MXPA01004438A - Inhibition of the formation of vascular hyperpermeability - Google Patents

Abstract

Vascular hyperpermeability in individuals is a prelude to a number of physiological events that are often deleterious. Among these events is the formation of edema, diapedesis, aberrant trans-endothelial exchange, extravasation, exudation and effusion, matrix deposition (often with abnormal stromal proliferation) and vascular hypotension. Vascular hyperpermeability and the subsequent events can be inhibited by the administration of a compound that inhibits the enzyme activity of the VEGF tyrosine kinase receptor known as KDR tyrosine kinase. Preferred administered compounds selectively inhibit the function of KDR tyrosine kinase but do not block the activity of Flt-1 tyrosine kinase which is another VEGF tyrosine kinase receptor.

Description

INHIBITION OF THE FORMATION OF VASCULAR H1PERMEABILITY BRIEF DESCRIPTION OF THE INVENTION Edema can be described with an increase in the volume of interstitial fluid. This is normally an abnormal condition for which relief is typically sought. This rather frequent condition arises because the fluid leaves the blood vasculature due to an increase in endothelial permeability, frequently associated with macromolecular extravasion, and finds a new residence in the interstitial spaces. There is a diversity of physiological and biochemical mechanisms that underlie edema and edematous state formation in an individual. An important mediator in one or more of these mechanisms is "vascular endothelial cell growth". This factor upregulates transport in vascular endothelial cells, and causes an increase in the permeability of numerous vascular beds including the skin, subcutaneous tissues, peritoneal wall, mesentery, diaphragm, trachea, bronchi, duodenum and uterus. Significant diapedesis, alterations in the exchange through the endothelium, extravasion and deposition of macromolecules in these sites and prolonged hypotension may accompany these effects of increased permeability. It is thought that these processes are a prelude that facilitates neovascularization.

VEGF is expressed by inflammatory T cells, macrophages, neutrophils and eosinophils, etc. in the sites of inflammation. This factor is up-regulated by hypoxia, some vasopressor hormones, growth factors, reproductive hormones and numerous inflammatory cytokines. Vascular patency mediated by VEGF has been implicated in disorders such as tumor ascites, endometriosis, adult respiratory distress syndrome (ARDS), hypotension related to postcardiopulmonary bypass and hyperpermeability that raises blisters, edematous responses to burns and trauma, endothelial dysfunction in diabetes, complications of ovarian hyperstimulation syndrome, and ocular edema. Thus, it is apparent that the inhibition of the appearance of VEGF activity would be especially beneficial in blocking the manifestation of the disorders listed above. In particular, agents that are capable of blocking VEGF-mediated hyperpermeability and edema and associated syndromes would be useful in alleviating those disorders. Protein Tyrosine kinases. Protein tyrosine kinases (PTKs) comprise a large and diverse class of proteins that have enzymatic activity. PTKs play an important role in the control of cell growth and differentiation (for review, see Schlessinger &Ullrich, 1992, Neuron 9: 383-391).

It has been shown that aberrant stimulation, expression or mutations in PTKs lead to uncontrolled cell proliferation (eg, malignant tubular growth) or to key defects in developmental, regulatory or repair processes. Consequently, the biomedical community has spent significant resources to discover the significant biological role of members of the PTK family, its role in differentiation processes, its involvement in tumorigenesis and other diseases, the biochemical mechanisms underlying its signal transduction pathways. activated with the stimulation of ligands and the development of new drugs. Tyrosine kinases can be of the receptor type (which has extracellular, transmembrane and intracellular domains) or the non-receptor type (being completely intracellular). Tyrosine receptor kinases (RTKs). The RIKs comprise a large family of transmembrane receptors with diverse biological activities. Currently, at least nineteen (19) different RTK subfamilies have been identified. The family of tyrosine kinase receptors (RTK) includes receptors that are crucial for the growth and differentiation of a variety of cell types (Yarden and Ullrich, Ann.Rev. Biochem. 57: 433-478, 1988; Ullrich and Schlessinger, Cell 61: 243-254, 1990). The intrinsic function of the RTKs is activated with the ligand binding, which results in phosphorylation of the receptor and multiple cell substrates, and subsequently in a variety of cellular responses (Ullrich &Schlessinger, 1990, CeJI 61: 203-212) . Thus, signal transduction mediated by the receptor tyrosine kinase is initiated by extracellular interaction with a specific growth factor (ligand), typically followed by receptor dimerization, stimulation of the intrinsic protein tyrosine kinase activity and transphosphorylation of the receptor. receiver. Whereby binding sites for intracellular signal transduction molecules are created and leads to the formation of complexes with a spectrum of cytoplasmic signaling molecules that facilitate the appropriate cellular response. (eg, cell division, differentiation, metabolic effects, changes in the extracellular microenvironment) see Schlessinger and Ullrich, 1993, Neuron 9: 1-20. Proteins with SH2 (src-2 homology) or phosphotyrosine binding domains (PTB) bind tyrosine kinase receptors and their substrates with high affinity to propagate signals within cells. Both domains recognize phosphotyrosine. (SH2: Fantle et al., 1992, Cell 69: 413-423, Songyant et al., 1994, Mol.Cell. Biol. 14; 2777-2785; Songyang et al., L993, Cell 72: 767-778; and Koch et al., 1991, Science 252: 668-618; Schoelson, Curr. Opin. Chem. Biol. (1997), 1 (2), 227-234; Coburn, Curr. Opin. Struct. Biol. (1997), 7 (6), 835-838). Several intracellular substrate proteins have been identified that associate with tyrosine kinase receptors (RTKs). They can be divided into two main groups: (1) substrates that have a catalytic domain; and (2) substrates that lack such a domain but serve as adapters and are associated with catalytically active molecules (Songyang et al., 1993, Cell 72: 767-778). The specificity of the interactions between the receptors or proteins and SH2 or PTB domains of their substitutes is determined by the amino acid residues that immediately surround the phosphorylated tyrosine residue. For example, differences in the affinities between the SH2 domains and the amino acid sequences surrounding the phosphotyrosine residues on particular receptors correlate with the differences observed in their substrate phosphorylation profiles (Songyang et al., 1993, Cell 72: 161 -118). The observations suggest that the function of each tyrosine kinase receptor is determined not only by its pattern of expression and ligand availability but also by the arrangement of the downstream signal transduction pathways that are activated by a particular receptor as well as the time and duration of those stimuli. Thus, phosphorylation provides an important regulatory step that determines the selectivity of signaling pathways recruited by specific growth factor receptors, as well as differentiation factor receptors. Various tyrosine kinase receptors have been suggested, and growth factors that bind to that play a role in angiogenesis, although some may indirectly promote angiogenesis (Mustonen and Alitalo, J. Cell Biol. 129: 895-898, 1995). . A tyrosine kinase receptor, known as "fetal liver kinase" (FLK-1), is a member of the type III subclass of the RTKs. An alternative designation for human FLK-1 is "receptor containing the kinase insert domain" (KDR) (Terman et al., Oncogene 6: 1611-83, 1991). Another alternative designation for FLK-1 / KDR is "vascular endothelial cell growth factor receptor 2" (VEGFR-2) since it binds VEGF with high affinity. The murine version of FLK-1 / VEGFR-2 has also been called NYK (Oe lrichs et al, Oncogene 8 (1): 11-15, 1993). DNAs encoding mouse, rat and human FLK-1, and encoded amino acid and nucleotide sequences have been isolated (Matthews et al., Proc. Nati, Acad. Sci. USA, 88: 9026-30.1991; Terman et al., 1991, supra; Terman et al., Biochem.

