit angiogenesis.

BACKGROUND OF THE INVENTION Angiogenesis is the formation of new blood vessels from the preexisting endothelial vasculature. Folkman, et al, J. Exp. Med. 133: 275-288, (1971). Most tumors require angiogenesis to sustain growth beyond a critical volume of 1-2 mm, when the supply of nutrients and metabolites becomes insufficient due to the limits of diffusion exchange. Folkman, J. Nat. Cancer Inst. 82: 4-6 (1990). Private tumors of angiogenesis remain inactive indefinitely, only to grow rapidly when a blood supply is acquired. Brem et al. , Cancer Res.36: 2807-2812 (1976). The degree of angiogenesis often increases with tumor progression. Dome et al, J. Pathol. 197: 355-362 (2002). In addition, it is also thought that the invasion and metastatic spread of tumors are events dependent on angiogenesis. Folkman, Ann Surg. 175: 409-416 (1972). The newly formed blood vessels provide a route for cancer cells to enter the circulatory system and spread to distant parts of the body. Fidler and Ellis, Cell 79: 185- 188 (1994). Because angiogenesis is an integral process in the growth and spread of tumors, it is an important approach to cancer therapy. Anti-angiogenesis therapy is effective not only for solid tumors, but also for hematopoietic tumors, leukemia and myeloma, Bellamy et al, Cancer Res. 59: 728-733 (1999); Rajkumar et al, Leukemia. 13: 469-472 (1999). It is thought that endothelial cells are the best targets for therapy than tumor cells because they have a longer generation time and more genetic stability than tumor cells. Endothelial cells are therefore less likely to "escape" from therapy by developing resistance to the drug for the therapy administered. Boehn-Vaiswanathan, Curr. Opin. Oncol. 12: 89-94 (2000). Other conditions suffered by mammals are also related to inappropriate angiogenesis. Wet macular degeneration occurs when blood capillaries grow inappropriately in the retina. Inappropriate angiogenesis has also been implicated as a fundamental characteristic of diabetic retinopathy, psoriasis and rheumatoid arthritis, among other conditions. Bussolino et al, Trends Biochem. Sci. 22: 251-256 (1997); Folkman, Nat. Med. 1: 27-31 (1995).

What is needed are additional therapies for inappropriate angiogenesis, particularly that which occurs during tumor formation. Such therapies can be useful in many conditions that exhibit inappropriate or unwanted formation of new blood vessels.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to compositions comprising cupredoxins, and their use to inhibit angiogenesis in cells, tissues, and mammalian animals, and particularly the angiogenesis that accompanies tumor development and particularly in humans. Specifically, the present invention relates to compositions comprising cupredoxin (s), and / or peptides that are variants, derivatives or structural equivalents of cupredoxins that retain the ability to inhibit angiogenesis in cells, tissues, or animals. mammals These compositions can be peptides or pharmaceutical compositions, among others. The compositions of the invention can be used to treat any pathological condition that has as a symptom or cause, inappropriate angiogenesis, and particularly inappropriate angiogenesis related to tumor development. One aspect of the invention is an isolated peptide which is a variant, derivative or structural equivalent of a cupredoxin and that can inhibit angiogenesis in mammalian cells. Cupredoxin can be azurine, pseudoazurin, plastocyanin, rusticianin, Laz and auracyanin, and specifically azurine. Cupredoxin can be from Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidan, Bordetella bronchiseptica, Methylomonas sp. , Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylella fastidiosa and Vibrio parahaemolyticus, and specifically Pseudomonas aeruginosa. The isolated peptide can be part of a SEQ ID NOS: 1, 3-19, or it can be a sequence for which SEQ ID NOS: 1, 3-19 have at least 80% amino acid sequence identity. The isolated peptide may be a truncation of cupredoxin. In these cases, the isolated peptide can be more than about 10 residues and no more than about 100 residues. The isolated peptide comprises residues 50-77 of the azurine of Pseudomonas aeruginosa, residues 50-67 or residues 36-88 or SEQ ID NOS: 20-24. In addition, the isolated peptide may consist of residues 50-77 of Pseudomonas aeruginosa, residues 50-67 or residues 36-88 or SEQ ID NOS: 20-24. Finally, the isolated peptide may comprise the equivalent residues; 50-77 residues of the azurine of Pseudomonas aeruginosa, residues 50-67 or residues 36-88. Another aspect of the invention is a composition Pharmaceutical comprising at least one cupredoxin or isolated peptide in a pharmaceutically acceptable carrier. This pharmaceutical composition may comprise at least two of the isolated cupredoxins or peptides. In addition, the pharmaceutical composition can be formulated for intravenous administration. In some embodiments, cupredoxin is from Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidan, Bordetella bronchiseptica, Methylomonas sp. , Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas fluorescein, Pseudomonas chlororaphis, Xylella fastidiosa or Vibrio parahaemolyticus, and specifically from Pseudomonas aeruginosa. Cupredoxin can be SEQ ID NOS: 1, 3-19. Another aspect of the invention are methods for treating a mammalian patient suffering from a condition related to inappropriate angiogenesis which comprises administering to the patient a therapeutically effective amount of the pharmaceutical composition. In some modality the patient is human. The patient may be suffering from cancer, and in particular cancer of melanoma, breast, pancreas, glioblastoma, astrocytoma, or lung cancer. In other embodiments, the patient may be suffering from a condition selected from the group consisting of macular degeneration, diabetic retinopathy, psoriasis or rheumatoid arthritis. In these methods, the pharmaceutical composition is administered by intravenous injection, intramuscular injection, subcutaneous injection, inhalation, topical administration, transdermal patch, suppository, vitreous or oral injection, and specifically by intravenous injection. In some embodiments of the methods, the pharmaceutical composition is co-administered with at least one other anti-cancer drug, and specifically at about the same time as the other anti-cancer drug. In other embodiments of the method, the pharmaceutical composition is coadministered with an anti-macular degeneration drug, a diabetic anti-retinopathy drug, an anti-psoriasis drug or an anti-rheumatoid arthritis drug. Another aspect of the invention is a kit comprising the pharmaceutical composition in a vial. This kit can be designed for intravenous administration. Another aspect of the invention is a method for studying angiogenesis or a condition related to inappropriate angiogenesis, comprising contacting the mammalian cells capable of developing angiogenesis with a cupredoxin or variant, derivative or structural equivalent of a cupredoxin and measuring the degree of angiogenesis. In some embodiments, the cells are human cells. In other embodiments, the mammalian cells are Human Umbilical Vascular Endothelial Cells (HUVECs). Another aspect of the invention is a vector of expression that codes for a variant, derivative or structural equivalent of a cupredoxin. These and other aspects, advantages, and features of the invention will be apparent from the following figures and detailed description of the specific embodiments.

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1. Amino acid sequence of azurine from Pseudomonas aeruginosa. SEQ ID NO: 2. Sequence of amino acids of P28, residues 50-77 of the azurine of Pseudomonas aeruginosa. SEQ ID NO: 3. Plastocyanin amino acid sequence from Phormidium laminosum. SEQ ID NO: 4. Amino acid sequence of rusticianin from Thiobacillus ferrooxidans. SEQ ID NO: 5. Amino acid sequence of pseudoazurin from Achromobacter cycloclastes. SEQ ID NO: 6. Amino acid sequence from Alcaligenes faecalis. SEQ ID NO: 7. Amino acid sequence of azurine from Achromobacter xylosoxidans ssp. denitrificans I. SEQ ID NO: 8. Amino acid sequence of azurine from Bordetella bronchiseptica. SEQ ID NO: 9. Amino acid sequence of azurin a from Methylomonas sp. J. SEQ ID NO: 10. Amino acid sequence of azurine from Neisseria meningi tidis Z2491. SEQ ID NO: 11. Amino acid sequence of azurine from Pseudomonas fluoresce. SEQ ID NO: 12. Amino acid sequence of azurine from Pseudomonas chlororaphis. SEQ ID NO: 13. Azurine amino acid sequence from Xylella fastidiosa 9a5c. SEQ ID NO: 14. Stelacianine amino acid sequence from Cucumis sa tivus. SEQ ID NO: 15. Auracyanin A amino acid sequence from Chloroflexus aurantiacus. SEQ ID NO: 16. Auracyanin B amino acid sequence from Chloroflexus aurantiacus. SEQ ID NO: 17. Amino acid sequence of cucumber basic protein from Cucumis sa tivus. SEQ ID NO: 18. Laz amino acid sequence from Neisseria gonorrhoeae F62. SEQ ID NO: 19. Amino acid sequence of azurine from Vibrio parahaemolyticus. SEQ ID NO: 20. Aminocyanin B amino acid sequence from 57 to 89 amino acids of Chloroflexus aurantiacus. SEQ ID NO: 21. Amino acid sequence of 51-77 amino acids of azurine from Pseudomonas syringae.

SEQ ID NO: 22. Amino acid sequence of 89-115 amino acids from Laz Neisseria meningi tidis. SEQ ID NO: 23. Amino acid sequence of 52-78 amino acids of azijin from Vijbrio parahaemolyticus. SEQ ID NO: 24. Amino acid sequence of 51-77 amino acids of Bordetella bronchiseptica azurin.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Figure 1 depicts confocal microscopy images of malignant and normal cells incubated with P28 labeled with Alexafluor® 568 and subsequently stained or stained with DAPI. The indicated cell lines were incubated in the absence (negative control) or presence (P28) of 20μM of P28 labeled with Alexafluor® 568 for 2h at 37 ° C. The images are indicative of the amount of cellular input observed. Figure IA represents the fluorescence of Alexafluor® 568 and the control fluorescence of melanoma, pancreatic, breast (BCA-I), breast (MCF-7), glioblastoma, astrocytoma, human lung and prostate cancer cells. Figure IB represents the fluorescence of Alexafluor® 568 and the fluorescence control of normal human fibroblast, pancreas and breast cells. Figure 1C depicts the fluorescence of Alexafluor® 568 and the fluorescence control of human umbilical vein endothelial cells (HUVEC).

Figure 2. Figure 2 depicts capillary tube formation by HUVEC cells plated on Matrigel® in the presence or absence of P28. The culture medium contained 20ng / ml of VEGF. Figure 2A shows the images of the HUVEC cells incubated for 4h at 37 ° C with O.lOμM, 0.30μM, 0.92μM, 2.77μM, 8.33μM, 25μM and 75μM of P28, and then stained with AM calcein and visualized using the fluorescence microscopy. In Figure 2B, the graph shows the average number of tubes formed in the treated peptide and in the control cells (untreated). Figure 3. Figure 3 represents the results of the HUVEC migration test of scraping injury. In Figures 3A-C the fixed cells stained for F-actin and nuclei are shown. In Figure 3A, HUVEC cells were scraped to 90% confluence using a 1 ml plastic pipet tip. In Figure 3B, the HUVEC cells were scraped and subsequently incubated in the culture medium containing 20ng / ml VEGF for 24h at 37 ° C in the absence of P28. In Figure 3C, the HUVEC cells were scraped and subsequently incubated for 24 h at 37 ° C in the presence of 25 μM of P28. The inserts of Figures 3A-C show the cell density in the area away from the recorded area. In Figure 3D, a bar graph indicates the average # of cells in 20 different fields (20X) of the scraped area in the treated P28 and control wells (Figure 3B and C).

