JP2002529421A - Methods for inhibiting vascular hyperpermeability - Google Patents

Abstract

  (57) [Summary] Vascular hyperpermeability in individuals is often a precursor to many deleterious physiological events. Among these events are edema, extravasation, abnormal transendothelial transfusion, extravasation, effusion, outflow, matrix deposition (often with abnormal proliferation), and decreased vascular blood pressure. Vascular hyperpermeability and subsequent events can be inhibited by administration of a compound that inhibits the enzymatic activity of the VEGF tyrosine kinase receptor, known as KDR tyrosine kinase. Preferred administered compounds selectively inhibit KDR tyrosine kinase, but do not block the activity of another VEGF tyrosine kinase receptor, Flt-1 tyrosine kinase.

Description

DETAILED DESCRIPTION OF THE INVENTION

Edema can be described as an increase in interstitial fluid (tissue fluid) volume. Normal,
This is an unusual condition that requires a buffer in principle. This condition often occurs because bodily fluids leave the vasculature due to increased endothelial permeability. This increase in endothelial permeability is often associated with extravasation of macromolecules, the interstitium (intertissue).
There is a new stay in the space.

[0002] There are a variety of physiological and biochemical mechanisms underlying edema and the formation of edematous conditions in individuals. An important mediator of one or more of these mechanisms is the growth (proliferation) of vascular endothelial cells. This factor up regulates the transport of vascular endothelial cells and increases its permeability in the vast endothelial bed, such as skin, subcutaneous tissue, peritoneal wall, mesentery, diaphragm, trachea, bronchi, duodenum and uterus . Significant extravasation at these sites, changes in transfusion across the endothelium, extravasation and macromolecular adhesion, and prolonged hypotension will be associated with these increased osmotic effects. These processes are considered precursors to promoting neovascularization. VEGF is expressed by ulcerative T cells, macrophages, neutrophils, eosinophils, and the like at sites of inflammation. This factor is upregulated by hypoxia, certain vasopressor hormones, growth factors, regenerative hormones and numerous inflammatory (ulcer) cell divisions. VEGF-mediated vascular permeability is associated with tumor ascites, endometriosis, adult respiratory distress syndrome (ARDS), hypotension and hyperpermeability blistering after myocardial bypass, edema response to burns and trauma, endothelial dysfunction in diabetes, ovary It is associated with diseases such as hyperstimulation syndrome and oedema of the eyes.

[0003] It is therefore clear that inhibition of VEGF production or activity would be beneficial, especially for the development of the diseases listed above. In particular, reagents capable of inhibiting VEGF-mediated hyperosmosis and edema and related syndromes would be useful for alleviating these diseases.

[0004] Protein tyrosine kinases: Protein tyrosine kinases (PTKs)
It is a large and diverse type of protein having enzymatic activity. PTKs show important roles in cell growth and differentiation (for review, see Schlessinger & Ullrich,
1992, Neuron 9: 383-391).

[0005] Aberrant stimulation, expression and mutations in PTKs have been shown to result in uncontrolled cell proliferation (eg, malignant tumor growth) or defects in underlying development, regulation or repair processes. As a result, biomedical organizations spend a great deal of effort
Specific biological roles of members of the TK family, their function in differentiation processes, their involvement in tumorigenesis and other diseases, signal transduction pathways activated by ligand stimulation and expression of novel drugs Discovered the biochemical mechanism that is the basis of the protein.

[0006] Tyrosine kinases can be of the receptor type (with extracellular, transmembrane and intracellular regions) or non-receptor type (entirely intracellular).