Biophys. Res. Comm. 187: 1579-86, 1992; Sarzani et al. , supra; and Millauer et al. , Cell 72: 835-846, 1993). Numerous studies such as those reported in Millauer et al. , supra, suggest that VEGF and FLK-l / KDR / VEGFR-2 are a pair of ligand receptors that play an important role in the proliferation of vascular endothelial cells, and the formation and outbreak of blood vessels, called vasculogenesis and angiogenesis, respectively . Another type III subclass of RTK designated "fms-like tyrosine kinase-1" (Flt-1) is related to FLK-1 / KDR (DeVris et al., Science 255; 989-991, 1992; Shibuya et al., Oncogene 5: 519-524, 1990). An alternative designation for flt-1 is "vascular endothelial cell growth factor receptor 1" (VEGRF-1). To date, members of the FLK-l / KDR / VEGFR-2 and flt-l / VEGFK-1 subfamilies expressed mainly on endothelial cells have been found. These subclass members are specifically stimulated by members of the vascular endothelial cell growth factor (VEGF) family of ligands (Klagsburn and D'Amore, Cytokine &Growth Factor Reviews 7: 259-270, 1996). Vascular endothelial cell growth factor (VEGF) binds to Flt-1 with higher affinity than FLK-1 / KDR and is mitogenic towards vascular endothelial cells (Terman et al., 1992, supra).; Mustonen et al. supra; DeVries et al. , supra). It is believed that Flt-1 is essential for endothelial organization during vascular development. The expression of Flt-1 is associated with early vascular development in mouse embryos, and with neovascularization during wound healing (Mustonen and Alitalo, supra). The expression of Flt-1 in adult organs such as kidney glomeruli suggests an additional function for this receptor that is not related to cell growth (Mustonen and Alitalo, supra). As previously established, recent evidence suggests that VEGF plays a role in the stimulation of pathological normal angiogenesis (Jakeman et al., Endocrinology 133: 848-859, 1993; Kolch et al., Breast Cancer Research and Treatment 36: 139 -155, 1995, Ferrara et al., Endocrine Reviews 18 (1), 4-25, 1997, Ferrara et al., Regulation of Angiogenesis (ed. LD Goldberg and EM Rosen), 209-232, 1997). In addition, VEGF has been implicated in the control and improvement of vascular permeability (Connolly, et al., J. Biol. Chem. 264: 20017-20024, 1989; Brown et al., Regulation of Angiogenesis (ed. and EM Rosen), 233-29, 1997). Different forms of VEGF that arise from the alternative splicing of mRNA have been reported, which include the four species described by Ferrara et al. (J. Cell, Biochem 47: 211-218, 1991). Both VEGF species secreted and predominantly associated with cells have been identified by Ferrara et al. supra, and the protein is known to exist in the form of disulfide-linked dimers. Several related VEGF homologs have recently been identified. However, their roles in normal affective and physiological processes have not yet been elucidated. In addition, members of the VEGF family are frequently coexpressed with VEGF in a number of tissues and are, in general, capable of forming heterodimers with VEGF. This property possibly alters the specificity of the receptor and the biological effects of the heterodimers and further complicates the elucidation of their specific functions as illustrated below (Korpelainen and Alitalo, Curr Opin Cell Cell, 159-164, 1998 and references cited in the same) . Placental growth factor (PLGF) has an amino acid sequence that has significant homology to the VEGF sequence (Park et al., J. Biol. Chem. 269: 25646-54, 1994, Maglione et al., Oncogene 8: 925-31, 1993). As with VEGF different species of P1GF arise from the alternative splicing of mRNA, and the protein exists in the dimeric form (Park et al., Supra). P1GF-1 and P1GF-2 bind to Flt-1 with high affinity, and P1GF-2 also binds avidly to neuropilin-1 (Migdal et al., J. Biol. Chem. 273 (35). -22272-22278) but none is linked to FLK-1 / KDR (Park et al., supra). It has been reported that P1GF potentiates vascular permeability and the mitogenic effect of VEGF on endothelial cells when VEGF is present at low concentrations (significantly due to heterodimeric formation) (Park et al., Supra).

VEGF-B occurs as two isoforms (167) and 185 residues) that also appears to bind to Flt-l / VEGFR-1. It can play a role in the regulation of extracellular matrix degradation, cell adhesion, immigration through modulation and activity of urokinase-like plasminogen activator and plasminogen activator inhibitor 1 (Pepper et al., Proc, Nati, Acad. Sci. USA (1998), 95 (20): 11709-11714). Originally VEGF-C was cloned as a ligand for VEGFR-3 / Flt-4 which is expressed mainly by lymphatic endothelial cells. In its fully processed form, VEGF-C can also bind KDR / VEGFR-2 and stimulate the proliferation and migration of endothelial cells in vi tro and angiogenesis in in vivo models (Lymboussaki et al., Am. J. Pathol, (1998), 153 (2): 395-403; Witzenbichler et al, Am. J. Pathol. (1998), 153 (2), 381-394). Overexpression of VEGF-C causes the proliferation and enlargement of only lymphatic vessels, while the blood vessels are affected. Unlike VEGF, the expression of VEGF-C is not induced by hypoxia (Ristimaki et al, J. Biol. Chem. (1998), 273 (14), 8413-8418). The most recently discovered VEGF-D is structurally very similar to VEGF- '.. It is reported that VEGF-D binds and activates at least two "V GFRs, VEGFR-3 / Flt-4 and KDR / VEGFR-2. as an inducible mitogen c-fos for fibroblasts and is more prominently expressed in the esenchymal cells of the lung and skin (Achen et al, Proc. Nati, Acad. Sci. USA (1998), 95 (2), 548-553 and references therein) It has been claimed that VEGF-C and VEGF-D induce increases in vascular permeability in vivo in a Miles test when injected into skin tissue (PCT / US97 / 14696; WO98 / 07832, itzenbichler et al. al., supra.) The physiological and significant role of these ligands in modulating vascular hyperpermeability and endothelial responses in tissues where they are expressed remain uncertain, based on discoveries emerging from other VEGF and VEGFRs homologues and precedents for heterodimerization. of ligand and receptor, the actions of such VEGF homologs may involve the formation of heterodimers by liquefying VEGF and / or receptor heterodimerization, or binding to a VEGFR not yet discovered (itzenbichler et al., supra). Also, recent reports suggest the possible involvement of neuropilin-1 (Migdal et al, supra) or VEGFR-3 / Flt-4 (Witzenbichler et al., Supra), and other receptors other than KDR / VEGFR-2 are responsible for the induction of vascular permeability (Stacker, SA, Vitali, A., Domagala, T., Nice, E., and ilks, AF, "Angiogenesis and Cancer" Conference, Amer. Assoc. Cancer Res., Jan. 1998, Orlando, FL; Williams, Diabetelogy 40: S118-120 (1997)). Development of Compounds to modulate the PTKs. In view of the putative importance of PTKs for the control, regulation and modulation of cell proliferation, as well as the diseases and disorders associated with abnormal cell proliferation, many attempts have been made to identify "inhibitors" of receptors and non-receptors. tyrosine kinase using a variety of proposals, including the use of mutant ligand (U.S. Application No. 4,966,849), soluble receptors and antibodies (Application No. WO94 / 10202; Kendall &Thomas, 1994, Proc. Nati. Acad. Sci 90 : 10105-09; Kim et al., 1993, Nature 362: 841-844) RNA ligands (Jellinek, et al., Biochemistry 33: 10450-56; Takano, et al., 1993, Mol. Biol. Cell 4 : 358A, Kinsella et al., 1992, Exp. Cell Res. 199: 56-62; Wright, et al., 1992, J. Cellular Phys. 152: 448-57) and tyrosine kinase inhibitors (WO 94/03427 WO 92/21660, WO 91/15495, WO 94/14808, US Patent No. 5,330,992, Mariani, et al., 1994, Proc. Am. Assoc. S. 35: 2268). More recently, attempts have been made to identify small molecules that act as tyrosine kinase inhibitors. For example, monocyclic, bicyclic or heterocyclic aryl bis compounds (PCT WO 92/20642) and vinylene-azaindol derivatives (PCT WO 94/14808) have generally been described as tyrosine kinase inhibitors. Styryl compounds (U.S. Patent No. 5,217,999), pyridyl compounds substituted with styryl (Patent North American NO. 5,302,606), some quinazoline derivatives (EP Application No. 0 566 266 Al); Expert Opin.

Ther. Pat. (1998), 8 (4); 475-478, seleoindoles and selenides (PCT WO 94/03427), tricyclic polyhydroxy compounds (PCT WO 92/21660) and iPCT benzylphosphonic acid compounds WO 91/15495) have been described as compounds for use as tyrosine kinase inhibitors for use in the treatment of cancer. Anilinociaolines (PCT WO 97/34846) and compu. Quinazoline derivatives (PCT WO 97/22596; PCT WO 97/42187) have been described as inhibitors of angiogenesis and vascular permeability. In addition, attempts have been made to identify small molecules that act as inhibitors of cerin / threonine kinase. In particular, bis (indolylmaleimide) compounds have been described as particular isoforms that inhibit PKC serine / threonine kinase whose dysfunction is associated with altered vascular permeability in VEGF-related diseases (PCT WO97 / 40830, PCT WO97 / 40831). The identification of effective macromolecules and small organic compounds that specifically inhibit tyrosine signal transduction by modulating the activity of receptor and non-receptor tyrosine kinases to regulate and modulate abnormal or inappropriate cell function, cell proliferation or differentiation is therefore desirable. . In particular, the identification of methods and compounds that specifically inhibit the function of tyrosine kinases that are essential for the formation of vascular hyperpermeability leading to edema, effusions, exudates and extravasation and macromolecular deposition as well as associated disorders would be beneficial.

SUMMARY OF THE INVENTION The invention is directed to the inhibition of vascular hyperpermeability by inhibiting the cellular signaling function of KDR tyrosine kinase. This invention also provides a method for inhibiting vascular hyperpermeability by selectively disrupting the kinase catalytic response of KDR / VEGFR-2 without significantly affecting the activity of Flt-1 / VEGFR1 or other tyrosine kinases. Agents that function according to this method have a distinct pharmacological advantage over current therapeutic approaches that encompass materials such as steroids and that are prone to numerous undesirable side effects. These methods of the present invention are also preferred over the use of less specific kinase inhibitors, including those that inhibit multiple VEGF receptors, since these methods will not directly disrupt the important normal physiological function of the other kinases. As a result of the invention of vascular endothelial hyperpermeability, the subsequent formation of edema, associated diapedesis, alterations in transendothelial molecular exchange, extravasation, exudates and effusions are also inhibited by the suspension of KDR tyrosine kinase activity. Since these latter events frequently lead to excessive matrix deposition, aberrant stromal proliferation and organic dysfunction, KDR tyrosine kinase inhibition is also useful in the treatment of numerous non-cancerous disorders that share these etiological characteristics. In addition, vascular hypotension that can be caused by an activated ligand related to VEGF that binds to the KDR tyrosine kinase receptor is also decreased by inhibiting KDR tyrosine kinase activity. This invention also provides a therapeutic response for the inhibition of vascular hyperpermeability and the formation of edema in individuals by administering a compound that specifically inhibits KDR tyrosine kinase activity.