The data represent the mean ± SEM. * indicates that the differences are statistically significant. Figure 4. Figure 4 represents the images of the location of the structural proteins of the cell with and without the P28 treatment. HUVEC cells were plated onto coated slides coated with Matrigel®, incubated in the culture medium containing 20ng / ml VEGF in the presence or absence of P28 peptide (25μM) for 4 and 24h, fixed, and processed by staining of CD31 / PECAM-1, paxilina, Fak (focal adhesion kinase), vinculin, WASP (Wiskott Aldrich Syndrome protein) and β-catenin. Each figure pertains to the detection of the particular structural protein: Figure 4A is CD31 / PECAM-1; Figure 4B is paxilin; Figure 4C is Fak; Figure 4D is WASP; Figure 4E is vinculin; and Figure 4F is β-catenin. Each figure is divided into four sections that show the image of the location of the fluorescent markers used. Each section is numbered to indicate the detected fluorescent marker: 1 = F-actin; 2 = DAPI; 3 = FITC - Protein of interest; 4 = merged image. The arrows indicate the location of the protein of interest. Figure 5. Figure 5 depicts Mel-2 cells that were treated with increasing concentrations of P28 for 24, 48, and 72 hours. The number of cells in the control and treated wells was accounted for using a counter Coulter The data represent the percentage of inhibition of cell growth when compared to control the crops at the time point. Figure 6. Represents the results when Mel-2 cells were injected subcutaneously into the left flank (approximately 1 million cells / animal). The animals received P28 at the dose indicated at the time of injection. Figure 6A shows the incidence of tumor occurrence after initiation of treatment with a graph indicating% of tumor-free animals in days after treatment with Mel-2 cells. Figure 6B shows the size of the tumor after the initiation of treatment with a graph indicating the average volume of tumors (cm3) in days after treatment with Mel-2 cells.

DETAILED DESCRIPTION OF THE INVENTION Definitions As used herein, the term "cell" includes either the singular or the plural of the term, unless specifically described as a "single cell". As used herein, the terms "polypeptide", "Peptide", and "protein" are used interchangeably to refer to a polymer of the amino acid residues. The terms apply for the amino acid polymers in the which one or more residues of the amino acid is an artificial chemical analogue of a naturally occurring corresponding amino acid. The terms also apply to naturally occurring amino acid polymers. The terms "polypeptide", "peptide", and "protein" are also inclusive of modifications including, but not limited to, glycosylation, lipid binding, sulfation, carboxylation of glutamic acid residue, hydroxylation and ADP ribosylation. It will be appreciated that the polypeptides are not always completely linear. For example, polypeptides can be branched as a result of ubiquitination and can be circular (with or without branches), generally as a result of post-translation events, including the event of natural processing and the events caused by human manipulation. It does not happen naturally. The circular, branched, and branched circular polypeptides can be synthesized by the natural non-translation process and also by completely syntc methods. As used herein, the term "pathological condition" includes anatomical and physiological deviations from normal that constitute a deterioration of the normal state of the living animal or of one of its parts, which interrupts or modifies the performance of bodily functions, and is a response to various factors (such as malnutrition, industrial risks, or weather), to specific infectious agents (such as worms, parasitic protozoa, bacteria, or viruses), to inherent defects of the organism (such as genetic abnormalities), or to combinations of these factors. As used herein, the term "condition" includes anatomical and physiological deviations from normal that constitute a deterioration of the normal state of the living animal or of one of its parts, which interrupts or modifies the performance of bodily functions. As used herein, the term "sufferer" includes currently exhibiting the symptoms of a pathological condition, having a pathological condition even without noticeable symptoms, in recovering from a pathological condition, or recovering from a pathological condition. As used herein, the term "treatment" includes preventing, ameliorating, halting, or reversing the progression or severity of the condition or symptoms associated with a condition being treated. As such, the term "treatment" includes medical, therapeutic administration, and / or prophylactic administration, as appropriate. As used herein, the term "inhibits cell growth" refers to the delay or cessation of cell division and / or cell expansion. This term also includes the inhibition of cellular development or Increases in cell death. As used herein, the term "inhibits angiogenesis" refers to the delay, cessation or reversal of the formation of blood vessels in a particular cell, tissue, or location of the body. Inhibition of angiogenesis may be due to direct or indirect effects on endothelial cells. Inhibition can also be at any stage of the angiogenesis process. For example, inhibition may be due to prevent a tumor from producing Vascular Endothelial Growth Factor (VEGF), • direct inhibition of endothelial cell proliferation and / or migration, acting as an antagonist of angiogenesis growth factors. , inhibition of endothelial specific integrin signaling / survival, or copper chelation. The inhibition of angiogenesis can be by any means whereby the formation of the blood vessels is delayed, stopped or reversed, including any means currently used by any anti-angiogenesis drug under development or on the market. As used herein, the term "inappropriate angiogenesis" refers to any occurrence or occurrence of angiogenesis that is undesirable. Inappropriate angiogenesis may be angiogenesis that is associated with a condition in a mammal. Inappropriate angiogenesis can be either the cause or the symptom of such a condition. Inappropriate angiogenesis in a broader sense can be any angiogenesis that is undesirable, although it may be within the realm of normal mammalian physiology. A "therapeutically effective amount" is an amount effective to prevent, ameliorate, arrest or reverse the development of, or to partially or fully alleviate, the existing symptoms of a particular condition for which the subject is being treated. The determination of a therapeutically effective amount is well within the ability of those skilled in the art. The term "substantially pure", as used herein, when used to modify a protein or other cellular product of the invention, refers to, for example, a protein isolated from the growth medium or cellular contents, in a form substantially free of, or not adulterated by, other proteins and / or active inhibitory compounds. The term "substantially pure" refers to a factor in an amount of at least about 75%, by dry weight, of the isolated fraction, or at least "75% substantially pure." More specifically, the term "substantially pure" refers to a compound of at least about 85%, by dry weight, of the active compound, or at least "85% substantially pure." More specifically, the term "substantially pure" is refers to a compound of at least about 95%, by dry weight, of the active compound, or at least "95% substantially pure". The term "substantially pure" can also be used to modify a synthetically produced protein or a compound of the invention, where, for example, the synthetic protein is isolated from the reagents and by-products of the synthesis reaction (s). The term "pharmaceutical grade", as used herein, when referring to a peptide or compound of the invention, is a peptide or compound that is substantially or essentially isolated from the components that normally accompany the material as it is in its natural state, including the synthesis reagents and by-products, and which is substantially or essentially isolated from the components that would impair its use as a pharmaceutical. For example, a "pharmaceutical grade" peptide can be isolated from any carcinogen. In some cases, the "pharmaceutical grade" can be modified by the proposed method of administration, such as "intravenous pharmaceutical grade", to specify a peptide or compound that is substantially or essentially isolated from any substance that would render the composition unsuitable for administration intravenously to a patient. For example, an "intravenous pharmaceutical grade" peptide can be isolated from detergents, such as SDS, and from antibacterial agents, such as azide.

The terms "isolated", "purified" or "biologically pure" refer to material that is substantially or essentially free of components that normally accompany the material as it is in its native state. Thus, the peptides isolated according to the invention preferably do not contain materials normally associated with the peptides in their in situ environment. An "isolated" region refers to a region that does not include the complete sequence of the polypeptide from which the region is derived. A nucleic acid, protein, or respective fragment thereof "isolated" has been substantially removed from its environment in vivo so that it can be manipulated by the skilled artisan, such as but not limited to nucleotide sequencing, restriction digestion, site-directed mutagenesis , and subcloning in the expression vectors for a nucleic acid fragment as well as for obtaining the protein or protein fragment in substantially pure amounts. The term "variant" as used herein with respect to a peptide, refers to variants of the amino acid sequence that may have replaced, deleted, or inserted amino acids compared to the wild-type polypeptide. The variants may be truncations of the wild-type peptide. Thus, a variant peptide can be produced by manipulating the genes that encode the polypeptide. A variant can be produced by altering the basic composition or the characteristics of the polypeptide, but not at least some of its fundamental activities. For example, a "variant" of azurine can be a mutated azurine that retains its ability to inhibit the growth of mammalian cancer cells. In some cases, a variant peptide is synthesized with non-natural amino acids, such as residues e- (3,5-dinitrobenzoyl) -Lys. Ghadiri & Fernholz, J. Am. Chem. Soc, 112: 9633-9635 (1990). In some embodiments, the variant has no more than 20 amino acids replaced, deleted or inserted compared to the wild-type peptide. In some embodiments, the variant has no more than 15 amino acids replaced, deleted or inserted compared to the wild-type peptide. In some embodiments, the variant has no more than 10 amino acids replaced, deleted or inserted compared to the wild-type peptide. In some embodiments, the variant has no more than 6 amino acids replaced, deleted or inserted compared to the wild-type peptide. In some embodiments, the variant has no more than 5 amino acids replaced, deleted or inserted compared to the wild-type peptide. In some embodiments, the variant has no more than 3 amino acids replaced, deleted or inserted compared to the wild-type peptide. The term "amino acid", as used herein, is refers to an amino acid radical comprising any naturally occurring or non-naturally occurring or synthetic amino acid residue, that is, any radical comprising at least one carboxyl and at least one amino residue directly attached by one, two, three or more carbon atoms, typically a carbon atom (). The term "derivative" as used herein with respect to a peptide refers to a peptide that is derived from the subject peptide. A derivation includes chemical modifications of the peptide such that the peptide still retains some of its fundamental activities. For example, a "derivative" of azurine may, for example, be a chemically modified azurine that retains its ability to inhibit angiogenesis in mammalian cells. Chemical modifications of interest include, but are not limited to, amidation, acetylation, sulfation, modification of polyethylene glycol (PEG), phosphorylation or glycosylation of the peptide. In addition, a derivatized peptide may be a fusion of a polypeptide or fragment thereof to a chemical compound, such as but not limited to, another peptide, drug molecule or other therapeutic or pharmaceutical agents or a detectable probe. The term "percent (%) amino acid sequence identity" is defined as the percentage of amino acid residues in a polypeptide that is identical to the amino acid residues in a candidate sequence when the two sequences are aligned. To determine the amino acid identity%, the sequences are aligned and, if necessary, spaces are introduced to achieve the maximum% sequence identity; Conservative substitutions are not considered as part of the sequence identity. Alignment procedures of the amino acid sequence to determine percent identity are well known to those skilled in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align the peptide sequences. In a specific modality, Blastp (available from the National Center for Biotechnology Information, Bethesda MD) is used using the filter default parameters of long complexity, expected 10, word size 3, existence 11 and extension 1 When the amino acid sequences are aligned, the% amino acid sequence identity of an A sequence of amino acids given to, with, or against a given B sequence of amino acids (which may alternatively be expressed as a given A sequence of amino acids having or comprises a certain% amino acid sequence identity a, with, or against a given B amino acid sequence) can be calculated as: % amino acid sequence identity = X / Y * 100 where X is the number of amino acid residues annotated as identical pairs by the alignment of the program or sequence alignment algorithm of A and B and Y is the total number of residues of amino acids in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the% amino acid sequence identity of B to A. When comparing the longest sequences to the shortest sequences, the shortest sequence will be the "B" sequence. For example, when comparing the truncated peptides to the corresponding wild-type polypeptide, the truncated peptide will be the "B" sequence.