[0007] Receptors (Receptors) Tyrosine Kinases (RTKs): Includes a large family of transmembrane receptors with diverse biological activities. Currently, at least 19 different RTK subfamilies have been identified. The receptor tyrosine kinase (RTK) family contains receptors that are important for the growth and differentiation of various cell types (Yarden & Ullrich, Ann. Rev. Biochem. 57: 433-478, 1988; Ullri
ch & Schlessinger, Cell 61: 243-245, 1990). The intrinsic function of RTKs is activated upon ligand binding, leading to phosphorylation of the receptor and a number of cell substrates, followed by a variety of cellular responses (Ullrich & Schlessinger, 1990, Cell 6).
1: 203-212). Thus, receptor tyrosine kinase-mediated signal transduction is initiated by extracellular contact with specific growth factors (ligands), which then typically undergo receptor dimerization, stimulation of intrinsic protein kinase activity and Receptor transphosphorylation takes place. Thereby, a binding site is created for an intramolecular signal transducing molecule and has a cytoplasmic signal molecule that facilitates appropriate cellular responses (eg, cell division, differentiation, metabolic effects, changes in the extracellular microenvironment). The formation of a complex results. See Schlessinger & Ullrich, 1992, Neuron 9: 1-20.

[0008] SH2 (src-homology-2) or phosphotyrosine binding (PTB)
The proteins with regions bind to activated tyrosine kinase receptors and their high affinity substrates and transmit signals to cells. Phosphotyrosine is found in both these regions. (SH2: Fantle et al., 1992, Cell 69: 413-423;
Songyang et al., 1994, Mol. Cell. Biol. 14: 2777-2785; Songyang et al., 1
993, Cell 72: 767-787; and Koch et al., 1991, Science 252: 668-678; Schoe
lson, Curr. Opin. Biol. (1997), 1 (2), 227-234; Cowburn, Curr. Opin. Struc.
t. Biol. (1997), 7 (6), 835-838). Several intracellular matrix proteins related to receptor tyrosine kinases (RTKs) were found to be homologous. These can be divided into two main groups: (1) substrates with catalytic regions; and (2) substrates without such regions but which function as adapters and associate with catalytically active molecules (Songyang et al. ., 1993, Cell 72: 767-778). The specificity of the interaction between the receptor or protein and the SH2 or PTB region of these substrates is determined by the amino acid residues immediately surrounding the phosphorylated tyrosine residue. For example,
Differences in binding affinity between the SH2 region and the amino acid sequence surrounding the phosphotyrosine residue on a particular receptor correlate with the differences observed in the phosphorylation behavior of substrates (Songyang et al. ., 1993, Cell 72: 767-778). From the observation results,
The function of each receptor tyrosine kinase is determined not only by its expression pattern and ligand efficacy, but also by the location of specific receptors and downstream signal transduction pathways that are activated by the timing and duration of these stimuli. It has been proposed that Thus, phosphorylation provides an important regulatory step, which determines the selectivity of specific growth factor receptors and signaling pathways increased by differentiation factor receptors.

[0009] Several receptor tyrosine kinases and their associated growth factors have been suggested to play a role in angiogenesis, although some may promote indirectly ( Munsonen & Alitaro, J, Cell Biol. 129: 895-899, 1995
). One such receptor tyrosine kinase is "fetal liver kinase 1" (F
Known as LK-1), it is a member of the Type III subfamily of RTKs. Another designation for human FLK-1 is "receptor containing kinase insert region" (KDR) (Terman et al., Oncogene 6: 1677-83, 1991). FLK-1 / KDR,
Another alternative designation is "vascular endothelial cell growth factor receptor 2" (VEGFR-2).
It is. This is because FLK-1 / KDR binds VEGF with high affinity. The murine version of FLK-1 / VEGFR-2 has also been referred to as NYK (Oelrichs et al., Oncogene, 8 (1): 11-15, 1993). Mouse, rat and human FLK-1 encoding DNAs have been isolated and their nucleotide and encoded amino acid sequences reported (Matthews et al., Proc. Natl. Acad. S.
ci. USA, 88: 9026-30, 1991; Terman et. al., 1991, supra; Terman et. al., B
iochem. Biophys. Res. Comm. 187: 1579-86, 1992; Sarzani et al., supra .;
And Millauer et al., Cell 72: 835-846, 1993). Huge research, for example Millauer
et al., supra., VEGF and FLK-1 / KDR /
VEGFR-2 has been suggested to play an important role in vascular endothelial cell proliferation, blood vessel formation and sprout formation (referred to as vasculogenesis and angiogenesis, respectively).