BRIEF DESCRIPTION OF THE INVENTION This invention describes a method for inhibiting vascular hyperpermeability by inhibiting the cellular signaling function of KDR tyrosine kinase. This invention also discloses a method for inhibiting vascular hyperpermeability through the use of agents that selectively inhibit the cell signaling function of KDR. Through the identification and use of highly selective KDR inhibitors that effectively block cell signaling KDR, and subsequently vascular hyperpermeability, according to the methods of this invention, the essential role of KDR in mediating the vascular hyperpermeability response to VEGF has been established. Such highly selective KDR inhibitors have shown efficiency in modulating vascular permeability, without the need to inhibit the function of the higher affinity receptor, VEGFR-1 / Fit-1. This property should allow better tolerance to therapy when current therapies or treatments are carried out with agents that less selectively disrupt the function of other non-KDR kinases. KDR tyrosine kinase is activated when the vascular endothelial cell growth factor (VEGF) or other activating ligand (such as HIV Tat protein, VEGF-C or VEGF-D) binds to a KDR tyrosine kinase receptor that lies on the surface of Vascular endothelial cells. Although naturally occurring kinase-activating mutations and interruptions have not yet been identified for KDR, they have been reported for EGFR and Tie-2 receptor kinase. As a result, KDR constitutive activation cases are also anticipated. KDR tyrosine kinase can also be referred to as FLK-1 tyrosine kinase, NYK tyrosine kinase or VEGFR-2 tyrosine kinase. In addition to stimulating angiogenesis, and endothelial cell migration and proliferation, VEGF induces hyperpermeability of blood vessels. As a result, the fluid that moves from the bloodstream passes the walls of the blood vessels into the interstitial spaces, thereby forming an area of edema. Diapedesis also frequently accompanies this response. Similarly, excessive vascular hyperpermeability can disrupt normal molecular exchange across the endothelium in critical tissues and organs (eg, lung and kidney), thereby causing organ dysfunction, macromolecular extravasation, and matrix deposition frequently with stromal proliferation. promoted. When it occurs in confined compartments, edema (for example cerebral edema) can lead to decreased organ function and damage. The function of . Cellular signaling KDR can be inhibited by a number of proposals: by blocking the production of activating ligand, by blocking the binding of activating ligand to the KDR tyrosine kinase receptor, by preventing dimerization and transphosphorylation of the receptor, by inhibiting the tyrosine enzymatic activity KDR kinase (inhibit the phosphorylation capacity of the enzyme) or by some other mechanism that interrupts its downstream signaling (D. Mukhopedhyay et al., Cancer Res. 58: 1278-1284 (1998) and references therein). According to the method described herein, such proposals that are selective for disrupting the KDR cell signaling function will reduce vascular hyperpermeability, as well as associated extravasation, subsequent edema formation and matrix deposition. There is a variety of compounds that have the KDR tyrosine kinase inhibition property requirement. Among these compounds are antibodies (hereinafter means to include constructs of single chain antibodies that bind the extracellular domain of the KDR receptor or the cell kinase enzyme portion or, alternatively, that binds the VEGF themselves.) These antibodies interfere with the binding from VEGF to the KDR tyrosine kinase receptor and / or, importantly, with the KDR tyrosine kinase signaling function The antibodies that bind to the KDR tyrosine kinase can act as VEGF antagonists or, more generally, VEGF activator antagonists. Alternatively these antibodies can block dimerization of the functional receptor or they can be KDR tyrosine kinase inhibitors.Antibodies that bind to VEGF or an activating ligand are VEGF neutralizing antibodies or activating ligand.It should be noted that such antibodies that neutralize VEGF can block the VEGF responses through the KDR and Flt-1 receivers and, you typically, they are specific for a simple activating ligand. In most cases, blockage of VEGF responses through Flt-1 receptors is neither necessary nor desirable. Since these VEGFRs have been reported to recognize different epitopes on VEGF, the desired specific blocking of KDR activation can be achieved through the use of specifically binding antibodies "masking" the KDR epitope of VEGF or another activating ligand. Other compounds that can inhibit KDR tyrosine kinase activity, and thereby decrease vascular hyperpermeability and edema formation, include peptides and organic molecules. Among the peptides is the soluble extracellular domain of KDR and fragments that bind KDR. Other useful peptides are VEGF mutants or VEGF related growth factors (eg, VEGF-C, VEGF-D or Tat HIT protein and fused proteins thereof) that bind to and block the additional ligand binding to this receptor but do not stimulate dimerization, activation or transphosphorylation of KDR tyrosine kinase. Such mutants can act as non-functional monomers or heterodimers, thereby blocking the binding of dimeric native VEGF or activating ligand. Similarly, other peptides or small molecules that block receptor dimerization and / or activation can be used successfully. These compounds also act as antagonists of activating ligands or are inhibitors of KDR tyrosine kinase activity. The preferred compounds are small organic molecules. In addition, molecules such as KDR-specific ribosines, antisense polynucleotides (such as antisense mRNA) or intracellular single chain antibodies (ScFv) that inhibit the biosynthesis or proper presentation of functional active KDR tyrosine kinase will effectively block the responses mediated by KDR to VEGF. These molecules can be introduced into the preformed cells or their production can be induced intracellularly (for example through the use of appropriate adenoviral, retroviral or vaculoviral vectors). The preferred compounds of this invention have the property of inhibiting the cellular signaling function of KDR without significantly inhibiting the cell signaling function Flt-1 (the tyrosine kinase Flt-1 is also referred to as the tyrosine kinase VEGFR-1). KDR tyrosine kinase and tyrosine kinase flt-1 are activated by binding VEGF to KDR tyrosine kinase receptors and to tyrosine kinase receptors Flt-1, respectively. Since the activity of tyrosine kinase Flt-1 can mediate important events in endothelial maintenance and vascular function, an inhibition of this enzymatic activity or associated transduced signals can lead to toxic or adverse effects. At least, such inhibition is unnecessary to block the induction of vascular hyperpermeability and the formation of edema, so that it is uneconomical and of no value to the individual. Preferred compounds of this invention are unique in that they inhibit the activity of a VEGF receptor tyrosine kinase (KDR) that is activated by activating ligands but does not inhibit other receptor tyrosine kinases, such as Flt-1, which are also activated by certain activating ligands. The most preferred compounds of this invention are, therefore, selective in their inhibitory activity of tyrosine kinase. It is known that VEGF contributes to vascular hyperpermeability and the formation of edema. VEGF is expressed by inflammatory T cells, macrophages, neutrophils and euoinophils, etc., at sites of inflammation. The production of this factor is rapidly regulated upwards by hypoxia, some vasopressor hormones, growth factors, reproductive hormones and numerous inflammatory cytokines. Indeed, vascular hyperpermeability and edema that is associated with the expression or administration of many other growth factors appears to be mediated through VEGF production. Vascular hyperpermeability, associated edema, altered transendothelial exchange and macromolecular extravasation, which is frequently accompanied by diapedesis, can result in excessive matrix deposition, aberrant stromal proliferation, fibrosis, etc. Consequently, VEGF-mediated hyperpermeability can contribute significantly to disorders with these etiological characteristics. For example: (1) VEGF is markedly increased in the skin epidermis of psoriatic lesion. This factor potently stimulates dermal endothelial cell proliferation and microvascular hyperpermeability associated with psoriasis. (2) After burns and severe scalds, many organs are frequently damaged. This seems to be manifested by an uncontrollable "disease mediator" that results from the his- tical damage of repercussion, swelling and edema of the visceral tissues, that is, endothelial cell damage. For burn victims, the wound caused by inhalation is one of the main causes of mortality. The tracheobronchial epithelium is molded and combined with a protein rich exudate to form an air passage mold that can lead to obstruction of this air passage. The combination of inhalation burn and hypoxia followed by exposure to a high concentration of oxygen (in an attempt to assist the individual) may worsen the situation by causing progressive changes in the lung, such as diffuse exudative formation, hemorrhage within the trachea and edema changes in the wall of blood vessels. Circulating levels of VEGF in serum increase dramatically (up to twenty times) in victims resulting from multiple burns and traumas and can be a major mediator of these complications (Grad et al, Clin. Chem. Lab. Med. 36: 319- 383, 1998). (3) Sunburns are also associated with the formation of edema. It is also known that the formation of VEGF is up-regulated after exposure to UV radiation. Other skin disorders where edema occurs include symptomatic erythedema that produces bladder (acrodynia), persistent acrodma, and bullous diseases such as erythema multiforme, bullous pemphigoid, and termatitis herpetiformis (ie, conditions of acute or chronic inflammation.) Edematous and rosaceous macules such as those associated with telangiectasia are additional disorders where edema is manifested. 4) Improved microvascular permeability and edema are common features of inflammatory and neoplastic disorders Brain tumors such as gliomas, where tumoral and peritumoral brain edemas and fluid-filled cysts are formed, and meningiomas with concomitant massive cerebral edema are Examples of such disorders Locally high levels of VEGF are associated with these disorders Induction of malignant ascites fluid and tumor effusions (especially pleural effusions and malignant pericardial effusions) are additional examples of such disorders that produce edema and are known to involve VEGF production. Additionally, the edema resulting from trauma to the head can produce conjunctions and decrease brain functions. Similarly, the communicating hydrocephalus has been shown to communicate cytokines such as IGF-1 and TGF-β1 known to modulate VEGF production. (5) Edema occurs in some types of chronic inflammation such as the formation of nasal polyps, cervical uterine polyps and gastric hyperplastic polyps. In such cases, inflammatory cells have been shown to play an important role in the development of these edematous states, at least in part through the production of VEGF. (6) Cytokine-activated eosinophils can be an important source of VEGF, thus contributing to the formation of tissue edema at sites of allergic inflammation. Edema and exudates are common complications that arise during allergic and delayed type hypersensitivity reactions; also frequently including anaphylaxis. VEGF is especially implicated in those reactions that do not respond to antihistamines or aspirins, and its upregulation has been observed in cases of poisoning with ivy, and contact dermatitis. In addition, tuberculosis, some viral infections, angioedema, urticaria (urticaria) and exercise-induced anaphylaxis are examples of such allergic and delayed-type hypersensitivity reactions that may also involve VEGF. Edema is also frequently formed as a result of drug sensitivity or hypersensitivity