General The present invention provides compositions comprising cupredoxin, and variants, derivatives and structural equivalents of cupredoxins, and methods for inhibiting angiogenesis and / or for inhibiting the growth of cancer cells in mammals. The present invention relates specifically to the compositions comprising cupredoxin, and its use in the inhibition of inappropriate angiogenesis that is associated with cancer and other conditions. The invention also relates to variants, derivatives and structural equivalents of cupredoxin that retain the ability to inhibit angiogenesis in mammals, and in particular angiogenesis associated with tumor development, and to compositions comprising the same. More particularly, the invention provides compositions comprising the azure of Pseudomonas aeruginosa, variants, derivatives and structural equivalents of azurine, and their use to treat patients with conditions related to inappropriate angiogenesis, and to angiogenesis related to tumor development, or to prevent infection in those at risk of them. Finally, the invention provides methods for studying the angiogenesis of mammalian cells, tissues and animals by contacting the cells with cupredoxin, or with a variant, derivative or structural equivalent thereof, before or after inducing angiogenesis and determining the variations in the development of blood vessels. Previously, it is known that a redox protein made by Pseudomonas aerugisnosa, the azurine cupredoxin, selectively enters J774 cells but not normal cells. Zaborina et al, Microbiology 146: 2521-2530 (2000). Azurine can also enter selectively in UISO-Mel-2 human melanoma or in human breast cancer cells MCF-7. Yamada et al. , PNAS 99: 14098-14103 (2002); Punj et al, Oncogene 23: 2367-2378 (2004). P. aeruginosa azurin preferentially enters the sarcoma cells of murine reticulum J774 cells, forms a complex and stabilizes tumor suppressor protein p53, improves the intracellular concentration of p53, and induces apoptosis. Yamada et al, Infection and Immunity 70: 7054-7062 (2002). Detailed studies of several domains of the azurine molecule showed that amino acids 50-77 (P28) (SEQ ID NO: 2) represented a domain (PTD) of protein transduction critical for internalization and subsequent apoptotic activity. Yamada et al, Cell. Microbial 7: 1418-31, (2005). Surprisingly, it is now known that sintered P28 not only enters a variety of malignant cell lines (melanoma (Mel-2), MCF-7, pancreatic, astrocytoma, glioblastoma, among others), but also in human endothelial cells of the non-cancerous umbilical vein (HUVEC). See Example 1. P28 enters these cells in a temperature-dependent manner, but does not enter normal cells (fibroblast, normal breast epithelium). As it is known that HUVEC cells instigate angiogenesis in human embryos, the entry of P28 into HUVEC cells prompted an examination of the effect of P28 on angiogenesis. HUVEC cells (20, 000 cells) were plated on wells coated with Matrigel® and incubated in medium containing 0-75μM of P28. The cultures were examined under light microscopy at 4h and 24h after treatment. Peptide P28 inhibited capillary tube formation of HUVEC in a dose-dependent manner, suggesting that P28 inhibits the capillary tube formation step of angiogenesis. See Example 2. In addition, P28 inhibited the migration of HUVEC cells onto Matrigel® in a scratch lesion migration assay, indicating that P28 also inhibits the migration stage of angiogenesis. See Example 3. Thus, in studies in vi tro with a model system of established angiogenesis, HUVEC cells on Matrigel®, P28 inhibits two critical stages in angiogenesis, capillary tube formation and cell migration. In addition, the cellular morphology of the HUVECs was also changed surprisingly by the addition of P28 to the growth medium. The addition of P28 to the HUVECs growing in Matrigel® prevented the normal changes related to angiogenesis in the cytoskeleton and other proteins that are associated with cell migration. See Example 4. In the cell with paxilina detected, paxilina was located mainly in the cell surface of the control cells, nevertheless it was found more often in the fibers F-actin in cells treated with P28 (Fig. 4B). In the cells with detected Fak, the Fak was located mainly on the cell surface of the control cells, whereas it was found more often on the F-actin fibers of the cells treated with P28, thus creating a less flexible cell and less mobile. The cell-cell binding CD-31 / PECAM-1 proteins were over-expressed and distinctly localized to the cell-cell junctions when the HUVECs were treated with P28, thus encouraging cell-to-cell contact. The WASP (Wiskott Aldrich Syndrome) actin branching and nucleation promotion factor, while normally found on the cell surface in HUVECs suffering from angiogenesis, was located in the nucleus of cells treated with P28, thus altering the branching and actin nucleation. Finally, the location of the ß-catenin inhibited by P28 to the nucleus, consequently, further inhibits the proliferation and cell migration in the HIVCs. Accordingly, several of the morphological marks of angiogenesis in HUVECs are reduced or eliminated by the presence of P28, further indicating that P28 has a direct effect on cells suffering from angiogenesis. P28 can specifically inhibit the growth of Mel-2 melanoma cells in vi tro in a concentration-dependent manner. See Figure 5. By Consequently, P28 is not only able to enter cancer cells in a specific way; It is also capable of directly inhibiting its growth. Tumor development proceeds in association with angiogenesis. P28 inhibited the growth of Mel-2 cells transplanted subcutaneously in athymic mice in a dose-dependent manner. See Example 6. The incidence of measurable tumors (diameter> 2mm) 30 days after treatment of 8 and 16 mg / kg by weight i.p. it was significantly lower in the treatment groups, compared to controls. In addition, the volume of the tumor was also significantly lower in the animals that received 16mg / kg of P28 compared to the control. Taken together, P28 has significant anti-tumor effects due to its selective entry into tumor cells and inhibition of its proliferation, and to suppresses angiogenesis related to tumor development. Due to the high degree of structural similarity between cupredoxins, it is likely that other cupredoxins also inhibit angiogenesis in mammals. Such cupredoxins can be found in, for example, bacteria or plants. It is contemplated that these other cupredoxins may be used in the compositions and methods of the invention. In addition, the variants, derivatives, and structural equivalents of the cupredoxins that retains the ability to inhibit angiogenesis in mammalian cells can also be used in the compositions and methods of the invention. These variants and derivatives may include, but are not limited to, truncations of a cupredoxin, conservative amino acid substitutions and protein modifications such as PEGylation and stabilization of all hydrocarbons in the α-helices. It also follows that other conditions related to inappropriate angiogenesis can be treated with cupredoxins, and in particular with azurine. For example, Avastin® (bevacizumab, Genentech, South San Francisco, CA), a recombinant humanized monoclonal IgGl antibody that binds to and inhibits the biological activity of human vascular endothelial growth factor (VEGF), is not only effective reducing angiogenesis associated with metastatic colorectal cancer, and is also highly effective in the treatment of inappropriate angiogenesis associated with neovascular age-related macular degeneration. Bashshur et al, Am J Ophthalmol. 142: 1, -9 (2006). Therefore, it is likely that P28, and other cupredoxins, and variants, derivatives and structural equivalents of cupredoxins, also inhibit inappropriate angiogenesis in conditions other than cancer, such as those associated with neovascular age-related macular degeneration. In addition, it is It is probable that cupredoxins, and variants, derivatives and structural equivalents of cupredoxins will be effective in the treatment of other conditions related to inappropriate angiogenesis, such as, but not limited to, diabetic retinopathy, psoriasis and rheumatoid arthritis.

Compositions of the Invention The invention is provided for peptides that are variants, derivatives or structural equivalents of cupredoxin that inhibit angiogenesis in mammalian cells, tissues and animals. The invention is also provided for peptides that are variants, derivatives or structural equivalents of cupredoxin that inhibit the growth of mammalian cancer cells. The invention is further provided for peptides that are variants, derivatives or structural equivalents of cupredoxin that specifically enter mammalian cancer cells. In some embodiments, the peptide is isolated. In some embodiments, the peptide is substantially pure or of pharmaceutical grade. In other embodiments, the peptide is in a composition comprising, or consisting essentially of, the peptide. In another specific embodiment, the peptide does not raise an immune response in a mammal, and more specifically a human. In some embodiments, the peptide is less than a full-length cupredoxin, and retains some of the functional characteristics of cupredoxins. Specifically, in some embodiments, the peptide retains the ability to inhibit angiogenesis in the HUVECs in Matrigel®. The invention also provides compositions comprising at least one peptide that is a cupredoxin, or variant, derivative or structural equivalent of a cupredoxin, specifically in a pharmaceutical composition. In specific embodiments, the pharmaceutical composition is designed in a particular mode of administration, for example, but not limited to, oral, intraperitoneal, intravenous, or intraocular. Such compositions may be hydrated in water, or may be dried (such as by lyophilization) for subsequent hydration. Such compositions may be in solvents other than water, such as but not limited to, alcohol. Due to the high structural homology between cupredoxins, it is contemplated that cupredoxins will have the same anti-angiogenesis activity as P28. In some embodiments, cupredoxin is, but is not limited to, azurine, pseudoazurin, plastocyanin, rusticianin, auracyanin or Laz. In particularly specific embodiments, azurine is derived from Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidans ssp. deni trificans I, Bordetella bronchiseptica, Methylomonas sp. , Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylella fastidiosa or Vibrio parahaemolyticus. In a very specific mode, azurine is from Pseudomonas aeruginosa. In other specific embodiments, cupredoxin comprises an amino acid sequence that is SEQ ID NO: 1, 3-19. The invention provides peptides that are variants of the amino acid sequence that have replaced, deleted, or inserted amino acids compared to wild-type cupredoxin. The variants of the invention may be truncations of the wild-type cupredoxin. In some embodiments, the peptide of the invention comprises a region of a cupredoxin that is less than the full length wild type polypeptide. In some embodiments, the peptide of the invention comprises more than about 10 residues, more than about 15 residues or more than about 20 residues of a truncated cupredoxin. In some embodiments, the peptide comprises no more than about 100 residues, no more than about 50 residues, no more than about 40 residues, no more than about 30 residues, or no more than about 20 residues of a truncated cupredoxin. In some embodiments, a cupredoxin has the peptide, and more specifically SEQ ID NOS: 1, 3-19, at least about 70% amino acid sequence identity, at least about 80% amino acid sequence identity, at least about 90% amino acid sequence identity, at least about 95% amino acid sequence identity or at least about 99% amino acid sequence identity. In specific embodiments, the cupredoxin variant comprises residues 50-77 of the azurine of P. aeruginosa, residues 50-67 of azurine, or residues 36-88 of azurine. In other embodiments, the cupredoxin variant consists of residues 50-77 of the azurine of P. aeruginosa, residues 50-67 of azurine, or residues 36-88 of azurine. In other specific embodiments, the variant consists of the equivalent residues of a cupredoxin deferent to azurine. It is also contemplated that other variants of cupredoxin having an activity similar to 50-77 azurine residues, 50-67 azurine residues, or azurine residues 36-88 can be designed. To do this, the amino acid sequence of cupredoxin subjected will be aligned to the azurine sequence of Pseudomonas aeruginosa using BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR), the relevant residues located in the azurine amino acid sequence of P. aeruginosa, and the equivalent residues found in the cupredoxin sequence subjected, and the equivalent peptide designed in this manner.