Another type III termed “fms-like tyrosine kinase-1” (Flt-1)
Subclass RTKs are involved in FLK-1 / KDR (DeVries et al., Science
255; 989-991, 1992; Shibuya et al., Oncogene 5: 519-524, 1990). flt-
Another name for one is "Vascular Endothelial Growth Factor Receptor 1" (VEGFR-1). To date, FLK-1 / KDR / VEGFR-2 and flt-1 / VEGFR
The -1 subfamily has been found to be first expressed on endothelial cells. Members of these subclasses are specifically stimulated by members of the family of ligands, the vascular endothelial growth factor (VEGF) family (Klagsburn and D'Amor, Cytokine & Growth Factor Reviews 7
: 259-270, 1996). Vascular endothelial cell growth factor (VEGF) is FLK-1 / KD
Binds Flt-1 with a higher affinity for R and is mitogenic for vascular endothelial cells (Terman et al., 1992, supra; Mustonen et al., Supra; De)
Vries et al., Supra). Flt-1 is believed to be essential for endothelial organization during vascular development. Flt-1 expression is associated with early angiogenesis in mouse embryos and with neovascularization during wound healing (Mu
stonen and Alitalo, supra). Expression of flt-1 in adult organs such as the kidney glomerulus suggests additional functions for this receptor that are not involved in cell growth (
Mustonen and Alitalo, supra).

As noted above, recent evidence suggests that VEGF plays a role in stimulating both normal and disease-induced angiogenesis (Lakeman et al., Endo).
crinology 133: 848-859, 1993; Koloch et al., Breast Cancer Research and T
reatment 36: 139-155, 1995; Ferrara et al., Endocrine Reviews 18 (1): 4-25
, 1997; Ferrara et al., Regulation of Angiogenesis (ed. LD Goldberg an
d EM Rosen), 209-232, 1997). In addition, VEGF has been implicated in controlling and increasing vascular permeability (Connolly, et al., J. Biol. Chem. 264: 20017-20024, 19).
89; Brown et al., Regulation of Angiogenesis (ed. LD Goldberg and EM
Rosen), 233-239, 1997).

[0012] Different forms of VEGF resulting from alternative conjugation of mRNA have been reported, including the four described in Ferrara et al., (J. Cell. Biochem. 47: 211-218, 1991). I have. Both the secreting species of VEGF and the preferentially cell-associated species are Ferrar
a et al., supra, whose protein is known to exist in the form of disulfide-bonded dimers.

[0013] Several related homologs of VEGF have recently been identified. However, their role in normal physiological and disease processes remains to be elucidated.
In addition, members of the VEGF family are expressed with VEGF in many tissues and are generally able to form heterodimers with VEGF. This property tends to alter the specificity and physiological effects of the heterodimeric receptor and further complicates the elucidation of its specific function, as described below (Korpelainen and
Altitalo, Curr. Opin. Cell Biol., 159-164, 1998 and references therein).

[0014] Placental growth factor (PIGF) has an amino acid sequence that shows important homologous relationships with the VEGF sequence (Park et al., J. Biol. Chem. 269: 25646-54, 1994; Maglione e).
tal., Oncogene 8: 925-31, 1993). As with VEGF, different types of PIGF are derived from alternative (one of the two) conjugations of mRNA, with two
It exists in a dimeric form (Park et al., Supra). PIGF-1 and PIGF-2 bind to Flt-1 with high affinity and PIGF-2 also binds to neuronal pilin-1 (neuropoin).
lin-1), but binds hard (Migdal et al., Biol, Chem. 273 (35): 22272-222).
78), does not bind to FLK-1 / KDR. When VEGF is present at low concentrations (referred to as for the formation of heterodimers), PIGF will
Have been reported to enhance both the vascular hyperpermeability and mitogenic effect of Parkin (Park et al.).
al., supra).