reactions, or in response to the administration of growth factor that up-regulates mixtures of VEGF or cytokines (eg, IGF-1, FGF-2, or IL-2). Radioanaphylaxis and radiodermatitis are associated with vascular hyperpermeability. (7) VEGF is involved in ocular neovascularization leading to retinoplatia and diabetic microangiopathy; blindness due to macular degeneration related to the neovascularization of the current is a major event that follows chemical burns, inflammation of the cornea and edema. Recent evidence implicates VEGF in the process after such ocular trauma. The feroxamine iron chelator has been used in the chemical treatment of cancer patients. However, this treatment often induces macular edema. The concentrations of this iron chelator that are achieved in patients induces a 3-5-fold increase in the expression of VEGF mRNA in all the normal and tumor cell lines studied, implicating VEGF as a likely mediator of edema formation . Increased intraocular pressures caused by the sole production of VEGF and edema can lead to inappropriate matrix depositions, eye distortions, disc changes, defects in the field of vision or can result in glaucoma, vascular hyperpermeability is frequently associated with conjunctivitis. (8) Chronic lung disease in neonates and adults results from lung injury and inadequate repair process. The production of VEGF has been reported in various animal models of lung injury. The destruction of pulmonary endothelial cells is also characteristic of the hyperoxic lung wound. During the recovery of hyperoxia, the VEGF mixture is up-regulated by alveolar type II cells and subsequently causes the pulmonary endothelial cells to proliferate and regenerate however, this result can cause disorganized exchange through the pulmonary endothelium and pulmonary edema. Asthma and bronchitis frequently involve bronchial vascular swelling, vascular injuritation, edema of the bronchial wall and exudates that result in thickening of the mucosa in the passage of air and narrowing of the bronchial lumen. Edema with protein exudates and aberrant stromal growth are typically intertwined with these phenomena. Through the related process, pulmonary edema is formed during adult respiratory distress syndrome. The causes of adult respiratory distress syndrome typically include pneumonia, inhalation of harmful substances, lung contusions, semi-drowned and aspiration of gastric contents. (9) Corticosteroids such as cortisone, hydrocortisone, dexamethasone or prednisolone, are among the most widely used therapeutics for edematous conditions. They are powerful inhibitors of VEGF expression. This property is now considered to contribute significantly to the well-known anti-edematous efficiency of such steroids. However, their pluripotent biological activities are also responsible for their undesirable side effects. The steroid hormones and their agonists and emtagonists also dramatically affect the production of VEGF, especially the reproductive tissues. The. Endometritis and endometriosis can occur during pregnancy, the menstrual cycle or sex hormone therapy. Swelling and cramping of menstruation are associated with vascular hyperpermeability. Tamoxifen is an agent that reduces the risk of breast cancer, it also increases uterine cell proliferation and the incidence of tumors. This analogous steroid, as well as estradiol, causes uterine edema and cell proliferation that have been shown to involve local increases in VEGF production. The ovarian hyperstimulation syndrome is a serious complication that affects the induction of ovulation. The most severe manifestation of these syndromes takes the form of massive ovarian enlargement and multiple cysts, ascites, hemoconcentration and fluid accumulation in third space. Increased capillary permeability initiated by VEGF release secreted by luteinized granulosa cells, etc. of the ovaries after stimulation with human chorionic gonadotropin is thought to play a key role in these syndromes. It has been shown that VEGF is over-expressed in the ovarian polycystic ovarian hysteretic stroma in the Stein-Leventhal syndrome. (10) A rapid differential induction of VEGF gene expression in neuronal and pial cells after transient occlusion of the middle cerebral artery has been demonstrated in animal models of attack. VEGF can contribute to the recovery of brain cells from ischemic insult, such as attack, head trauma or cerebral infarction, by boosting. neovascularization, but brain damage can also be exacerbated by the concomitant formation of cerebral edema. Malaria can also induce edema as a result of cerebral hypoxia induced by VEGF. Cerebral edema associated with brain tumor and fluid-filled cysts arise because the tumor capillaries lack the normal blood-brain barrier function. The VEGF released by the glioma cells in itself most likely accounts for the pathognomonic histopathology and clinical features of glioblastoma tumors in patients that include increased cerebral edema. Carpal tunnel syndrome is accompanied by improved nerve hydration and, frequently, by the subsequent deposition of increased extracellular matrix (capture neuropathy). Increased VEGF levels in the tissues surrounding the nerve can cause nerve capture by inducing vascular permeability, the flow of fluids and the position of the stroma within the perineural tissues. (11) VEGF production in tissue is upregulated dramatically in response to hypoxia. Consequently, the observation that in regions of necrosis, ischemia, infarction, occlusion, anemia, circulatory declines, or other oxygen deprivation, VEGF levels are increased and vascular hyperpermeability, edema and extravasation are common. The low oxygen pressure that is responsible for "altitude sickness" also induces the rapid production of VEGF which is the probable cause of life-threatening cerebral and pulmonary edema (HACE and HAPE) that can occur if a person does not acclimatize (12) The overproduction of VEGF is equally implicated in pericardial and pleural effusions caused by vascular hyperpermeability resulting from radiation injury, rheumatoid diseases, lupus, myocardial infarction, trauma or drug reaction. Not surprisingly, the overproduction of VEGF in association with pericardial and pleural effusions is commonly observed at autopsy in patients with lung or breast carcinomas, lymphomas and leukemias. The amounts of VEGF also rise significantly in the synovial fluid of inflamed joints of individuals with rheumatoid arthritis. Decay and fractures, although associated with some inflammation and vascular hyperpermeability that is beneficial to promote angiogenesis and healing, may be accompanied by undesirable, painful and excessive edema. Similarly, the involvement of VEGF is anticipated in conditions such as synovitis or meniscus injury with effusion (eg, "water in the knee"). (13) Ulcerations associated with circulatory restriction (eg, decubitus, gravitational, and varicose ulcers) are often accompanied by edema and protein exudates. . Diabetic complications frequently arise as a result of elevated circulating glucose levels (hyperglycemia) and the formation of advanced glycation end products (AGE), often accompanied by decreased circulation. These conditions, alone or in combination, are known to stimulate VEGF production and, consequently, vascular hyperpermeability can lead to numerous diabetic complications. (14) Due to the significant constitutive production of endogenous VEFG by kidney podocytes and the effects of vascular hyperpermeability known of high levels of VEGF renal disorders such as micro albuminuria, proteinuria, oliguria, electrolyte imbalance (frequently found as diabetic complications) ) and nephrotic syndrome (especially when they result from burns induced by hypoxia, shock or trauma) and can be treated according to the method of the present invention. (15) The extravasation of proteins and diapedesis, which commonly accompanies edema and leads to excessive matrix deposition and stromal proliferation, contributes to the progression of other disorders. These disorders include hyperviscosity syndrome, liver cirrhosis, fibrosis, keloid and unwanted scar tissue formation. The inhibition of VEGF mediated hyperpermeability will prevent such progression of the disease. (16) It is known that significant amounts of VEGF isoforms are stored in platelets, Mast cells, etc. and in extra cellular matrices. In certain situations, these VEGF / VPF stores can be released quickly and contribute to acute vascular hyperpermeability. From these various examples, it is readily apparent that edema occurs under a variety of physiological conditions and VEGF / VPF or a related analogue is strongly involved in the formation of edema and extravasation. The compounds of this invention minimize the edematous state associated with macular edema, aphakic / pseudoacachistic cystoid edema, retinoblastoma, ocular ischemia, disease or ocular inflammatory infection, choroidal melanoma, edematous side effect induced by iron chelation therapy, pulmonary edema, pleural effusion, pericardial effusion, myocardial infarction, rheumatoid diseases, tissue edema at sites of trauma or allergic inflammation, polyp edema at sites of chronic inflammation , cerebral edema, cysts filled with brain tumor fluid, communicating hydrocephalus, edema associated with organ damage resulting from a burn and edema resulting from inhalation damage. The compounds of this invention also decrease in the edematous state associated with skin burns, blister, erythema multiforme, edematous macules and other skin disorders, brain tumors, ascites and various effusions associated with cancers, carpal tunnel syndrome, "disease "of heights, allergies and hypersensitivity reactions, radio anaphylaxis, radio dermatitis, glaucoma, conjunctivitis, adult respiratory distress syndrome, asthma, bronchitis, ovarian hyperstimulation syndrome, polycystic ovary syndrome, menstrual swelling and cramps, attack, trauma of head, infarction or cerebral occlusion, ulcerations, sprains, fractures, effusions associated with synovitis, diabetic complications, liver cirrhosis, and the administration of growth factors. The compounds of this invention can also be used to treat microalbuminuria, proteinuria, oliguria, electrolyte imbalance, nephrotic syndrome, hyperviscosity syndrome, exudates, fibrosis, keloid and unwanted scar tissue formation. The compounds of this invention can be administered in combination with one or more additional pharmaceutical agents that inhibit or prevent!) The production of VEGF, attenuated intracellular responses. .-. ~ to VEGF, inhibit vascular hyperpermeability, r;;! -ir inflammation or inhibit or prevent the formation of edema. The compounds of the invention can be administered before, subsequent to, or concurrently with the additional pharmaceutical agent, any course of administration is appropriate. Additional pharmaceutical agents include but are not limited to anti-edema stereos, NSAIDs, rash inhibitors, anti-TNF agents, anti-IL1 agents, antihistamines, PAF antagonists, COX-1 inhibitors, CX2m inhibitors, NO synthase inhibitors, PKC, and PI3, kinase inhibitors. The compounds of the invention and the additional pharmaceutical agents act either additively or synergistica Thus, administration of such a combination of substances that inhibits vascular hyperpermeability and / or inhibits the formation of edema may provide greater relief from the deleterious effects of vascular hyperpermeability or edema than the administration of either a single substance. Since edema formation frequently results from extravasation of fluid from the bloodstream, hypotension frequently occurs as extravasation occurs. Hypotension can also occur as a result of VEGF binding or the VEGF activator to VEGF receptors in vascular endothelial cells. The compounds of this invention minimize the development of hypotension by, its occurrence, by inhibiting the cell signaling function of KDR which is a consequence of the VEGF linkage (or other activating ligand) to this receptor. The compounds of this invention inhibit hypotension in individuals when administered to the individual.