In one embodiment of the invention, the cupredoxin variant contains at least 57 to 89 amino acids of auracyanin B from Chloroflexus aurantiacus (SEQ ID NO: 20). In another embodiment of the invention, the cupredoxin variant contains at least 51-77 amino acids of azurine from Pseudomonas syringae (SEQ ID NO: 21). In another embodiment of the invention, the cupredoxin variant contains at least 89-115 amino acids from Laz Neisseria meningi tidis (SEQ ID NO: 22). In another embodiment of the invention, the cupredoxin variant contains at least 52-78 amino acids of Vibrio parahaemolyticus azurine (SEQ ID NO: 23). In another embodiment of the invention, the cupredoxin variant contains at least 51-77 amino acids of Bordetella bronchiseptica azurin (SEQ ID NO: 24). • The variants also include peptides produced with synthetic amino acids that do not naturally exist. For example, naturally-occurring amino acids can be integrated into the variant peptide to extend or optimize the half-life of the composition in the bloodstream. Such variants include, but are not limited to, D, L-peptides (diastereomer), (for example Futaki et al, J. Biol. Chem. 276 (8): 5836-40 (2001); Papo et al, Cancer Res. 64 (16) -5779-86 (2004), Miller et al, Biochem Pharmacol 36 (1): 169-76, (1987), peptides containing rare amino acids (for example Lee et al, J. Pept. Res. 63 (2): 69-84 (2004)), non-natural amino acids containing olefins followed by fixation by staples of hydrocarbons (eg Schafmeister et al, J. Am. Chem. Soc. 122: 5891-5892 (2000); Walenski et al, Science 305: 1466-1470 (2004)), and peptides comprising the residues e- (3,5-dinitrobenzoyl) -Lys. In other embodiments, the peptide of the invention is a derivative of a cupredoxin. The cupredoxin derivatives are chemical modifications of the peptide such that the peptide still retains some of its fundamental activities. For example, a "derivative" of azurine can be a chemically modified azurine that retains its ability to inhibit angiogenesis in cells, tissues, or mammalian animals. Chemical modifications of interest include, but are not limited to, stabilization of hydrocarbons, amidation, acetylation, sulfation, modification of polyethylene glycol (PEG), phosphorylation and glycosylation of the peptide. In addition, a derivatized peptide can be a fusion of a cupredoxin, or variant, derivative or structural equivalent thereof to a chemical compound, such as but not limited to, another peptide, drug molecule or other pharmaceutical or therapeutic agent or a probe perceptible. Derivatives of interest include chemical modifications by which the half-life in the bloodstream of the peptides can be extended or optimized and compositions of the invention, such as by various methods well known to those in the art, including but not limited to, peptides formed in a circle (eg Monk et al, BioDrugs 19 (4): 261-78, (2005); DeFreest; et al., J. Pept. Res. 63 (5): 409-19 (2004)), N- and C- terminal modifications (e.g. Labrie et al., Clin. Invest. Med. 13 (5): 275-8, (1990)), and non-natural amino acids containing olefins followed by staple fixation of hydrocarbons (eg Schafmeister et al, J. Am. Chem. Soc. 122: 5891-5892 (2000); Walenski et al. , Science 305: 1466-1470 (2004)). In another embodiment, the peptide is a structural equivalent of a cupredoxin. Examples of studies that determine significant structural homology between cupredoxins and other proteins include Toth et al. (Developmental Cell 1: 82-92 (2001)). Specifically, the significant structural homology between a cupredoxin and the structural equivalent is determined using the VAST algorithm. Gibrat et al, Curr Opin Struct Biol 6: 377-385 (1996); Madej et al, Proteins 23: 356-3690 (1995). In specific embodiments, the VAST p-value of a structural comparison of a cupredoxin to the structural equivalent is less than about 10-3, less than about 10 ~ 5, or less than about 10"7. In other embodiments, structural homology significant between a cupredoxin and the structural equivalent is determined using the DALI algorithm. Hol & Sander, J. Mol. Biol. 233: 123-138 (1993). In the specific modalities, the DALÍ Z count for a structural comparison in pairs is at least approximately 3.5, at least approximately 7.0, or at least approximately 10.0. It is contemplated that the peptides of the composition of the invention may be more than one of a variant, derivative and / or structural equivalent of a cupredoxin. For example, the peptides may be a truncation of the azurine that has been PEGylated, thus making them a variant and a derivative. In one embodiment, the peptides of the invention are synthesized with non-natural a, -disubstituted amino acids containing olefin-carrying bonds, followed by a "staple" of all hydrocarbons by the ruthenium-catalyzed olefin metathesis. Scharmeister et al, J. Am. Chem. Soc. 122: 5891-5892 (2000); Walensky et al, Science 305: 1466-1470 (2004). Additionally, peptides that are structural equivalents of azurine can be fused to other peptides, thereby making a peptide that is a structural equivalent and a derivative. These examples are merely to illustrate and not to limit the invention. The variants, derivatives or structural equivalents of cupredoxin may or may not bind copper. In some modalities, cupredoxin, or variant, derivative or structural equivalent thereof has some of the functional characteristics of P. aeruginosa azurine, and specifically P28. In a specific embodiment, cupredoxins and variants, derivatives and structural equivalents of cupredoxins that can inhibit angiogenesis in cells, tissues, or mammalian animals, and specifically but not limited to, HUVECs. The invention is also provided for cupredoxins and variants, derivatives and structural equivalents of cupredoxin that may have the ability to inhibit the growth of mammalian cancer cells, and specifically but not limited to melanoma, breast, cancer cells. pancreas, glioblastoma, astrocytoma, or lung. The invention is also provided for cupredoxins and variants, derivatives and structural equivalents of cupredoxin that may have the ability to enter mammalian cancer cells compared to non-cancer equivalent cells, specifically, but not limited to, cancer cells. of melanoma, breast, pancreas, glioblastoma, astrocytoma, or lung. The inhibition of angiogenesis or growth of cancer cells is any decrease, or decrease in the rate of increase, of that activity that is statistically significant compared to the control treatments. The income in the cells is any speed of entry in cells that is statistically significant compared to the rate of entry into normal equivalent cells. Because it is now known that cupredoxins can inhibit angiogenesis in cells, tissues, or mammalian animals, and specifically in the HUVECs that grow in Matrigel®, it is now possible to design variants and derivatives of cupredoxins that retain this antiangiogenesis activity. Such variants, derivatives and structural equivalents can be produced, for example, by creating a "library" of various variants, derivatives and structural equivalents of the cupredoxins and subsequently testing each for the anti-angiogenesis activity, and specifically the anti-angiogenesis in the HUVECs. using one of the many methods known in the art, such is the exemplary method in Examples 2 and 3. It is contemplated that the variants and derivatives resulting from the cupredoxins with anti-angiogenesis activity can be used in the methods of the invention, instead of or in addition to cupredoxins. In some specific embodiments, the cupredoxin or variant, derivative or structural equivalent inhibits capillary tube formation in HUVEC cells to a degree that is statistically different from an untreated control. A peptide can be tested for this activity using the capillary tube formation test described in Example 3 or in Sulochana et al. , J. Biol. Chem. 280: 27936-27948 (2005).

Other methods to determine if capillary tube formation is inhibited are well known in the art and can also be used. In some specific embodiments, the cupredoxin or variant, derivative or structural equivalent inhibits the migration of the HUVEC in a scratch lesion migration assay to a degree that is statistically different from an untreated control. A peptide can be tested for this activity using the capillary tube formation test described in Example 4. Other methods to determine if the migration of HUVEC is inhibited are well known in the art and can also be used.

Cupredoxins These small blue copper proteins (cupredoxins) are electron transfer proteins (10-20 kDa) that participate in the bacterial electron transfer chains or are of unknown function. The copper ion is only bound by the protein matrix. A special distorted trigonal plane array for two histidine ligands and a cysteine ligand around copper gives rise to the very peculiar electronic properties of the metal site and to an intense blue color. Several cupredoxins have been characterized in a crystallographic manner in medium to high resolution.

Cupredoxins in general have low sequence homology but high structural homology. Gough & Clothia, Structure 12: 917-925 (2004); De Rienzo et al, Protein Science 9: 1439-1454 (2000). For example, the amino acid sequence of azurine is 31% identical to that of auracyanin B, 16.3% to that of rusticianin, 20.3% to that of plastocyanin, and 17.3% to that of pseudoazurin. See Table 1. However, the structural similarity of these proteins is more pronounced. The p-value of VAST for the comparison of the structure of azurin to auracyanin B is 10 ~ 7"4, azurin to rusticianin is 10" 5, azurin to plastocyanin is 10 ~ 5'6, and azurin to pseudoazurin is 10" 4-1 All cupredoxins possess a beta-cylindrical or beta-intercalated fold of eight-stranded Greek key and have a highly conserved site argument, De Rienzo et al, Protein Science 9: 1439-1454 (2000). prominent hydrophobic, due to the presence of many long-chain aliphatic residues such as methionines and leucines, is present around the copper site in azurines, amycinins, cyanobacterial plastocyanins, cucumber basic protein and to a lesser extent, pseudoazurin and plastocyanins Eukaryotic patches Id. Hydrophobic patches are also found to a lesser extent in the stelacianine and rusticianin copper sites, but have different characteristics. Id.

Table 1. Sequence and alignment of the structure of azurine (1JZG) from P. aeruginosa to other proteins using the VAST algorithm x Lined length: The number of equivalent pairs of C-alpha atoms superimposed between the two structures, that is, how many residues have been used to calculate the 3D overlay. 2P-VAL: The p-value of VAST is a measure of the importance of the comparison, expressed as a probability. For example, if the value p is 0.001, then the inequalities are 1000 to 1 against seeing a pair of this quality for pure chance. The p-value of VAST is adjusted for the purposes of multiple comparisons using the assumption that there are 500 independent and unrelated types of domains in the MMDB database. The p-value shown thus corresponds to the p-value for the comparison of pairs of each pair of domains, divided by 500. 3Conteo: The counting of structure similarity of VAST. This number is related to the number of overlapping secondary structure elements and the quality of that overlay. The higher VAST counts correlate with greater similarity. 4RMSD: The residue in superscript of the mean square root in Angstroms. This number is calculated after the optimal superposition of two structures, such as the square root of the mean square distances between the C-alpha equivalent atoms. Note that the RMSD value is scaled with the degree of structural alignments and that this size should be taken into consideration when using RMSD as a descriptor of overall structural similarity. 5 Main protein of C. elegans sperm tested as an efrin antagonist in the maturation of oocytes. Kuwabara, Genes and Development 17: 155-161 (2003).

Azurine Azurines are proteins that contain copper from 128 amino acid residues that belong to the family of cupredoxins involved in the transfer of electrons in certain bacteria. The azurines include those from P. aeruginosa (PA) (SEQ ID NO: 1), A. xylosoxidans, and A. deni trifi cans. Murphy et al. , J. Mol. Biol. 315: 859-871 (2002). The amino acid sequence identity between the azurines varies between 60-90%, these proteins showed a strong structural homology. All azurines have a ß-intercalated radical or motif with a characteristic Greek key and the only copper atom is always placed in the same region of the protein. In addition, azurines have an essentially neutral hydrophobic patch that surrounds the copper site. Id.

Plastocyanins Plastocyanins are soluble proteins of cyanobacteria, algae and plants that contain one molecule of copper per molecule and are blue in their oxidized form. They occur in the chloroplast where they function as electron carriers. From the determination of the poptocyanine structure of the poplar in 1978, the structures of the plastocyanins in the algae (Scenedesmus, Enteromorpha, Chl amy dome) and plant (French bean) have been determined either by crystallographic methods or NMR methods, and the poplar structure has been refined at the resolution of 1.33 Á. SEQ ID NO: 3 shows the amino acid sequence of plastocyanin from Phormidium laminosum, a thermophilic cyanobacterium. Despite the divergence of sequences between algae plastocyanins and vascular plants (eg, 62% sequence identity between Chlamydomonas and poplar proteins), three-dimensional structures are conserved (eg, deviation of 0.76 A rms in the position of C alpha between proteins) of Chlamydomonas and the Alamo). Structural features include a distorted tetrahedral copper binding site at one end of an eight-strand antiparallel beta-cylinder, a pronounced negative patch, and a flat hydrophobic surface. The copper site is optimized for its electron transfer function, and negative and hydrophobic patches are proposed to be involved in the recognition of physiological reaction partners. The chemical modification experiments, cross-linking, and site-directed mutagenesis have confirmed the importance of negative and hydrophobic patches in the binding interactions with cytochrome f, and have validated the model of two functionally significant electron transfer trajectories involving plastocyanin. A putative electron transfer pathway is relatively short (approximately 4A) and involves the His-87 copper ligand exposed to the solvent in the hydrophobic patch, while the other is longer (approximately 12-15 A) and involves the Tyr-83 residue almost conserved in the negative patch. Redinbo et al, J. Bioenerg.

Biomembr. 26: 49-66 (1994).