VEGF-B is produced as two isoforms (167 and 185 residues) that also appear to bind to Flt-1 / VEGFR-1. It plays a role in regulating extracellular matrix (matrix) degradation, cell adhesion and migration by modulating the expression and activity of urokinase-type plasminogen activator and plasminogen activator inhibitor 1. (Pepper et al., Proc. Natl. Acad. Sci. US
A. (1998), 95 (20): 11709-11724).

[0016] VEGF-C is originally expressed by VEGFR-
3 / Flt-4 was cloned as a ligand. In this fully processed form, VEGF-C also binds KDR / VEGFR-2 and can stimulate endothelial cell proliferation and migration in vivo and stimulate angiogenesis in an in vivo model. (Lymboussaki et al., Am. J. Pathol. (1998), 153 (2): 395-403;
Witzenbichler et al., Am. J. Pathol. (1998), 153 (2), 391-394). VEGF
Overexpression of -C results in proliferation and expansion of lymphatic vessels only, while vessels are unaffected. Unlike VEGF, VEGF-C expression is not caused by hypoxia (Ristimalki et al, J. Bio ;. Chem. (1998), 273 (14), 8413-8418).
.

The recently discovered VEGF-D is structurally very close to VEGF-C. VEG
FD comprises at least two VEGFRs, VEGFR-3 / Flt-4 and K
It has been reported to bind and activate DR / VEGFR-2. This was originally
Cloned as a fibroblast c-fos-inducible mitogen,
Most prominently expressed in mesenchymal cells of the lung and skin (Achen et al., Proc.
Acad. USA (1998), 95 (2), 548-553; and references therein).

VEGF-C and VEGF-D are required to induce increased vascular permeability in vivo in the Miles assay when injected into the skin (PCT / US
97/14696; WO 98/07832, Witzenbichler et al., Supra.).
The physiological role and importance of these ligands in modulating vascular hyperpermeability and endothelial response in tissues remains elusive.

Based on the discovery of the neogenesis of VEGF and other homologs of VEGFRs, and the precedent of heterodimers of ligands and receptors, the action of such VEGF homologs is
It may involve the formation of F-ligand heterodimers and / or heterodimerization of the receptor, or binding to a previously undiscovered VEGFR (Witzenbi).
chler et al.supra). In a recent report, neuronal pilin-1 (Migdal et al.
supra) or VEGFR-3 / Flt-4 (Witzenbichler et al. supra) and the possibility that receptors other than KDR / VEGFR-2 are responsible for inducing vascular permeability ( Stacker, SA, Vitali, A., Domagala, T.,
Nice, E., and Wilks, AF, "Angiogenesis and Cancer" Conference, Amer.
Assoc. Cancer Res., Jan. 1998, Orlando, FL; Williams, Diabetelogia 40: S
118-120 (1997)).

Generation of compounds that modulate the PTKs. With respect to the control, regulation and regulation of cell proliferation, as well as the putative importance of PTKs for diseases and disorders associated with abnormal cell proliferation, receptor and non-receptor tyrosine kinases "inhibitors" can be identified as mutant ligands ( US Application No. 4966849), soluble receptors and antibodies (Application No. WO9)
4/10202; Kendall & Thomas, 1944, Proc. Natl. Acad. Sci 90: 10705-
09; Kim et al., 1993, Nature 362: 841-844), RNA ligands (Jellinek, et.
al., Biochemistry 33: 10450-56; Takano, et al., 1993, Mol. Bio. 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 (WO94).
WO03 / 21427; WO92 / 21660; WO91 / 15495; WO94 / 14
808; 5330992; Mariani, et al., 1994, Proc. Am. A
ssoc. Cancer Res. 35: 2268), and many attempts have been made to identify them using research methods.