Pharmaceutical Formulations The compounds of this invention can be administered to a human patient by themselves or in pharmaceutical compositions where they are mixed by suitable carriers or excipients in doses to treat or improve vascular hyperpermeability, edema and associated disorders. Mixtures of these compounds can also be administered to the patient as a simple mixture or in suitably formulated pharmaceutical compositions. A therapeutically effective dose additionally refers to that amount of the compound or compounds sufficient to result in the prevention of edema, hyperpermeability associated with VEGF and / or progression of hypotension related to VEGF. Techniques for the formulation and administration of the compounds of the current application can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition.

Routes of Administration Suitable routes of administration may, for example, include parenteral, oral, eye drops, rectal, transmucosal, topical or intestinal administration; including injections. intramuscular, subcutaneous, intramedullary, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Alternatively, one can administer the compound in a local form rather than in a systematic manner, for example, by injecting the compound directly into an edematous site, often in a depot or sustained release formulation. In addition, one can administer the drug in a selected drug delivery system, for example, in a liposome coated with antibody specific for endothelial cell.

Composition / Formulation The pharmaceutical compositions of the present invention can be manufactured in a form that is itself known, for example, by means of conventional mixing, dissolving, granulating, dragee-making, spraying, emulsifying, encapsulating, entrapping or lyophilizing processes. . The pharmaceutical compositions for use in accordance with the present invention can thus be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds within the preparations that can be used pharmaceutically. The appropriate formulation depending on the selected administration route. For injection, the agents of the invention can be formulated in aqueous solutions, preferably in physiologically compatible regulators such as Hanks' solution, Ringer's solution, or physiological saline regulator. For transmucosal administration, appropriate penetrants to the barrier must be permeated to be used in the formulation. Such penetrants are generally known in the art. For oral administration, the compounds can be easily formulated by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers allow the compounds of the invention to be formulated as tablets, lozenges, dragees, capsules, liquids, gels, syrups, slurry, suspensions, and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by combining the active compound with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methyl cellulose, hydroxypropylmethyl cellulose, sodium carboxymethyl cellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as polyvinylpyrrolidone crosslinker, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, varnish solutions, and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Pharmaceutical preparations that can be used orally include push and fit capsules made of gelatin, as well as sealed soft capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push and fit capsules may contain the active ingredients in admixture with the filler such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. All formulations for oral administration should be in doses suitable for such administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation of pressurized packets or a nebulizer, with the use of a suitable propellant, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane , carbon dioxide or other suitable gas. In the case of pressurized aerosol, the dosage unit can be determined by providing a valve to supply a measured content. Capsules and cartridges of eg gelatin for use in an inhaler or isuflator can be formulated containing a powder mixture of the compound and a suitable powder base such as lactose or starch. The compounds can be formulated for parenteral administration by injection, for example bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, for example in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water soluble form. Additionally, suspensions of the active compounds can be prepared as appropriate suspensions for oily injection. Suitable lipophilic solvents and vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Suspensions for aqueous injection may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain stabilizers or suitable agents that increase the solubility of the compounds to allow the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg sterile pyrogen-free water, before use.

The compounds can also be formulated in rectal compositions such as suppositories or retention enemas, for example, containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds can also be formulated as a deposit preparation. Said long-lasting formulations can be administered by implantation (for example subcutaneously or intramuscularly or by intramuscular injection). Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or sparingly soluble derivatives, for example, as a sparingly soluble salt. An example of a pharmaceutical carrier for the hydrophobic compounds of the invention is a cosolvent system comprising benzyl alcohol, a non-polar surfactant, an organic water miscible polymer and an aqueous phase. The cosolvent system can be the VPD cosolvent system. The VPD is a solution of 3% w / v of benzyl alcohol, 8% w / v of the non-polar polysorbate 80 surfactant, and 65% w / v of polyethylene glycol 300, carried by volume to absolute ethanol. The VPD cosolvent system (VPD: 5W) consists of VPD diluted 1: 1 with 5% dextrose in aqueous solution. This cosolvent system suitably dissolves the hydrophobic compounds, and in itself produces low toxicity in the systemic administration. Naturally, the proportions of a cosolvent system can be varied considerably without destroying its characteristic solubility and toxicity. In addition, the identity of the cosolvent components can be varied: for example, other non-polar surfactants of low toxicity can be used in place of polysorbate 80; the size of the polyethylene glycol fraction can be varied; other biocompatible polymers can replace polyethylene glycol, for example polyvinylpyrrolidone; and other sugars or polysaccharides can be substituted for dextrose. Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of vehicles or hydrophobic drug delivery carriers. Some organic solvents such as dimethyl sulfoxide can also be used, but usually at the cost of increased toxicity. Alternatively, the compounds can be delivered using a sustained release system such as semipermeable matrices of solid hydrophobic polymers. they contain the therapeutic agent. Various sustained release materials have been established and are well known to those skilled in the art. Sustained-release capsules can, depending on their chemical nature, release the compounds for a few weeks for up to 100 days. Depending on the chemical nature and biological stability of the therapeutic reagent, additional strategies for stabilization of the protein may be employed. The pharmaceutical compositions may also comprise suitable solid phase or gel carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Many of the organic molecule compounds of the invention can be provided as salts with pharmaceutically compatible contractions. The pharmaceutically compatible salts can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. The salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.

Effective Dosage Pharmaceutical compounds suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve their intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent the development of or to alleviate the existing symptoms of the subject being treated. The determination of effective amounts is well within the ability of those skilled in the art. The effective dose of the compound inhibits the cellular signaling function of KDR sufficiently to suppress vascular hyperpermeability without causing significant adverse effects due to the inhibition of Flt-1 or other tyrosine kinase functions. Some compounds that have such activity can be identified by in vitro tests that determine the dose-dependent inhibition of KDR tyrosine kinase. Preferred compounds have an IC5o against KDR that is significantly lower than IC5o against Flt-1 or other PTK's determined under similar conditions of [ATP] / Km (ATP) and a substrate (ideally, ~ 100x selective for tyrosine KDR kinase). For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cellular tests. For example, a dose can be formulated in cellular and animal models to achieve a circulating concentration range that includes the IC50 as determined in cellular tests (ie, the concentration of test compound that achieves maximum mean inhibition of cell signaling function KDR, normally in response to VEGF or other activating stimulus). The determination of the cellular IC50 in the presence of 3 to 5% of serum albumin can approximate the binding effects of the plasma protein on the compound. Such information can be used to more accurately determine useful doses in humans. Additionally, the most preferred compounds for systemic administration effectively inhibit the KDR cell signaling function in intact cells at levels that are surely obtainable in plasma. A therapeutically effective dose refers to that amount of the compound that results in the improvement of symptoms in a patient. The toxicity and therapeutic efficiency of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example to determine the maximum tolerated dose (MTD) and ED50 (effective dose for 50% maximum response). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio between MTD and ED50. Compounds that exhibit high therapeutic indices are preferred. The data obtained from these cell culture tests and animal studies can be used to formulate a dosage range for use in humans Dosage of such compounds lies preferably within a range of circulating concentrations that include the ED5o with little or no toxicity. The dosage may vary within this range depending on the dosage form employed and the route of administration used.The exact formulation, route of administration and dosage may be selected by the particular physician in view of the condition of the patient. (See for example, Fingí et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 pl) In the treatment of crisis, the administration of an acute bolus or an infusion close to the BAT may be required to obtain a rapid response. Dosage and dosage range can be adjusted individually to provide plasma levels of an active portion that is sufficient to maintain modulating effects KDR, or minimum effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data; for example the concentration needed to achieve 50-90% inhibition of tyrosine kinase KDR using the tests described herein. The dosages necessary to achieve MEC will depend on the individual characteristics and the route of administration. However, HPLC tests or bioassays can be used to determine plasma concentrations. The dosing intervals can also be determined using the MEC value. The compounds should be administered at a regimen that maintains the plasma levels above the ECM for 10-90% of the time, preferably between 30-90% and more preferably 50-90% until the desired improvement of the symptoms is achieved. In cases of local administration or selective taking, the effective local concentration of the drug may not be related to the plasma concentration. The amount of the composition administered will of course be dependent on the subject being treated, the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

Packaging The compositions may, if desired, be presented in a package or dispensing device that may contain one or more unit dosage forms containing the active ingredient. The package can for example comprise a thin sheet of metal or plastic, such as an ampoule package. The package or distributor device can be accompanied by instructions for administration. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier can also be prepared, placed in a suitable place, and labeled for the treatment of an indicated condition. Suitable conditions indicated on the label may include the treatment of edema, inhibition of vascular hyperpermeability and extravasation, stromal deposition, minimization of VEGF-related hypotension, and the like.