Rusticianins Rusticianins are single chain polypeptides containing blue copper, obtained from a Thiobacillus (now called Acidi thiobacillus). The X-ray crystal structure of the oxidized form of the highly oxidizing cupredoxin rusticianin and extremely stable from Thiobacillus ferrooxidans (SEQ ID NO: 4) has been determined by the anomalous multi-wavelength diffraction and refined to the resolution of 1.9Á. The rusticianins are composed of a beta-interleaved core fold of a six- and seven-strand b-sheet. Like other cupredoxins, the copper ion is coordinated by a stacking of four conserved residues (His 85, Cysl38, Hisl43, Met 148) placed in a distorted tetrahedron. Walter, R.L. et al, J. Mol. Biol. 263: 730-51 (1996).

Pseudoazurins Pseudoazurins are a family of single chain polypeptides containing blue copper. The amino acid sequence of the pseudoazurin obtained from Tlc roinoJbacter cycloclastes is shown in SEQ ID NO: 5. Analysis of the X-ray structure of pseudoazurin shows that it has a structure similar to azurines although there is low sequence homology between these proteins. There are two main differences between the global structure of pseudoazurines and azurines. There is an extension of the carboxy term in the pseudoazurines, relative to the azurines, which consist of two alpha-helices. In the bluffs of the middle region of the peptide they contain an extended loop, shortened in the pseudoazurins that form a spoiler containing a short a-helix. The only major differences in the site of the copper atom are the conformation of the MET-side chain and the length of the Met-S copper bond, which is significantly shorter in pseudoazurin than in azurine.

Phytocyanins Proteins identifiable as phytocyanins include, but are not limited to, the basic protein of cucumber, stelacianin, mavicianin, umecianin, a cupredoxin of cucumber skin, a blue copper protein putative in pods of peas, and a blue copper protein from Arabidopsis thaliana. In all but the basic cucumber protein and the pea pod protein, the axial methionine ligand normally found in the blue copper sites is replaced by glutamine.

Auracyanin Three small blue copper proteins designated auracyanin A, auracyanin B-1, and auracyanin B-2 have been isolated from the photosynthetic, green, thermophilic photosynthetic Chloroflexns aurantiacns bacteria. The two B-forms are glycoproteins and have almost identical properties to each other, but are different from form A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates the molecular masses of the monomer apparent as 14 (A), 18 (B-2 ), and 22 (Bl) kDa. The amino acid sequence of auracyanin A has been determined and has shown that auracyanin A is a 139 residue polypeptide. Van Dreissche et al, Protein Science 8: 947-957 (1999). His58, Cysl23, Hisl28, and Metl32 are separated in a manner that is expected if they are the evolutionary conserved metal ligands as in the small known copper proteins plastocyanin and azurine. The prediction of secondary structure also indicates that auracyanin has a general beta-cylinder structure similar to that of azurine from Pseudomonas aeruginosa and from plastocyanin from poplar leaves. However, auracyanin appears to have sequence characteristics of both classes of small copper protein sequences. The overall similarity with a consensus sequence of azurine is approximately same as that with a plastocyanin consensus sequence, namely 30.5%. The 1-18 N-terminal sequence regions of auracyanin are remarkably rich in glycine and hydroxy amino acids. Id. See the exemplary amino acid sequence SEQ ID NO: 15 for the A chain of auracyanin from Chloroflexus aurantiacus (Access to NCBI Protein Data Bank No. AAM12874). The auracyanin B molecule has a standard cupredoxin fold. The crystal structure of auracyanin B has been studied from Chloroflexus aurantiacus. Bond et al, J. Mol. Biol. 306: 47-67 (2001). With the exception of an additional N-terminal strand, the molecule is very similar to that of the bacterial cupredoxin, azurine. As in other cupredoxins, one of the ligands Cu lies on the 4-strand of the polypeptide, and the other three lie along a large loop between strands 7 and 8. The geometry of the Cu site is described with reference to the amino acids that spaced between the last three ligands. The Cu binding domain characterized in a crystallographic manner of auracyanin B is probably bound to the periplasmic side of the cytoplasmic membrane by an N-terminal tail that exhibits a significant sequence identity with the known bonds in several other associated electron transfer proteins. with the membrane. The amino acid sequences of the B forms are presented in McManus et al. J. Biol. Chem. 267: 6531-6540 (1992). See the exemplary amino acid sequence SEQ ID NO: 16 for the B chain of auracyanin from Chloroflexus aurantiacus (Access to NCBI Protein Data Bank No. IQHQA).

Stelacianin Stelacianins are a subclass of phytocyanins, a ubiquitous family of plant cupredoxins. An exemplary sequence of a stelacianin is included herein as SEQ ID NO: 14. The crystal structure of umecianin, a stelacianin of radish root, is also known (Koch et al, J. Am. Chem. Soc. 127: 158 -166 (2005)) and cucumber stelacianin (Hart et al, Protein Science 5: 2175-2183 (1996)). The protein has a global fold similar to the other phytocyanines. The tertiary structure of the ectodomain of the ephrin protein B2 gives a significant similarity to the stelacianin. Toth et al, Developmental Cell 1: 83-92 (2001). An exemplary amino acid sequences of a stelacianin is found in the National Center for Biotechnology Information Access to Protein Data Bank No. 1JER,? EC ID NO: 14.

Cucumber basic protein An exemplary amino acid sequence of a cucumber basic protein is included here as SEQ ID NO: 17. The crystal structure of the cucumber basic protein (CBP), a blue copper protein of type 1, has been refined in resolution of 1.8Á. The molecule resembles other blue copper proteins in having a beta-cylinder structure with a Greek key, except that the cylinder is open on one side and is best described as a "beta-intercalation" or "beta-taco". Guss et al, J. Mol. Biol. 262: 686-705 (1996). The tertiary structure of the ectodomain of the ephrin protein B2 gives a high similarity (deviation rms 1.5Á for the 50 to carbons) to the basic protein of cucumber. Toth et al, Developmental Cell 1: 83-92 (2001). The Cu atom has the NNSS 'co-ordination of normal blue copper with the Cu-N bond lengths (His39) = 1.93 A, Cu-S (Cys79) = 2.16 A, Cu-N (His84) = 1.95 A, Cu-S (Met89) = 2.61 A. A disulfide bond, (Cys52) -SS- (Cys85), seems to play an important role in the stabilization of the molecular structure. The fold of the polypeptide is typical of a sub-family of blue copper proteins (phytocyanines) as well as Ra3 of ragweed allergen, without metalloprotein, with which CBP has a high degree of sequence identity. The proteins currently identifiable as phytocyanins are CBP, stelacianin, mavicianin, umecianin, a cupredoxin from cucumber cover, a putative blue copper protein in pods of peas, and a blue copper protein a from Arabidopsis thaliana. In all but CBP and peas protein, the axial methionine ligand normally found in blue copper sites is replaced by glutamine. An exemplary sequence for the basic cucumber protein is found in Access to the NCBI Protein Data Bank No. 2CBP, SEQ ID NO: 17.

Methods of Use The invention provides methods for treating a mammalian patient suffering from cancer, recovering from cancer, recovering from cancer or at risk of cancer, comprising administering to the patient at least one polypeptide that is a cupredoxin, or a variant, derivative or structural equivalent thereof, as described above. Specifically, cancers that can be treated with the compositions of the invention include, but are not limited to, melanoma, breast, pancreatic, glioblastoma, astrocytoma, or lung cancer. The invention further provides methods for treating patients suffering from, recovering from, recovered from or at risk of having other conditions related to inappropriate angiogenesis, comprising administering to the patient at least one polypeptide that is a cupredoxin, or a variant, derivative or structural equivalent thereof. These conditions include, but are not limited to, macular degeneration related to neovascular age, diabetic retinopathy, psoriasis and rheumatoid arthritis. In the specific modalities, the patient is human. The invention further includes methods for studying angiogenesis comprising contacting the mammalian cells with a composition comprising cupredoxin, or a variant, derivative or structural equivalent thereof. In some embodiments, the cells are HUVECs, while in others they are other cells that suffer from angiogenesis. The methods of the invention further include methods for studying conditions related to inappropriate angiogenesis comprising contacting the mammalian cells with a composition comprising cupredoxin, or a variant, derivative or structural equivalent thereof. In these methods, the cells may be those that suffer from angiogenesis in mammalian patients suffering from the condition. Compositions comprising a cupredoxin or a variant, derivative or structural equivalent thereof can be administered to the patient by many routes and in many regimens that will be well known to those in the art. In the specific modalities, the cupredoxin, or the variant, derivative or structural equivalent thereof, is administered • intravenously, intramuscularly, subcutaneously or intraocularly. The compositions can be administered to the patient by any means that delivers the peptides to the site of inappropriate angiogenesis. In one embodiment, the methods may comprise co-administering to a patient a unit dose of a composition comprising a cupredoxin or a variant, derivative or structural equivalent of cupredoxin and a unit dose of a composition comprising another anti-cancer drug, in any order, administered at about the same time, or within about a given time followed by administration of the other, for example, about one minute to about 60 minutes after the administration of the other drug, or about 1 hour to about 12 hours after of the administration of the other drug. Such drugs include, for example, those listed herein and specifically 5-fluorouracil; They inferred; Methotrexate; Tamoxifen; and Vincrinstine. The above examples are provided for illustration only, many other such compounds are known to those skilled in the art. The compounds of the invention can also be used in conjunction with radiation therapy and surgery. Other drugs suitable for treating cancer include, but are not limited to, alkylating agents such as nitrogen mustards, alkyl sulfonates, nitrosoureas, ethylene imines, and triazenes; antimetabolites such as folate antagonists, purine analogues, and pyrimidine analogues; antibiotics such as anthracyclines, bleomycins, mitomycin, dactinomycin, and plicamycin; enzymes such as L-asparaginase; famesyl-protein transferase inhibitors; 5. alpha inhibitors. -reductasa; 17.beta inhibitors. -hydroxysteroid dehydrogenase type 3; hormonal agents such as glucocorticoids, estrogens / antiestrogens, androgens / antiandrogens, progestins, and luteinizing hormone releasing hormone antagonists, octreotide acetate; microtubule disrupting agent, such as ecteinascidins or their analogs and derivatives; microtubule stabilizing agents such as taxanes, for example, paclitaxel (Taxol ™), docetaxel (Taxotere ™), and their analogs, and epothilones, such as epothilones A-F and their analogues; plant-derived products, such as vinca alkaloids, epipodophyllotoxins, taxanes; and topiosomerase inhibitors; inhibitors of the transferase-prenyl protein; and miscellaneous agents such as hydroxyurea, procarbazine, mitotane, hexamethylmelamine, platinum coordination complexes such as cisplatin and carboplatin; and other agents used as anti-cancer and cytotoxic agents such as biological response modifiers, growth factors; immune modulators and monoclonal antibodies. The representative examples of these kinds of Anti-cancer and cytotoxic agents include but are not limited to mechlorethamine hydrochloride, cyclophosphamide, chlorambucil, melphalan, ifosfamide, busulfan, carmustine, lomustine, semustine, streptozocin, thiotepa, dacarbazine, methotrexate, thioguanine, mercaptopurine, fludarabine, pentastatin, cladribine, cytarabine, fluorouracil, doxorubicin hydrochloride, daunorubicin, idarubicin, bleomycin sulfate, mitomycin C, actinomycin D, safracin, saframycin, quinocarcin, discodermolide, vincristine, vinblastine, vinorelbine tartrate, etoposide, etoposide phosphate, teniposide, paclitaxel, tamoxifen, estramustine, sodium estramustine phosphate, flutamide, buserelin, leuprolide, pteridines, diineses, levamisole, aflacon, interferon, interleukins, aldesleukin, filgrastim, sargramostim, rituximab, BCG, tretinoin, irinotecan hydrochloride, betamethasone, gemcitabine hydrochloride, altretamine, and a library and any analogue or derivatives thereof. Preferred members of these classes include, but are not limited to, paclitaxel, cisplatin, carboplatin, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, metopterin, mitomycin C, ecteinascidin 743, or pofiromycin, 5-fluorouracil, 6-mercaptopurine, gemcitabine , arabinoside cytosine, podophyllotoxin or podophyllotoxin derivatives such as etoposide, etoposide phosphate or teniposide, melphalan, vinblastine, vincristine, leurosidine, vindesine and leurosine. Examples of anti-cancer and other cytotoxic agents useful for co-ading with the compositions of the invention include the following: epothilone derivatives as found in German Patent No. 4138042.8; WO 97/19086, WO 98/22461, WO 98/25929, WO 98/38192, WO 99/01124, WO 99/02224, WO 99/02514, WO 99/03848, WO 99/07692, WO 99/27890, WO 99/28324, WO 99/43653, WO 99/54330, WO 99/54318, WO 99/54319, WO 99/65913, WO 99/67252, WO 99/67253 and WO 00/00485; cyclin-dependent kinase inhibitors as found in WO 99/24416 (see also US Patent No. 6,040,321); and transferase-prenyl protein inhibitors as found in WO 97/30992 and WO 98/54966; and agents such as those described generically and specifically in U.S. Patent No. 6,011,029 (the compounds of which may be used in conjunction with any NHR modulator (including, but not limited to, those of the present invention) such as AR modulators, modulators ER, with LHRH modulators, or with surgical techniques In another embodiment, the cupredoxin, variant, derivative or structural equivalent thereof can be co-administered with a drug for the treatment of age-related macular degeneration.