[0021] More recently, attempts have been made to identify small molecules that function as tyrosine kinase inhibitors. For example, compounds of bis monocyclic, bicyclic or heterocyclic aryl (PCT-WO92 / 20642) and vinylene-azaindole derivatives (P
CT-WO94 / 14808) is generally described as a tyrosine kinase inhibitor. Styryl compounds (US Pat. No. 5,217,999), styryl-substituted pyridyl compounds (US Pat. No. 5,302,606), and specific quinazoline derivatives (E
P application no. 0566266 A1); Expert Opin. Ther. Pat. (1998), 8 (4): 4.
75-478), seleoindole and selenide (PCT-WO94 / 03427),
Tricyclic polyhydroxyl compounds (PCT-WO92 / 21660) and benzylphosphonic acid compounds (PCT-WO91 / 15495) have been described as tyrosine kinase inhibitor compounds for use in the treatment of cancer. Anilinosinoline
nocinnoline) (PCT-WO97 / 34876) and quinazoline derivative compounds (
PCT-WO 97/22596; PCT-WO 97/42187) are described as inhibiting angiogenesis and inhibiting vascular permeability.

In addition, attempts have been made to identify small molecules that act as serine / seleonin kinase inhibitors. In particular, bis (indolylmaleimide) compounds have been described as inhibiting certain PKC serine / threonine kinase variants whose dysfunction is associated with altered vascular permeability in VEGF-mediated diseases (PCT-WO97).
/ 40830; PCT-WO 97/40831).

Thus, an effective macromolecule that specifically inhibits tyrosine signal transduction by regulating the activity of receptor and non-receptor tyrosine kinases to regulate and regulate abnormal or inappropriate cell function Identification of molecules and small organic compounds is desirable.
In particular, it would be beneficial to be able to identify methods and compounds that specifically inhibit tyrosine kinase function, which is essential for the formation of edema, effusion, and macromolecular extravasation and deposition, as well as related disorders.

[Summary of the Invention] The present invention is to inhibit vascular hyperpermeability by inhibiting the cell signal function of KDR tyrosine kinase. The present invention also relates to KDR / VEGFR-2.
Provides a method of inhibiting vascular hyperpermeability by selectively disrupting the catalytic kinase response of Flt-1 / VEGFR1 or other tyrosine kinases without significantly affecting the activity. Reagents that function according to this method have pharmacological advantages over current treatment methods that use materials such as steroids that are prone to enormous side effects. These methods of the invention, including those that inhibit multiple VEGF receptors, are also preferred for the use of less specific kinase inhibitors. Because these methods would not directly perturb the important normal physiology of other kinases. Inhibition of vascular endothelial hyperpermeability results in subsequent edema formation, associated extravasation (leakage), trans-endothelial
Changes in molecular exchange, bleeding, exudation from blood vessels or lymph vessels and exudation from tissues or capillaries are also inhibited by suppression of tyrosine kinase activity of KDR. Because these latter things often result in excessive matrix deposition, aberrant matrix proliferation and organ dysfunction, the inhibition of KDR tyrosine kinases in the treatment of enormous non-cancerous diseases with these pathogenic features. It is also useful. In addition, the reduction in blood pressure that can be caused by VEGF-active ligands that bind to the KDR tyrosine kinase receptor is also minimized by inhibiting the activity of KDR tyrosine kinase.

The present invention also provides therapeutic means for inhibiting vascular hyperpermeability and inhibiting edema formation in an individual by administering a compound that specifically inhibits the activity of KDR tyrosine kinase.

DETAILED DESCRIPTION OF THE INVENTION The present invention discloses a method of inhibiting vascular hyperpermeability by inhibiting the cell signaling function of KDR tyrosine kinase. The present invention also discloses a method of inhibiting vascular hyperpermeability by utilizing a reagent that selectively inhibits the cell signaling function of KDR. In accordance with the present invention, the identification and exploitation of highly selective KDR inhibition, which effectively blocks KDR cell signaling and then vascular hyperpermeability, establishes an essential role for KDR in mediating the vascular permeability response of VEGF did. Such highly selective KDR inhibitors have demonstrated efficacy in regulating vascular permeability without inhibiting the function of the high affinity receptor, VEGFR-1 / Flt-1. This property should provide better tolerance for treatment compared to current therapies or treatments that use reagents that selectively block the function of other non-KDR kinases.