EXAMPLES I. PTK In vitro Tests The following in vi tro tests can be used to determine the level of activity and effect of the different compounds of the present invention in one or more of the PTKs. Similar tests can be designed along the same lines for other tyrosine kinase using techniques well known in the art. A. Production of KDR Tyrosine Kinase Using Baculovirus System: The coding sequence for the intracellular domain of human KDR (aa789-1354) was generated by PCR using the cDNAs isolated from HUVEC cells. A poly-His6 sequence was introduced into the N-terminus of this protein as well. This fragment was cloned into the transfection vector of pVL1393 in Xba 1 and not in site 1. Recombinant baculovirus (BV) was generated through co-transfection using the BaculoGold transfection reagent (PharMingen). The recombinant BV plate was purified and verified through Western analysis. For protein production, SF-9 cells were grown in an SF-900-II medium at 2 x 106 / ml, and infected at 0.5 plaque-forming units per cell (MOI). The cells were harvested at 48 hours after infection B. Purification of KDR SF-9 cells expressing (His) 6KDR (aa789-1354) were used when adding 50 ml of Triton X-100 lysis buffer (20mM Tris, pH 8.0, 137mM NaCl, 10% glycerol, 1% Triton X-100, lmM PMSF, lOμg / ml aprotinin, lμg / ml leupeptin) to the cellular pill of cell culture. The lysate was centrifuged at 19,000 rpm in a Sorval SS-34 rotor for 30 minutes at 4 ° C. The cell lysate was applied to a sepharose chelating column with 5 ml of NiCl2, equilibrated with 50 mM of HEPES, pH7.5, 0.3 M NaCl. KDR was eluted using the same buffer containing 0.25 M imidazole. Fractions from the column were analyzed using SDS-PAGE and an ELISA test (subsequently) which measures the kinase activity. The purified KDR was exchanged within 25mM HEPES, pH7.5, 25 mM NaCl, 5 mM DTT regulator and stored at -80 ° C. C. Production and Purification of Human Tie-2 Kinase The coding sequence for the intracellular domain of human Tie-2 (aa775-1124) was generated by PCR using human placental cDNA isolates as a template. A poly-His6 sequence was introduced into the N-terminus and this construct was cloned into the transfection vector pVL 1939 in Xba 1 and Not in site 1. Recombinant BV was generated through co-transfection using the reagent BaculoGold transfection (PharMingen). It was purified by plaque and the recombinant BV was verified through Western analysis. For protein production, SF-9 insect cells were grown in an SF-900-II medium at 2 x 106 / ml, and infected at MOI of 0.5. The purification of the labeled His kinase used in the selection was analogous to that described for KDR. D. Production and Purification of Human Flt-1 Tyrosine Kinase. The baculoviral expression vector pVL1393 (Phar Mingen, Los Angeles, CA) was used. A nucleotide sequence encoding poly-His6 was placed 5 'in the region of the nucleotide encoding the entire intracellular kinase domain of human Flt-1 (amino acid 786-1338). The nucleotide sequence encoding the kinase domain was generated through PCR using cDNA libraries isolated from HUVEC cells. The histidine residues allowed affinity purification of the protein as an analogous form to that of KDR (part B) and ZAP70 (part F) SF-9 insect cells were infected at a multiplicity of 0.5 and harvested 48 hours after of the infection.

E. Tyrosine kinase sources Lck and EGFR Lck or truncated forms of Lck were obtained commercially (eg Upstate. Biotechnology Inc., Saranac Lake, NY or Santa Cruz Biotechnology, Inc., Santa Cruz CA) or purified from natural sources or recombinants: known methods using conventional methods. EGFR was purchased from Sigma (Cat # E-3641; ~ 500 units / 50μl) and the ligand: purchased from Oncogene Research Products / Calbiochem (Cat # PF011-100) F. Tyrosine Production ZAP70 kinase The baculoviral expression vector pVL1393 was used (Phar Mingen, Los Angeles, CA). A nucleotide sequence encoding poly-His6 was placed 5 'in the region of the nucleotide encoding complete ZAP70 (amino acid 1-619). The nucleotide sequence encoding the ZAP70 coding region was generated through PCR using cDNA libraries isolated from immortalized T cells Jurkat. The histidine residues allowed affinity purification of the protein (see part B). The LVPRGS bridge constitutes a recognition sequence for the proteolytic cleavage by thrombin, thereby allowing the removal of the affinity tag of the enzyme. The SF-9 insect cells were infected at a multiplicity of 0.5 and harvested 48 hours after infection.

G. Purification of ZAP70 SF-9 cells were used in a regulator containing 20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol, 1% Triton X-100, lmM PMSF, lμg / ml leupeptin, lOμg / ml aprotinin and 1 mM sodium orthovanadate. The soluble lysate was applied to a chelating column of Sepharose Hi Trap (Pharmacia) equilibrated in 50 mM HEPES, pH7.5, 0.3 M NaCl.

The fused protein was eluted with 250 mM of imidazole. The recovered enzyme was stored in a regulator containing 50 mM HEPES, pH7.5, 50 mM NaCl, and 5 mM DTT. H. Enzyme Linked Enzyme-Linked Immunosorbent Test (ELISA) for RTKs Enzyme-linked immunosorbent assays (ELISA) were used to detect and measure the presence of tyrosine kinase activity. The ELISA was conducted according to known protocols which are described in, for example, Voller, et al., 1980, "Enzyme -Linked I munosorbent Assay," In: Manual of Clinical Immunology, 2d. ed. , edited by Rose and Friedman, pp 359-371 7? M. Soc. Of Microbiology, Washintong, D.C. The described protocol was adapted to determine the activity with respect to a specific RTK. For example, a preferred protocol for conducting the ELISA experiments for KDR is provided below. The adaptation of this protocol to determine the activity of a compound for other members of the RTK family, as well as non-receptor tyrosine kinase, is well within the capabilities of those in the art. For purposes of determining inhibitor selectivity, a universal PTK substrate (eg, poly (Glu 4 Tyr) random copolymer, 20,000-50,000 MW) was employed in conjunction with ATP (typically 5 μM) at concentrations of approximately two apparent Km in the proof.

IN VITRO ELISA FOR KDR The following procedure was used to test the in vivo effect of the compounds of this invention on the KDR tyrosine kinase activity: Regulators and solutions: PGT: Poly (Glu, Tyr) 4: 1 Store the powder at -20 ° C. Dissolve the powder in phosphate buffered saline (PBS) for a 50 mg / ml solution. Store aliquots of 1 ml at -20 ° C. When making the plates, dilute to 250 μg / ml in Gibco PBS. Reaction Regulator: 100mM Hepes, 20mM MgCl2, 4mM MnCl2, 5mM DTT, 0.02% BSA, 200μM NaV04, pH 7.10 ATP: Stored aliquots of 100mM at -20 ° C.

Diluted at 20μM in water. Wash Regulator: PBS with 0.1% Tween 20 for 1 ng / μl for a total of 50 ng per well in the reactions. Store on ice. - Make 4x ATP solution at 20μM of 100 mM standard stored in water. Store on ice. - Add 50μl of the enzyme solution per well (typically 5-50 ng of enzyme / well depending on the specific activity of the kinases). - Add 25μl 4x inhibitor. - Add 25μl 4x of ATP for the inhibitor test. Incubate for 10 minutes at room temperature. - Stop the reaction to add 50μl of 0.05 N HCl per well. - Wash plate. ** Final Concentrations for Reaction: ATP: 5μM 5% DMSO 3. Antibody Link - Dilute aliquot of 1 mg / ml PY20-HRP antibody (Pierce) (one phosphotyrosine antibody) to 50 ng / ml in 0.1% BSA in PBS by a two-stage dilution (lOOx), then 200x). - Add lOOμl of Ab per well. Incubate one hour at room temperature. Incubate 1 hour at 4 ° C. - Wash plate 4x 4. Color Reaction - Prepare TMB substrate and add lOOμl per well. - Observe OD at 650 nm until 0.6 is reached. - Stop with 1M phosphoric acid. Shake on the plate reader. - Read OD immediately at 450 nm Optimal incubation times and enzymatic reaction conditions vary slightly with the enzyme preparations and are determined empirically for each batch. Analogous test conditions were used for Flt-1, Tie-2, EGFR and ZAP70. For Lck, the Reaction Regulator used was 100 mM MOPSO, pH 6.5, 4 mM MnCl2, 20 mM MgCl2, 5 mM DTT, 0.2% BSA, 200 mM NaV04 under analogous test conditions. PKC Kinase Source The catalytic ubiquity of PKC can be obtained commercially (Calbiochem). PKC Kinase Test A radioactive kinase test was used following a published procedure (Yasuda, I., Kirshimoto, A., Tanaka, S., Tominaga, M., Sakurai, A., Nishizuka, Y. Biochemical and Biophysical Research Communication 3: 166, 1220-1227 (1990)). Briefly, all reactions were performed in a regulatory kinase consisting of 50 mM Tris-HCl pH7.5, 10mM MgCl2, 2mM DTT, 1mM EGTA, 100mM ATP, 8μM peptide, 5% DMSO and 33P ATP (8Ci / mM). The compound and the enzyme were mixed in the reaction vessel and the reaction was initiated by the addition of ATP and the substrate mixture. After determination of the reaction by the addition of 10 μl of high buffer (5 mM ATP in 75 mM phosphoric acid), a portion of the mixture was labeled on the phosphocellulose filters. The labeled samples were washed three times in 75 mM phosphoric acid at room temperature for 5 to 15 minutes. The incorporation of radio brand was quantified by counting by liquid scintillation.