Such drugs include, but are not limited to, Lucentis® (Genetech, South San Francisco CA, Ranibizumab, vitreous injection), Macugen® (OSI Pharmaceuticals, Melville NY, pegaptanib, vitreous injection), Retaane® (Alcon, Fort Worth TX). , Anecortave, posterior juxtascleral injection), AdPEDF (GenVec, Gaithersburg MD, anti-angiogenic gene therapy, intravitreal or sub-Tenon injection), EVIZON® (Genaera, PIymouth Meeting, PA, anti-angiogenic aminosterol, Squalamin, intravenous injection) , Prodrug AAdPEDF4 Combretastatin (OXiGENE, Waltham MA, CA4P, vascular target agent), Cand5 (Acuity Pharmaceuticals, Inc., Philadelphia PA, Vascular Endothelial Growth Factor (VEGF) targeting Sirna-027® siRNA (Sirna Therapeutics, San Francisco, CA, Receptor-1 Vascular Endothelial Growth Factor (VEGFR-I) that targets siRNA), Celecoxib with PDT (Celebrex®, oral anti-inflammatory drug), and Envision® TD (Control Delivery Systems, Wa tertown, MA, sustained-release steroid implant of the luocinolone implant in vitreous). In another embodiment, the cupredoxin, the variant, derivative or structural equivalent thereof can be coadministered with a drug for the treatment of diabetic retinopathy. Surgical treatment is also contemplated as a co-treatment with the compositions of the invention.

In another embodiment, the cupredoxin, the variant, derivative or structural equivalent thereof can be coadministered with a drug for the treatment of psoriasis. Such drugs include, but are not limited to, Amevive®, Raptiva®, Enbrel®, Humira®, Remicade®, Cyclosporine, Neoral®, Methotrexate, Soriatane®, Accutane®, Hydrea®, mycophenolate mofetil, sulfasalazine, and 6-thioguanine . In another embodiment, the cupredoxin, variant, derivative or structural equivalent thereof can be coadministered with a drug for the treatment of rheumatoid arthritis. Such drugs include, but are not limited to, Methotrexate (Rheumatrex®, Folex PFS®), Sulfasalazine (Azulfidine®), Leflunomide (Arava®), Gold salts (aurothiomalate, auranofin [Ridaura®]), D-penicillamine, Hydroxychloroquine. (Plaquenil®), Azatioprine (Imuran®), Cyclosporine (Neoral®), Etanercept (Enbrel®), Infliximab (Remicade®), Adalimumab (Humira®), Anakinra (Kineret®), Abatacept (Orencia®), Prednisone (Deltasone) ®, Meticorten®, Orasone®), Betamethasone (Celestone®), nonsteroidal anti-inflammatory drugs (NSAIDs), and COX-2 inhibitors (celecoxib, Celebrex®).

Pharmaceutical Compositions Comprising Cupredoxin, O Variant, Derivative, or Structural Equivalent of the Same Pharmaceutical compositions comprising cupredoxin or the variant, derivative or structural equivalents thereof, may be manufactured in any conventional manner, for example, by the process of mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping, or conventional lyophilizing. Cupredoxin or variants, derivatives and structural equivalents thereof substantially pure or of pharmaceutical grade can be combined rapidly with a pharmaceutically acceptable carrier well known in the art. Such carriers allow the preparation to be formulated as a tablet, pill, lozenge, capsule, liquid, gel, syrup, slurry, suspension, and the like. Suitable carriers or excipients may also include, for example, fillers and cellulose preparations. Other excipients may include, for example, flavoring agents, coloring agents, viscosity scavengers, thickeners, and other acceptable additives, adjuvants, or binders. In some embodiments, the pharmaceutical preparation is substantially free of preservatives. In other embodiments, the pharmaceutical preparation may contain at least one preservative. The General methodology on pharmaceutical dosage forms is found in Ansel et al, Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams &Wilkins, Baltimore MD (1999)). The composition comprising a cupredoxin or a variant, derivative or structural equivalent thereof used in the invention can be administered in a variety of ways, including by injection (eg, intradermal, subcutaneous, intramuscular, intraperitoneal and the like), by inhalation, by topical administration, by suppository, using a transdermal patch or through the mouth. General information on drug delivery systems can be found in Ansel et al, Id .. In some embodiments, the composition comprising a cupredoxin or a variant, derivative or structural equivalent thereof can be formulated and used directly as injectables, for Subcutaneous and intravenous injection, among others. The injectable formulation, in particular, can be advantageously used to treat patients who are at risk of, who probably have or who have a condition related to inappropriate angiogenesis. The composition comprising a cupredoxin or a variant, derivative or structural equivalent thereof can also be taken orally after mixing with protective agents such as polypropylene glycols or similar coating agents. When the administration is by injection, the cupredoxin or the variant, derivative or structural equivalent thereof can be formulated in the aqueous solutions, specifically in compatible buffers. physiologically such as the Hanks solution, the Ringer's solution, or the physiological saline buffer. The solution may contain formulating agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the cupredoxin or variant, derivative or structural equivalent thereof may be in powdered form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. In some embodiments, the pharmaceutical composition does not comprise an adjuvant or any other added substance to enhance the immune response stimulated by the peptide. In some embodiments, the pharmaceutical composition comprises a substance that inhibits an immune response to the peptide. When the administration is by intravenous fluids, the intravenous fluids for use of administration of the cupredoxin or the variant, derivative or structural equivalent thereof can be composed of crystalloids or colloids. Crystalloids as used herein are aqueous solutions of mineral salts or other water-soluble molecules. The colloids as used herein contain larger insoluble molecules, such as gelatin. Intravenous fluids can be sterile. Crystalloid fluids that can be used for intravenous administration include but are not limited to, normal saline solution (0.9% sodium chloride solution), Ringer's lactate or Ringer's solution, and a 5% dextrose solution in water sometimes called D5W, as described in Table 2.

Table 2. Composition of the Common Crystalloid Solutions * E1 Ringer's Lactate also has 28 mmol / L lactate, 4 mmol / L K + and 3 mmol / L Ca2 +.

When administration is by inhalation, the cupredoxin or the variant, derivative or structural equivalent thereof, may be provided in the form of an aerosol spray of pressurized packets or a nebulizer with the use of a suitable propellant, for example, dichlorodifluoromethane , trichlorofluoromethane, carbon dioxide or other suitable gas. In the case of an aerosol Pressurized, the dosage unit can be determined by providing a valve to deliver a measured quantity. Capsules and cartridges, for example, of gelatin, for use in an inhaler or insufflator can be formulated containing a powder mixture of the proteins and a suitable powder base such as lactose or starch. When the administration is by topical administration, the cupredoxin or variant, derivative or structural equivalent thereof can be formulated as solutions, gels, ointments, creams, jellies, suspensions, and the like, as is well known in the art. In some modalities, the administration is by means of a transdermal patch. When the administration is by suppository (eg, rectal or vaginal), the cupredoxin compositions or variants and derivatives thereof can also be formulated in compositions containing conventional suppository bases. When the administration is oral, a cupredoxin or a variant, derivative or structural equivalent thereof can be rapidly formulated by combining the cupredoxin or the variant, derivative or structural equivalent thereof with pharmaceutically acceptable carriers well known in the art. A solid carrier can be employed, such as mannitol, lactose, magnesium stearate, and the like; such carriers allow the cupredoxin and variants, derivatives or structural equivalent thereof are formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. For solid oral formulations such as, for example, powders, capsules and tablets, suitable excipients include fillers such as sugars, cellulose preparation, granulation agents, and binding agents. Other suitable carriers, as is well known in the art, also include multivalent carriers, such as the bacterial capsular polysaccharide, a dextran or a genetically engineered vector. In addition, sustained release formulations that include a cupredoxin or a variant, derivative or structural equivalent thereof allow the release of the cupredoxin or variant, derivative or structural equivalent thereof over extended periods of time, such that without the formulation of sustained release, the cupredoxin or variant, derivative or structural equivalent thereof would be removed from the system of a subject, and / or degraded by, for example, proteases and by simple hydrolysis before a therapeutic effect is aroused or enhanced. The half-life in the bloodstream of the peptides of the invention can be extended or optimized by various methods well known to those in the art. The Peptide variants of the invention may include, but are not limited to, several variants that may increase their stability, specific activity, longevity in the bloodstream, and / or may decrease the immunogenicity of cupredoxin, retaining the ability of the peptide to inhibit angiogenesis, to enter mammalian cancer cells and / or to inhibit the growth of mammalian cancer cells. Such variants include, but are not limited to, those that decrease the hydrolysis of the peptide, decrease peptide deamidation, decrease oxidation, decrease immunogenicity, increase the structural stability of the peptide or increase the size of the peptide. Such peptides also include peptides formed in a circle (see Monk et al, BioDrugs 19 (4): 261-78, (2005)).; DeFreest et al. , J. Pept. Res. 63 (5): 409-19 (2004)), D, L-peptides (diastereomer), Futaki et al, J. Biol. Chem. Feb 23; 276 (8): 5836-40 (2001); Papo et al, Cancer Res. 64 (16): 5779-86 (2004); Miller et al, Biochem. Pharmacol. 36 (1): 169-76, (1987)); peptides containing rare amino acids (see Lee et al, J. Pept. Res. 63 (2): 69-84 (2004)), terminal N- and C- modifications (see Labrie et al, Clin. Invest. Med. (5): 275-8, (1990)), fixation by staples of hydrocarbons (see Schafmeister et al, J. Am. Chem. Soc. 122: 5891-5892 (2000); Walenski et al, Science 305: 1466- 1470 (2004)) and PEGylation. Of particular interest are the d- isomerization (substitution) and modification of peptide stability via amino acid substitution L- or substitution D-. In various embodiments, the pharmaceutical composition includes carriers and excipients (including but not limited to buffers, carbohydrates, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, gelling agents, suspending agents, thickening agents and / or preservatives), water, oils, saline solutions, aqueous dextrose and glycerol solutions, other pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents, wetting agents and the like. It will be recognized that, while any suitable carrier known to those of ordinary skill in the art may be employed to administer the compositions of this invention, the type of carrier will vary, depending on the mode of administration. The compounds can also be encapsulated within liposomes using well-known technology. Biodegradable microspheres can also be used as the carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are described, for example, in U.S. Patent Nos. 4,897,268; 5,075,109; 5,928,647; 5,811,128; 5,820,883; 5,853,763; 5,814,344 and 5,942,252. The pharmaceutical compositions can be sterilized by conventional well-known sterilization techniques, or they can be sterile filtered. The resulting aqueous solutions can be packaged for use as is, or they can be lyophilized, the lyophilized preparation being combined with a sterile solution before administration.