[0027] KDR tyrosine kinase is expressed by vascular endothelial cell growth factor (VEGF) or other active ligands (eg, HIV Tat protein, VEGF-C or VEGF-
D) is activated when it binds to the KDR tyrosine kinase receptor present on the surface of vascular endothelial cells. Spontaneous kinase activating mutations have not yet been identified for KDR, but they have been reported for EGFR and Ti-2 receptor kinase. Therefore, the example of the configuration activation of KDR is also ahead of schedule. KDR tyrosine kinase is FLK-1 tyrosine kinase, NYK tyrosine kinase or VE.
Also called GFR-2 tyrosine kinase.

In addition to stimulating angiogenesis and endothelial cell migration and proliferation, VEGF induces vascular hyperpermeability. As a result, bodily fluids pass through the blood vessel wall from the bloodstream,
It moves into the interstitial space, thereby forming an edema area. Extravasation (vascular) is also associated with this response. Similarly, excessive vascular hyperpermeability prevents normal molecular exchange across critical tissues and organs (eg, lungs and kidneys), thereby leading to organ dysfunction, macromolecular extravasation, and enhanced matrix growth. Causes frequent substrate deposition. Edema (eg, cerebral edema), if it occurs in a confined compartment, will result in impairment and damage to organ function.

The KDR cell signaling function can be inhibited by a number of approaches: blocking the production of the activating ligand, blocking the binding of the activating ligand to the KDR tyrosine kinase receptor, Prevention of dimerization and transphosphorylation, inhibition of the enzyme activation of KDR tyrosine kinase (inhibition of the phosphorylation capacity of the enzyme), or some other mechanism that inhibits downstream signaling (D. Mukhopedhyay et al.
al., Cancer Res. 58: 1278-1284 (1998) and references therein).
According to the methods described herein, the approach chosen to inhibit KDR cell signaling function will reduce vascular hyperosmosis, as well as associated extravasation or extravasation, followed by edema formation and matrix deposition.

There are various compounds with essential KDR tyrosine kinase inhibitory properties. Among these compounds, antibodies are those that bind to the extracellular KDR receptor region or cell kinase enzyme moiety (hereafter including the single-chain antibody configuration), or those that bind to VEGF itself. These antibodies interfere with VEGF binding to the KDR tyrosine kinase receptor and / or, importantly, KDR tyrosine kinase cell signaling function. Antibodies that bind to KDR tyrosine kinase will serve as VEGF antibodies, and more generally, as VEGF activator antagonists. Alternatively, these antibodies would block functional receptor dimerization or be KDR tyrosine kinase inhibitors. Antibodies that bind to VEGF or activating ligand are VEGF
Neutralizing antibodies for GF or activating ligand. Such VEGF neutralizing antibodies are known as K
It is noteworthy that it blocks the VEGF response via both the DR and Flt-1 receptors and will typically be specific for a single activating ligand. In most cases, blocking the VEGF response via FLt-1 is neither necessary nor desirable. As these VEGFRs are reported to recognize different epitopes on VEGF, the desired specific inhibition of KDR activation specifically binds to the KDR-binding epitope of VEGF or other activating ligand, resulting in a "mask" This can be achieved by the use of antibodies to

Other compounds that can inhibit KDR tyrosine kinase activity, thereby minimizing vascular hyperpermeability and edema formation, include peptides and organic molecules. Among the peptides, it is the soluble extracellular region of KDR and KDR binding fragments. Other useful peptides include VEGF and VEGF-related growth factors (
E.g., VEGF-C, VEGF-D or HIV-Tat protein and its fusion proteins), which bind and block another ligand that binds to this receptor, but dimerize, activate Or it does not stimulate the KDR tyrosine kinase transphosphorylation reaction. Such variants serve as monomers or non-functional heterodimers, thereby blocking the binding of native dimeric VEGF or activating ligand. Similarly, other peptides or small molecules that block receptor dimerization and / or activation can be successfully used. These compounds may also act as activating ligand antagonists or may be inhibitors of KDR tyrosine kinase activity. Small organic molecules are the preferred compounds.