Estrogen Receptor Binding Assay The binding of 1 nM radiolabelled 17β-estradiol to the human estrogen receptor in the cytosol of MCF-7 mammary carcinoma cells was determined after incubation for 20 h at 4 ° C using the reaction conditions of Shein et al. , Cancer Res. 45: 4192 (1985) (incorporated herein by reference). After incubation, the cytosol fractions were mixed with a carbon suspension coated with dextran for 10 minutes at 4 ° C, centrifuged, and the supernatant was collected. The remaining bound radioactivity in the carbon supernatant was measured with a scintillation counter (LS 6000, Beckman) using a liquid scintillation cocktail (formula 989 Packard). The compounds were tested simultaneously in eight concentrations in duplicate to obtain a competition curve to quantify the inhibitory activity. The radio-specific ligand binding to the estrogen receptor was defined as the difference between the total binding and non-specific binding determined in the presence of an excess of unlabeled 17β-estradiol (6 μM).

Results The following inhibitory concentrations of a representative compound were obtained with the structural formula: They were obtained: These results demonstrate that the compounds of the present invention and exemplified herein have remarkable inhibitory activity for KDR tyrosine kinase and are particularly selective as KDR tyrosine kinase inhibitors.

II. Cellular RTK tests The following nuclear tests were used to determine the level of activity and the effect of the different compounds of the present invention on KDR. Similar tests can be designed along the same lines for other tyrosine kinases using appropriate antibody reagents and techniques such as immunoprecipitations and Western stress well known in the art. A. KDR Phosphorylation Induced by VEGF in Human Umbilical Vein Endothelial Cells (HUVEC) As Measured by Western Blots. 1. HUVEC cells (from assembled donors) were purchased from Clonetics (San Diego, CA) and cultured according to the manufacturer's instructions. Only the first steps (3-8) were used for this test. Cells were grown in 100 mm boxes (Falcon for tissue culture, Becton Dickinson, Plymouth, England) using complete EBM media (Clonetics). 2. To evaluate the inhibitory activity of a compound, the cells were trypsinized and plated at 0.5-1.0 x 10 5 cells / well in each well of 6-well cluster plates (Costar, Cambridge, MA). 3. After 3-4 days of seeding, the plates were 90-100% confluent. The medium was removed from all wells, the cells were washed with 5-10 ml of PBS and incubated 18-24h with 5 ml of EBM base medium without added supplements (ie, serum deprivation). 4. Serial dilutions of 1 ml inhibitors of EBM media (25 μM, 5 μM, or 1 μM) final cell concentration were added and incubated for 1 hour at 37 ° C. Human recombinant VEGF (165) (R & D Systems) was then added to all wells in 2 ml of EBM media at a final concentration of 50ng / ml and incubated at 37 ° C for 10 minutes. Control cells that were untreated or treated with VEGF were only used to assess the base phosphorylation and the induction of VEGF phosphorylation. 5. All wells were then washed with 5-10 ml of cold PBS containing one mM sodium orthovanadate (Sigma) and the cells were lysed and scraped in 200μl of regulator RIPA (50mM Tris-HCl) pH7, 150mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1mM EDTA) containing protease inhibitors (in 1 mM PMSF, lμg / ml aprotinin, lμg / ml pepstatin, lμg / ml leupeptin, 1 mM Na vanadate, 1 mM Na fluoride) and lμg / ml DNase (all chemists from Sigma Chemical Company, St Louis, MO). The lysate was centrifuged at 14,000 rpm for 30 minutes to remove nuclei. 6. Equal amounts of protein were then precipitated by the addition of cold ethanol (-20 ° C) (2 volumes) for a minimum of one hour or a maximum overnight. The tablets were reconstituted in Laemli sample buffer containing 5% β-mercaptoethanol (BioRad, Hercules, CA) and boiled for 5 minutes. The proteins were resolved by polyacrylamide gel electrophoresis (6%, 1.5 mm Novex, San Diego, CA) and transferred onto a nitrocellulose membrane using the Novex system. After blocking with bovine serum albumin (3%), the proteins were probed overnight with polyclonal anti-KDR antibody (C20, Santa Cruz Biotechnology, Santa Cruz, CA) or with anti-phosphotyrosine monoclonal antibody (4G10, Upstate Biotechnology , Lake Placid, NY) at 4 ° C. After washing and incubation for 1 hour with F (ab) 2 HRP conjugate of goat anti-rabbit IgG or goat anti-mouse bands were visualized using the emission chemiluminicensity system (ECL) (Amersham Life Sciences, Ariington Heigth, IL). Results The inhibitory concentrations of a com / this representative I with the structural formula: where : This compound has also demonstrated KDR tyrosine kinase selectivity (see section I). These results demonstrate that the suitable compounds of the present invention have remarkable inhibitory activity for the tyrosine-induced phosphorylation of VEGF of KDR tyrosine kinase in endothelial cells.

III. Uterine Edema Model This test measures the ability of the compounds to inhibit the acute increase in uterine weight in mice that occurs in the first few hours after stimulation with estrogen. This early emergence of uterine weight gain is known to be due to edema caused by increased permeability of the uterine vasculature. Cullinan-Bove and Koss (Endocrinology (1993), 233: 829-837) demonstrated a close temporal relationship of uterine edema stimulated by estrogen with increased expression of VEGF to mRNA in the uterus. These results have been confirmed by the use of neutralizing monoclonal antibody to VEGF that significantly reduces the acute increase in uterine weight after estrogen stimulation.

(WO / 97/42187). Consequently, this system can serve as an in vivo model for the inhibition of VEGF-mediated hyperpermeability and edema. Materials: All hormones were purchased from Sigma (St, Louis, MO) or Cal Biochem (La Jolla, CA) as lyophilized powder and prepared according to the supplier's instructions. Vehicle components (DMSO, Cremaphor EL) were purchased from Sigma (St, Louis, MO). Mice (Balb / c, 8-12 weeks old) were purchased from Taconic (Germantown, NY) and housed in a facility for pathogen-free animals according to the Institutional Animal Care and Use Committee Guidelines. Method: Day 1: Balb / c mice were given an intraperitoneal (i.p.) injection of 12.5 units of pregnant mare serum gonadotropin (PMSG). Day 3: Mice received 15 units of human chorionic gonadotropin (hCG) i.p. Day 4: Mice were randomized and divided into groups 5-10. Test compounds were administered by routes i.p., and i.v., or p.o. depending on the solubility and vehicle at doses that vary to 1-200 mg / kg. The vehicle control group received only vehicle and two groups were left untreated. Typically 30 minutes later, to the experimental groups, one vehicle and one of those without treatment were given an i.p. of 17β-estradiol (500 μg / kg). After 2-3 hours the animals were sacrificed by CO 2 inhalation. After a midline incision, each uterus was isolated and removed by cutting just below the cervix and at the junctions of the uterus and oviduct. The fat and connective tissue are removed with care so as not to disturb the integrity of the uterus before weighing it. The average weights of the treated groups were compared to the groups without treatment or treated with vehicle. The significant weights were determined by the Student test. The unstimulated control group was used to observe the estradiol response. Results Percent inhibition of uterine edema after stimulation with estradiol for a representative compound with the structural formula: It was obtained for three routes of administration at 100 mg / kg.

It has been demonstrated herein that compound I is KDR-selective for the inhibition of in vivo kinase activity and effective for blocking the cellular autophosphorylation of KDR in response to a VEGF stimulation. These results demonstrate that suitable compounds of the present invention such as compound I that selectively inhibits KDR function effectively blocks the formation of edema. The results also show that the i.v. of i.p. they are particularly effective for this compound. Significantly, similar results of anti-edemic efficiency have also been obtained with numerous structurally distinct selective inhibitors of KDR function. EQUIVALENTS While this invention has been particularly shown and described with references to the preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention. as defined by the appended claims. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein. Such equivalents are intended to be encompassed within the scope of the claims.