Administration of the Cupredoxin or Variant, Derivative or Structural Equivalent of the Same The cupredoxin or the variant, derivative or structural equivalent thereof can be administered formulated as pharmaceutical compositions and can be administered by any suitable route, for example, by oral, buccal, inhalation, sublingual, rectal, vaginal, transurethral, nasal, topical, percutaneous, that is, transdermal or parenteral (including intravenous, intramuscular, subcutaneous and intracoronary) or vitreous administration. The pharmaceutical formulations thereof can be administered in any effective amount to achieve their intended purpose. More specifically, the composition is administered in a therapeutically effective amount. In specific embodiments, the therapeutically effective amount is generally about 0.01-20 mg / day / kg of body weight. The compounds comprising the cupredoxin or the variant, derivative or structural equivalent thereof are useful for the treatment and / or prophylaxis of the related conditions in inappropriate angiogenesis, alone or in combination with other active agents. The appropriate dosage, of course, will vary depending on, for example, the cupredoxin compound or variant, derivative or structural equivalent thereof, the host, the mode of administration and the nature and severity of the conditions being treated. However, in general, it is indicated that satisfactory results are obtained in humans at daily dosages of approximately 0.01-20 mg / kg of body weight. A daily dosage indicated in humans is in the range of about 0.7 mg to about 1400 mg of a cupredoxin compound or variant, derivative or structural equivalent thereof conveniently administered, for example, in daily doses, weekly doses, monthly doses, and / or continuous doses. Daily doses may be in discrete dosages from 1 to 12 times per day. Alternatively, doses may be administered every other day, every third day, every fourth day, every fifth day, every sixth day, every week, and similarly in increments of days up to 31 days or more. Alternatively, the dosage can be continuoususing patches, i.v. administration and similar. The exact formulation, the route of administration, and the dosage are determined by the attending physician in view of the patient's condition. The amount of the dosage and the range can be adjusted individually to provide the plasma levels of the active cupredoxin or variant, derivative or structural equivalent thereof that are sufficient to maintain the therapeutic effect. Generally, the desired cupredoxin or variant, derivative or structural equivalent thereof is administered in a mixture with a selected pharmaceutical carrier with respect to the intended route of administration and standard pharmaceutical practice. In one aspect, the cupredoxin or variant, derivative or structural equivalent thereof is supplied as DNA such that the polypeptide is generated in itself. In one embodiment, the DNA is "naked," as described, for example, in Ulmer et al, (Science 259: 1745-1749 (1993)) and reviewed by Cohen (Science 259: 1691-1692 (1993)). The uptake of naked DNA can be increased by covering the DNA on a carrier, for example, biodegradable beads that are then transported efficiently in the cells. In such methods, DNA can be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacterial and viral expression systems. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. For example, see WO90 / 11092, WO93 / 24640, WO 93/17706, and U.S. Patent No. 5,736,524. The vectors, used to transport genetic material from organism to organism, can be divided into two general classes: The cloning vectors are plasmid or replication phage with regions that are essential for propagation in an appropriate host cell and in which the host can be inserted. Foreign DNA; Foreign DNA reproduces and propagates as if it were a component of the vector. An expression vector (such as a plasmid, yeast, or animal virus genome) is used to introduce the foreign genetic material into a host cell or tissue to transcribe and translate the foreign DNA, such as the DNA of a cupredoxin. In the expression vectors, the introduced DNA is operably linked to the elements such as the promoters that signal the host cell to highly transcribe the inserted DNA. Some promoters are exceptionally useful, such as inducible promoters that control gene transcription in response to specific factors. Uniting operably a cupredoxin and the polynucleotide of the variants and derivatives thereof to an inducible promoter can be controlled for the expression of cupredoxin and variants and derivatives thereof in response to specific factors. Examples of classical inducible promoters include those that are sensitive to Inferno-a, heat shock, heavy metal ions, and steroids such as glucocorticoids (Kaufman, Methods Enzymol 185: 487-511 (1990)) and tetracycline. Other desirable inducible promoters include those which are not endogenous to the cells in which the structure is being introduced, but which are sensitive in those cells when the induction agent is delivered exogenously. In general, useful expression vectors are often plasmids. However, other forms of expression vectors are contemplated, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses). The choice of vector is dictated by the organism or cells that are used and the desired destination of the vector. In general, the vectors comprise signal sequences, origins of replication, marker genes, polylinker sites, enhancer elements, promoters, and transcription termination sequences.

Kits or Equipment Comprising Cupredoxin, or Variant, Derivative or Structural Equivalent thereof In one aspect, the invention provides regimes or kits comprising one or more of the following in a package or container: (1) a biologically active composition comprising at least one cupredoxin or a variant, derivative or structural equivalent thereof; (2) an antiviral or anti-bacterial drug, specifically an anticancer drug, a macular anti-degenerative drug, an anti-psoriasis drug or an anti-rheumatoid arthritis drug. When a kit is supplied, the different components of the composition can be packaged in separate containers, if appropriate, and mixed immediately before use. Such packaging of the components separately can allow long-term storage without losing the functions of the active components. The reagents included in the kits can be supplied in containers of any kind such that the life of the different components is conserved and not adsorbed or altered by the container materials. For example, sealed glass ampoules may contain lyophilized cupredoxin and variants, derivatives and structural equivalents thereof, or buffers that have been packaged under a non-reactive, neutral gas, such as nitrogen. The vials can consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, etc., ceramics, metal or any other material typically employed to retain similar reagents. Other examples of suitable containers include simple bottles that can be made of similar substances such as ampoules, and shells, which can comprise sheet-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, bottles, bottles, syringes, or the like. The containers may have a sterile access port, such as a bottle having a stopper that can be punctured by a hypodermic injection needle. Other containers may have two compartments that are separated by a rapidly removable membrane that, when removed, allows the components to mix. The removable membranes can be made of glass, plastic, rubber, etc. Kits or equipment can also be supplied with instructional materials. The instructions may be printed on paper or other substrate, and / or may be provided as an electronic readable medium, such as a floppy disk device, CD-ROM, DVD-ROM, Zip disk, videotape, audio tape, flash memory, etc. Detailed instructions may not be physically associated with the kit; in Instead, a user can go to a web site (on the Internet) specified by the manufacturer or distributor of the kit, or can be provided as an email.

Modification of Cupredoxin and Variants, Derivatives and Structural Equivalents of the same Cupredoxin or the variant, derivative or structural equivalents thereof can be chemically modified or genetically altered to produce variants and derivatives as explained above. Such variants and derivatives can be synthesized by standard techniques. In addition to naturally occurring allelic variants of cupredoxin, changes can be introduced by mutation in the cupredoxin-encoding sequence that results in alterations in the amino acid sequences of the encoded cupredoxin that does not significantly alter the ability of cupredoxin to inhibit angiogenesis. A "non-essential" amino acid residue is a residue that can be altered from the wild type sequences of cupredoxin without altering the biological activity, while an "essential" amino acid residue is required for such biological activity. For example, the amino acid residues that are conserved between Cupredoxins are predicted to be particularly non-compliant to the alteration, and therefore are "essential." The amino acids for which conservative substitutions can be made that do not change the activity of the polypeptide are well known in the art. Useful conservative substitutions are shown in Table 3, "Preferred Substitutions." The conservative substitutions with which an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the invention so long as the substitution does not materially alter the biological activity of the compound.

Table 3: Preferred substitutions Non-conservative substitutions that affect (1) the structure of the main structure of the polypeptide, such as a ß-laminar or a-helical conformation, (2) the charge, (3) the hydrophobicity, or (4) the volume of the Side chain of the target site can modify the function of the cytotoxic factor. The waste is divided into groups based on the common properties of the side chain as denoted in Table 4. Non-conservative substitutions cause exchanging a member of one of these classes for another class. Substitutions can be introduced at conservative substitution sites or more specifically at non-conserved sites. Variant polypeptides can be produced using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter, Biochem J. 237: 1-7 (1986), Zoller and Smith, Methods Enzymol 154: 329-350 (1987)), cassette mutagenesis, restriction selection mutagenesis (Wells et al. , Gene 34: 315-323 (1985)) or other known techniques can be performed on the cloned DNA to produce the variant DNA of cupredoxin. Known mutations of the cupredoxins can also be used to create the variant cupredoxin to be used in the methods of the invention. For example, the mutants C112D and M44KM64E of azurine are known to have cytotoxic activity and stop growth, which is different from native azurine, and such altered activity may be useful in the methods of treatment of the present invention. One embodiment of the methods of the invention utilizes cupredoxin and the variants and derivatives thereof that retain the ability to inhibit angiogenesis. In another embodiment, the methods of the present invention utilize cupredoxin variants such as mutant M44KM64E, which has the ability to cause cell growth arrest. A more complete understanding of the present invention can be obtained by reference to the following specific Examples. The Examples are described only for purposes of illustration and are not intended to limit the scope of the invention. Changes in the form and substitution of equivalents are contemplated when circumstances may suggest or become convenient. Although specific terms have been used here, such terms are projected in a descriptive sense and not for purposes of limitations. The modifications and variations of the invention as set forth hereinbefore can be made without departing from the spirit and scope thereof, and, consequently, only such limitations must be imposed as indicated by the attached modalities.

EXAMPLES Example 1. Entry of P28 into the endothelial cells of the human umbilical vein. P28 was labeled with 20μM of Alexafluor® 568 (Molecular Probes, Eugene, OR). The cell lines indicated were grown on coated slides coated with cell culture overnight at 37 ° C. The pre-heated medium containing the labeled peptide was added at the indicated concentrations. After incubation with the labeled peptide, the coated slides were washed 3X with PBS and fixed in formalin for 5 minutes. Coated slides were subsequently mounted in medium containing 1.5 μg mi-1 of DAPI for nuclear staining (VECTASHIELD®, Vector Laboratories, Burlingame, CA). The analysis was performed with a confocal microscope (Model LC510, Cari Zeiss, Thornwood, NY). The P28 effectively entered the malignant cell lines that originate from melanoma, breast, pancreas, glioblastoma, astrocytoma, and lung (Figure IA). P28 was also efficiently entered into the HUVEC cells (Figure 1C). No significant admission was observed in other "normal" cell lines that originate from skin, breast and pancreas fibroblasts (Figure IB). Therefore, in addition to entering specifically into mammalian cancer cells, P28 also enters specifically into HUVEC cells. This experiment shows that the 50-77 azurine peptide of P. aeruginosa has activity that inhibits capillary tube formation in endothelial cells, a step in angiogenesis. The peptide 50-77 of azurine of P. aeruginosa can therefore be used to control angiogenesis and can therefore be used as a cancer treatment, and as a treatment of other conditions related to inappropriate angiogenesis.