In addition, KDR-specific ribozymes, antisense polynucleotides (eg, antisense mRNA), or intercellular single-chain antibodies (S _c F) that inhibit the biosynthesis or appropriate expression of active functional KDR tyrosine kinase _v ) and other molecules effectively block the KDR-mediated response of VEGF. These molecules can be inserted into preformed cells or their production can be induced intracellularly (eg, by use of an appropriate adenovirus, retrovirus or baculovirus vector).

A preferred compound of the present invention has the property of inhibiting the cell signal function of KDR without significantly inhibiting the cell signal function Flt-1 (Flt-1
Tyrosine kinases are also called VEGFR-1 tyrosine kinases). Both KDR tyrosine kinase and Flt-1 tyrosine kinase are activated by VEGF binding to the KDR tyrosine kinase receptor and the Flt-1 tyrosine kinase receptor, respectively. Since Flt-1 tyrosine kinase activity mediates key events in endothelial maintenance and vascular function, this enzyme activity or related transduction signals may result in toxic or adverse effects. Even in very small cases,
Such inhibition is not necessary to block the induction of vascular hyperpermeability and the formation of edema, so it is useless and worthless to the individual. Preferred compounds of the invention are characteristic for inhibiting the activity of one VEGF receptor tyrosine kinase (KDR). This KDR is activated by an activating ligand,
However, it does not activate other receptor tyrosine kinases (eg, Flt-1). Other tyrosine kinases are also activated by certain activating ligands. For this reason, the most preferred compounds of the invention are those that are specific in their tyrosine kinase inhibitor activity.

VEGF is known to contribute to vascular hyperpermeability and edema formation. V
EGF is expressed at sites of inflammation by inflammatory T cells, macrophages, neutrophils, eosinophils (eosinophils), and the like. The production of this factor is immediately upregulated by hypoxia, certain blood pressure increasing hormones, growth factors, regenerative hormones and numerous inflammatory cytokines. Indeed, vascular hyperpermeability and edema associated with the development or administration of many other growth factors appear to be mediated by the production of VEGF. Macromolecular leakage with vascular hyperpermeability, associated edema, altered transendothelial exchange and frequent transmigration can result in excessive matrix deposition, abnormal matrix (stromal) proliferation, fibrosis, and the like. Thus, VEGF-mediated hyperpermeability can contribute profoundly to diseases with these pathogenic features. For example: (1) VEGF is significantly increased in the epidermis of lesional psoriatic skin. This factor strongly stimulates microvascular hyperpermeability associated with skin endothelial cell proliferation and psoriasis.

(2) Major organs are frequently injured following burns and severe burns. This is likely to be accentuated by ischemia reperfusion injury, swelling and edema of internal organs, an uncontrolled "mediator disease" resulting from endothelial cell injury. For burn victims, inhaled wounds are one of the primary causes of mortality. The tracheobronchial epithelium becomes rotted and binds with protein-rich exudates, forming airway templates that can lead to airway obstruction. Exposure to high oxygen concentrations (when rescuing an individual) following inhalation burns and the complications of hypoxia causes progressive changes in the lungs (eg, extensive exudation, bleeding into the trachea and edema changes in the vessel wall) This can make the situation worse. In victims of burns and multiple trauma, circulating plasma VEGF levels increase dramatically (
And up to 20-fold), and this level may be a major mediator of these complications (Grad et al., Clin. Chem. Lab. Med. 36: 379-383, 1998).

(3) Sunburn is also associated with edema formation. It is also known that VEGF production is upregulated after UV light exposure. Other skin disorders that edema produces include blistering red edema (tip [limb] pain), persistent acroderma, and bullous disorders such as erythema polymorphism, bullous pemphigoid and There is herpes dermatitis (ie, acute or chronic inflammation). Spots of edema and roseacea, such as those associated with telangiectasia, are another disease in which edema is prominent.