Claims (29)

1. CLAIMS 1. A method for inhibiting vascular hyperpermeability in an individual comprising the step of administering to the individual a compound that inhibits the cellular signaling function of KDR.
2. 2. The method of claim 1, in donation to inhibition of the cell signaling function KDK < Selective for the KDR signaling function.
3. 3. The method of claim 1, wherein the KDR cell signaling function is stimulated by the binding of an activating ligand to the KDR receptor portion.
4. 4. The method of claim 3, wherein the inhibition of the KDR cell signaling function is selective for the KDR signaling function.
5. The method of claim 1, wherein the inhibition of KDR signaling function is a process selected from the group consisting of blocking the production of an activating ligand, modulating the binding of activating ligand to the KDR tyrosine kinase receptor, disrupting receptor dimerization, block transphosphorylation of KDR, inhibit KDR tyrosine kinase activity, decrease the recruitment of intracellular KDR substrates, and interrupt downstream signaling initiated by KDR tyrosine kinase phosphorylation activity.
6. 6. The method of claim 5, wherein the inhibition of the KDR cell signaling function is selective for the KDR signaling function.
7. 7. The method of claim 1, wherein the compound inhibits. KDR kinase catalytic activity.
8. 8. The method of claim 1, wherein the compound is an antagonist of KDR tyrosine kinase activation.
9. The method of claim 1, wherein the compound selectively inhibits the phosphorylation of KDR kinase substrates.
10. The method of claim 1, wherein the compound is selective for KDR tyrosine kinase.
11. The method of claim 10, wherein the compound is selected from the group of peptides, anti-bodies, and organic molecules, and wherein the compound binds to the KDR tyrosine kinase.
12. The method of claim 11, wherein the administration of the compound inhibits the formation of an affective state selected from the group consisting of macular edema, aphakic / pseudophakic cystoid macular edema, retinoblastoma, ocular ischemia, inflammatory eye disease or infection, melanoma. choroidal, edematous side effects induced by iron chelation therapy pulmonary edema, myocardial infarction, rheumatoid diseases, anaphylaxis, tissue edema at sites of trauma and allergic inflammation, allergies, hypersensitive reactions, poly edema at sites of chronic inflammation, cerebral edema , cysts filled with brain tumor fluid, communicating hydrocephalus, carpal tunnel syndrome, organ damage resulting from a burn, burn inhalation wound, skin burns, blisters associated with sunburn, irritation or infection erythema multiforme, macules edematous and other disorders of the. skin, brain tumors, tumor effusions, carcinomas of lung or breast, ascites, pleural effusions, pericardial effusions, "disease" of the heights, radio anaphylaxis, radio dermatitis, glaucoma, conjunctivitis, choroidal melanoma, adult respiratory distress syndrome, asthma , bronchitis, ovarian hyperstimulation syndrome, polycystic ovary syndrome, menstrual swelling, menstrual cramps, attack, head trauma, cerebral infarction or occlusion hypotension, ulcerations, sprains, fractures, effusions associated with synovitis, diabetic complications, hyperviscosity syndrome, liver cirrhosis, micro albuminuria, proteinuria, oliguria, electrolyte imbalance, nephrotic syndrome, exudates, fibrosis, keloid, and the administration of growth factors.
13. The method of claim 10, wherein adverse effects associated with an alteration in the cellular signaling function of tyrosine kinases other than KDR are avoided when the compound is administered.
14. 14. The method of claim 1, wherein the compound is selected from the group consisting of single chain antibodies, ribosomes specific for KDR and antisense polynucleotides, wherein the compound is introduced or produced intracellularly thereby inhibiting the proper presentation of the functional KDR tyrosine kinase.
15. The method of claim 1, wherein the compound is administered in combination with a pharmaceutical agent selected from the group consisting of an antiedemic steroid, a Ras inhibitor, anti-TNF agents, anti-IL1 agents, an antihistamine, an antagonist of PAF, a COX-1 inhibitor, a C0X-2 inhibitor, a NO synthase inhibitor, a non-steroidal anti-inflammatory agent (NSAID), a PKC inhibitor and a Pl3 kinase inhibitor.
16. 16. A method to inhibit a process or physiological state in an individual, the process or physiological state selected from the group consisting of formation of edema, diapedesis, extravasation, effusion, exudation, ascites formation, matrix deposition and vascular hypotension, in wherein the inhibition comprises the administration of a compound that inhibits the function of KDR cell signaling.
17. 17. The method of claim 16, wherein the compound is selective for KDR tyrosine kinase.
18. 18. The method of claim 17, wherein the compound is selected from the group consisting of peptide, antibodies and organic molecules, wherein the compound binds to the KDR tyrosine kinase.
19. The method of claim 18, wherein administration of the compound inhibits the formation of affective state selected from the group consisting of macular edema, cystoid / pseudoapadic cystoid edema, retinoblastoma, ocular ischemia, inflammatory eye disease or infection, choroidal melanoma , edematous side effects induced by iron chelation therapy pulmonary edema, myocardial infarction, rheumatoid diseases, anaphylaxis, tissue edema at sites of trauma and allergic inflammation, allergies, hypersensitive reactions, poly edema at sites of chronic inflammation, cerebral edema, cysts filled with brain tumor fluid, communicating hydrocephalus, carpal tunnel syndrome, organ damage resulting from a burn, burn inhalation wound, skin burns, blisters associated with sunburn, irritation or infection erythema multiforme, edematous macules and other skin disorders, - brain tumors, tumor effusions, carcinomas of the lung or breast, ascites, pleural effusions, pericardial effusions, "disease" of the heights, radio anaphylaxis, radio dermatitis, glaucoma, conjunctivitis, choroidal melanoma, adult respiratory distress syndrome, asthma, bronchitis, ovarian hyperstimulation, polycystic ovarian syndrome, menstrual swelling, menstrual cramps, attack, head trauma, cerebral infarction or occlusion, hypotension, ulcerations, sprains, fractures, effusions associated with synovitis, diabetic complications, hyperviscosity syndrome, liver cirrhosis, micro albuminuria , proteinuria, oliéruria, electrolyte imbalance, nephrotic syndrome, exudates, fibrosis, keloid, and the administration of growth factors.
20. The method of claim 16, wherein the compound inhibits the catalytic activity of KDR kinase.
21. The method of claim 16, wherein the compound is an antagonist of KDR tyrosine kinase activation.
22. The method of claim 16, wherein the compound selectively inhibits the phosphorylation of KDR kinase substrates.
23. 23. The method of claim 16, wherein the compound is selective for KDR tyrosine kinase.
24. The method of claim 16, wherein the KDR cell signaling function is stimulated by the binding of an activating ligand to the KDR receptor portion.
25. 25. The method of claim 24, wherein the selective compound for tyrosine kinase KDR.
26. 26. The method of claim 16, wherein the compound is selected from the group consisting of single chain antibodies, ribosomes specific for KDR and antisense polynucleotides, wherein the compound is introduced or produced intracellularly thereby inhibiting proper presentation of tyrosine kinase KDR.
27. The method of claim 16, wherein the inhibition of the cell signaling function of KDR is a process selected from the group consisting of blocking the production of an activating ligand, modulating the binding of activating ligand to the receptor tyrosine kinase KDR, disorganize receptor dimerization, block trans-phosphorylation of KDR, inhibit KDR tyrosine kinase activity, decrease the recruitment of intracellular KDR substrates, and interrupt downstream signaling initiated by KDR tyrosine kinase phosphorylation activity.
28. The method of claim 16, wherein adverse effects associated with an alteration in the cellular signaling function of tyrosine kinases other than KDR are avoided when the compound is administered.
29. 29. The method of claim 16, wherein the compound is administered in combination with a pharmaceutical agent selected from the group consisting of an anti-edematous steroid, a Ras inhibitor, anti-TNF agents, anti-IL1 agents, an antihistamine. , a. PAF antagonist, a C0X-1 inhibitor, a C0X-2 inhibitor, a NO synthase inhibitor, a non-steroidal anti-inflammatory agent (NSAID), a PKC inhibitor and a Pl3 kinase inhibitor. SUMMARY OF THE INVENTION Vascular hyperpermeability and subsequent events such as macular edema, retinoblastoma, ocular ischemia, inflammatory eye disease or infection, choroidal melanoma, edematous side effects induced by iron chelation therapy pulmonary edema, myocardial infarction, rheumatoid diseases, anaphylaxis, allergies, hyperst.-nsible reactions, cerebral edema, cysts filled with brain tumor fluid, communicating hydrocephalus, carpian tunnel syndrome.), organ damage resulting from a burn, irritation or infection, erythema multiforme, edematous macules. ?, and other skin disorders, brain tumors, tumor effusions, lung or breast carcinomas, ascites, pleural effusions, pericardial effusions, "disease" of the heights, radio anaphylaxis, radio dermatitis, glaucoma, conjunctivitis, melanoma, choroidal syndrome of respiratory distress in adults, asthma, bronchitis, hyperstimulation syndrome or varicose, polycystic ovary syndrome, menstrual swelling, menstrual cramps, attack, head trauma, cerebral infarction or occlusion hypotension, ulcerations, sprains, fractures, effusions associated with synovitis, diabetic complications, hyperviscosity syndrome, liver cirrhosis, micro albuminuria, proteinuria, oliguria, electrolyte imbalance, nephrotic syndrome, exudates, fibrosis, keloid, can be inhibited by the administration of a compound that inhibits the enzymatic activity of the tyrosine kinase receptor VEGF known as KDR tyrosine kinase. The preferred compound 4,5-dihydro-3-pyridin-4-yl-l (2) H-benzo [g] indazole selectively inhibits the function of the KDR tyrosine kinase but does not block the activity of tyrosine kinase Flt-1 which is another VEGE tyrosine kinase receptor.