Example 2. Effects of P28 on the formation of the capillary tube of HUVEC in Matrigel®. Matrigel® matrix (Becton Dickinson Biosciences, San Jose CA) is a solubilized base membrane preparation, extracted from EHS mouse sarcoma, a tumor rich in ECM proteins. Its main component is laminin, followed by collagen IV, heparan sulfate proteoglycans, and entactin 1. At room temperature, the Matrigel® Matrix polymerizes to produce the biologically active matrix material that resembles the cell-based membrane of a mammal The cells behave as they do in vivo when they are grown in the Matrigel® Matrix. It provides a physiologically relevant environment for studies of cell morphology, biochemical function, migration or invasion, and gene expression. The Matrigel® Matrix serves as a substrate for endothelial cell invasion and in vitro tube formation assays. The effects of P28 on capillary tube formation of HUVEC cells using Matrigel® were investigated. HUVEC cells were plated (15,000 cells / well) on 8-well chamber slides coated with Matrigel® with 20ng / ml of VEGF and in the presence or absence of the peptide. P28 concentrations of OμM (control), O.lOμM, 0.30μM, 0.92μM, 2.77μM, 8.33μM, 25μM and 75μM were used. Cells were stained 4h and 24h after treatment with AM calcein, and capillary tube formation was examined using a fluorescence microscope (Figure 2A). The results show that as little as O.lO.μM prevents capillary tube formation by HUVEC cells by approximately 50% (Figure 2A). P28 therefore inhibits tube formation of HUVEC cells, and therefore will also inhibit capillary tube formation related to angiogenesis.

Example 3. Effects of P28 on the motility of the HUVEC. The effects of P28 on the motility of the HUVEC were investigated with the scratch injury migration test. The HUVEC cells were plated on 60mm tissue culture dishes and allowed to reach 90% confluence. After removing the medium, the cell layers were injured using a 1 ml sterile plastic pipet tip. The plates were rinsed with the culture medium. The medium with 20ng / ml of VEGF alone, or the medium with 20ng / ml of VEGF and containing the peptide P28 is added later to the plates. A plate was scratched as mentioned above and set immediately to mark the exact area of the lesion. Figure 3A. After 24 h, the cultures were fixed and stained for F-actin and nuclei using the stain Faloidina and Hoechst. The scratched areas were examined using a fluorescence microscope and photographed. The number of cells that migrated in the scraped area was counted in the control (Figure 3B) and in the dishes treated with peptide (Figure 3C). The number of HUVECs that migrated in the scratch lesion in the cells treated with P28 was approximately half of those that migrated in the scratch lesion in the control. Figure D. Consequently, the presence of P28 inhibited the motility of the HUVECs suffering from angiogenesis.

Example 4. Effects of P28 on the structural proteins of HUVEC. The effects of P28 on the structural proteins of HUVEC were studied in order to gain a better understanding of the way in which P28 affects these cells. HUVEC cells plated on coated slides coated with Matrigel® were incubated with 20ng / ml of VEGF in the presence or absence of 25 μM of P28 peptide for 4h or 24h. After incubation, the rinses were rinsed cells in PBS, fixed in buffered formalin and permeabilized in 0.2% triton in PBS. The cells were incubated with the indicated antibodies for 90 min., and if necessary, they were incubated with a specific secondary antibody, and subsequently they were mounted in DAPI containing the mounting medium. The analysis was performed with a confocal microscope (model LC510, Cari Zeiss). The proteins examined are as follows: CD-31 (protein present in the intercellular junctions that is necessary for cell-cell binding), Fak (focal adhesion kinase), Paxilin, Vinculin (critical adhesion assembly proteins), WASP (Wiskott Aldrich Syndrome protein, required for the nucleation and elongation of F-actin fibers), β-catenin (required for cell survival, regulation of cell surface proteins). In the cells with CD31 / PECAM1 detected, the location of the pronounced CD31 / PECAM was found at the cell / cell junctions in the cells treated with P28 compared to the control (Figure 4A). In the paxilina cell detected, paxilina was located mainly in the cell surface of the control cells, nevertheless it was found more often in F-actin fibers in the cells treated with P28 (Figure 4B). In the cells with detected Fak, the Fak was located mainly on the cell surface of the control cells, while it was found more often in the F-actin fibers of the cells treated with P28 (Figure 4C). In the cells with WASP detected, in 4h the location of the WASP was mainly nuclear in the control cells, whereas the WASP was located in the nucleus and on the cell surface in the cells treated with P28 (Figure 4 D). In 24h, the WASP was mainly located on the cell surface in the control cells, whereas it was located mainly in the nucleus in the cells treated with P28 (Figure 4D). In the cells with vinculin detected, the vinculin was located mainly on the cell surface in the control cells, whereas the vinculin was located more often in the fibers of F-actin in the cells treated with P28 (Figure 4E). In the cells with ß-catenin detected, in 4h, the location of the β-catenin was mainly cytoplasmic with some on the cell surface in the control cells, whereas the β-catenin was mainly localized on the cell membrane with some in the perinuclear space in cells treated with P28. Within 24 hours, the location of β-catenin was mainly in the cell membrane and in the nucleus in the control cells, whereas β-catenin was located in the cell membrane and in the perinuclear area in the cells treated with P28. Consequently, the presence of P28 prevented the structural changes normally found in the HUVECs that suffer from angiogenesis.

Example 5. Inhibition of in-vitamin growth of human melanoma cells by P28. The ability of P28 to inhibit the growth of Mel-2 cells of human melanoma in vi tro was determined. The Mel-2 cells were plated in 24-well culture plates at 10,000-12,000 cells / well and allowed to attach to the plate overnight. The cells were subsequently incubated at 37 ° C in the medium alone (MEM-E with 10% FBS) or in medium containing the P28 peptide. P28 was added in 5 μM, 50 μM, and 100 μM. The number of cells in each well was counted to Oh, 24h, 48h and 72h. The number of cells in each well was counted using a Coulter counter at the indicated time. The results show that P28 inhibits the growth of Mel-2 cells in a concentration-dependent manner. P28 inhibited the growth of the Mel-2 cell by approximately 50% in 100 μM and 24 h (Figure 5). These results indicate that P28 inhibits the growth of cancer cells, specifically human melanoma-2 cells.

Example 6. Anti-tumor activity in vivo of the P28 peptides. One million mel-2 cells were injected subcutaneously into the dorsal flank of 3-4 week old nude mice (n = 13 per group). The animals received daily i.p injections of PBS alone, 8mg, or 16mg per kg of body weight (by weight) of the P28 peptide in PBS. The animals were examined daily for the development of palpable tumors. Once the tumor developed, the size of the tumor was measured using a calibrator and the tumor volume was determined. P28 inhibited tumor incidence and growth in mice. With the treatment of 16mg / kg b.w., approximately 50% of the animals were tumor free 40 days after the mel-2 cells were injected, while only approximately 95% of the control animals had tumors 22 days after the mel-2 cells were injected ( Figure 6A). P28 also inhibited tumor growth by approximately 30% in 20 days after treatment with 16 mg / kg b.w. of P28 (Figure 6B). These results indicate that P28 can cause the delay and prevent the development of tumors, as well as retard the growth of existing tumors in vivo, and that therefore would make an effective therapeutic for the prevention of cancer and treatment in humans.

Claims (34)

1. CLAIMS Having described the invention as above, property is claimed as contained in the following. 1. An isolated peptide that is a variant, derivative or structural equivalent of a cupredoxin; and characterized in that it can inhibit angiogenesis in mammalian cells. The isolated peptide of claim 1, characterized in that the cupredoxin is selected from the group consisting of azurine, pseudoazurin, plastocyanin, rusticianin, Laz and auracyanin. 3. The isolated peptide of claim 2, characterized in that the cupredoxin is azurine. The isolated peptide of claim 1, characterized in that the cupredoxin is from an organism selected from the group consisting of Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidan, Bordetella bronchiseptica, Methylomonas sp. , Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylella fastidiosa and Vibrio parahaemolyticus 5. The isolated peptide of claim 4, characterized in that it is from Pseudomonas aeruginosa 6. The isolated peptide of claim 1, characterized in that it is part of a peptide selected from the group consisting of SEQ ID NOS: 1, 3-19. The isolated peptide of claim 1, for which a sequence selected from the group consisting of SEQ ID NOS: 1, 3-19 has at least 80% amino acid sequence identity. 8. The isolated peptide of claim 1, characterized in that it is a truncation of cupredoxin. 9. The isolated peptide of claim 8, characterized in that the peptide is more than about 10 residues and no more than about 100 residues. The isolated peptide of claim 8, characterized in that the peptide comprises a sequence selected from the group consisting of residues 50-77 of azurine of Pseudomonas aeruginosa, residues 50-67 of azurine of P. pseudomonas aeruginosa, residues 36-88 of azure from Pseudomonas .aeruginosa, and SEQ ID NOS: 20-24. The isolated peptide of claim 10, characterized in that the peptide consists of a sequence selected from the group consisting of residues 50-77 of azurine of Pseudomonas aeruginosa, residues 50-67 of azurine of Pseudomonas aeruginosa, residues 36-88 of Pseudomonas aeruginosa azurine and SEQ ID NOS: 20-24. The isolated peptide of claim 1, characterized in that the peptide comprises the residues equivalents of a cupredoxin as a region of the azure of Pseudomonas aeruginosa selected from the group consisting of residues 50-77, residues 50-67 and residues 36-88. 13. A pharmaceutical composition, characterized in that it comprises at least one cupredoxin or peptide of claim 1 in a pharmaceutically acceptable carrier. 14. The pharmaceutical composition of claim 13 characterized in that it comprises at least two of the cupredoxins or peptides. 15. The composition of claim 13, characterized in that the pharmaceutical composition is formulated for intravenous administration. The composition of claim 13, characterized in that the cupredoxin is from an organism selected from the group consisting of Pseudomonas aeruginosa, Alcaligenes faecalis, Achromobacter xylosoxidan, Bordetella bronchiseptica, Methylonionas sp. , Neisseria meningitidis, Neisseria gonorrhea, Pseudomonas fluorescens, Pseudomonas chlororaphis, Xylella fastidiosa and Vibrio parahaemolyticus 17. The composition of claim 15, characterized in that cupredoxin is from Pseudomonas aeruginosa. 18. The composition of claim 13, characterized in that the cupredoxin is selected from the group which consists of SEQ ID NOS: 1, 3-19. A method for treating a mammalian patient suffering from a condition related to inappropriate angiogenesis, characterized in that it comprises administering to the patient a therapeutically effective amount of the composition of claim 13. 20. The method of claim 19, characterized in that the patient is human. 21. The method of claim 19, characterized in that the patient suffers from cancer. The method of claim 21, characterized in that the cancer is selected from melanoma, breast, pancreas, glioblastoma, astrocytoma, and lung. 23. The method of claim 19, characterized in that the patient has a condition selected from the group consisting of macular degeneration, diabetic retinopathy, psoriasis and rheumatoid arthritis. The method of claim 19, characterized in that the pharmaceutical composition is administered by a mode selected from the group consisting of intravenous injection, intramuscular injection, subcutaneous injection, inhalation, topical administration, transdermal patch, suppository, vitreous and oral injection. 25. The method of claim 24, characterized in that the mode of administration is by intravenous injection. 26. The method of claim 21, characterized in that the pharmaceutical composition is coadministered with at least one other anti-cancer drug. The method of claim 24, characterized in that the pharmaceutical composition is administered at about the same time as another anti-cancer drug. The method of claim 19, characterized in that the pharmaceutical composition is coadministered with at least one drug selected from the group consisting of an anti-macular degeneration drug, a diabetic anti-retinopathy drug, an anti-psoriasis drug and a drug. anti-rheumatoid arthritis. 29. A kit or equipment comprising the composition of claim 13 in a vial. 30. The kit or equipment of claim 29, characterized in that the kit is dned for intravenous administration. 31. A method for studying angiogen or a condition related to inappropriate angiogen, characterized in that it comprises contacting the cells of mammals capable of developing angiogen with a cupredoxin or peptide of claim 1; and measure the degree of angiogen. 32. The method of claim 31, characterized in that the cells are human cells. 33. The method of claim 30, characterized in that the cells are HUVECs. 34. An expression vector, characterized in that it encodes the peptide of claim 1.