(4) Enhanced microvascular permeability and edema are common features of inflammatory and neoplastic diseases. Brain tumors such as gliomas, in which neoplastic and peritumoral cerebral edema, and fluid-filled sac (vesicles) are formed, and meningeal species with massive cerebral edema,
An example of such a disease. Partial high levels of VEGF have been associated with these diseases. Induction of malignant ascites fluid and tumor exudation (particularly malignant pleural and pericardial exudation)
Is another example of such an edema-forming disease and is known to involve VEGF production. In addition, edema resulting from head trauma imparts concussion and impairs brain function. Similarly, associated hydrocephalus has been found to involve known cytokines that regulate VEGF production, such as IGF-1 and TGF-β1.

(5) Edema occurs in some types of chronic inflammation, such as the formation of nasal polyps, cervical polyps and hyperplastic polyps of the stomach. In such cases, inflammatory cells have been found to play an important role in the development of these edematous conditions, at least in part through the production of VEGF.

(6) Cytokine-activated eosinophils can be an important source of VEGF, thereby contributing to tissue edema formation at sites of allergic inflammation. Edema and effusion are common complications that occur during allergic and delayed-type hypersensitivity reactions, often with hypersensitivity. VEGF is particularly associated with these responses not responding to antihistamines or aspirin, and its upregulation is observed in cases of toxic ivy and contact dermatitis. In addition, tuberculosis, certain viral infections, angioedema, hives (
Rash) and exercise-induced hypersensitivity are examples of such allergic and delayed-type hypersensitivity reactions that can be associated with VEGF. Edema may also occur as a result of drug-sensitive or hypersensitivity reactions, or VEGF up-regulated growth factors or cytokines (eg, IGF-1
, FGF-2 or IL-2). Radiosensitivity and radiation dermatitis are associated with vascular hyperpermeability.

(7) VEGF has been implicated in ocular neovascularization leading to diabetic retinopathy and microangiopathy, blindness due to age-related macular degeneration, and neonatal blindness resulting from hyperoxia exposure. In many instances, these conditions precede plaques or other ocular edema. VEGF has been identified as iris, cornea and retinal neovascularization in cases of ocular ischemia and angioedema. VEGF-induced vascular hyperpermeability contributes to vascular-retinal barrier damage in various ocular diseases with extravasation (extravasation) and matrix deposition that underlie blood vessel (vascular) formation. Corneal neovascularization is a major outcome following chemical burn, corneal inflammation and edema. Recent evidence implicates VEGF in the process following such eye trauma. The iron chelator deferoxamine has been used for critical treatment of cancer patients. However, this treatment often causes macula edema. The concentration of iron chelates reached in the patient was determined by VEGF in all normal and tumor cell lines studied, including VEGF as a suitable mediator of edema formation.
-A 3-5 fold increase in mRNA expression. Inappropriate skin deposition, eye distortion, due to increased intraocular pressure caused by overproduction of VEGF and edema,
The optic disc (optic disc), which results in visual field defects and can lead to glaucoma. Vascular hyperpermeability is also often associated with conjunctivitis.

(8) Chronic lung disease in newborns and adults is caused by lung injury and inappropriate recovery processes. VEGF production has been reported in several animal models with lung injury. Endothelial cell destruction in lung disease is also characteristic of hyperoxic lung injury. During recovery from hyperoxia, VEGF is upregulated by alveolar type II cells, which in turn causes proliferation and regeneration of lung endothelial cells. However, this result prevents exchange across lung disease endothelium and lung disease edema. Asthma and bronchitis often include bronchial vasodilation, vascular hyperemia, edema and exudation of the bronchial walls, which thicken the airway mucosa and narrow the bronchial lumen. Edema with protein exudation and abnormal substrate growth is typically associated with these phenomena. By a related process, pulmonary disease edema is formed in adult dyspnea syndrome. Causes of adult respiratory distress syndrome are typically pneumonia, inhalation of harmful substances, lung contusion, immersion and inhalation near the contents of the stomach, and the like.

(9) Corticosteroids such as cortisone, hydrocortisone, dexamethasone or prednisolone are among the most widely used therapeutic agents for edema conditions. These are strong inhibitors of VEGF expression. This property is
It is believed that such steroids contribute significantly to the well-known anti-edema efficacy. However, these pluripotent biological activities are also responsible for unwanted side effects. Steroid hormones and their agonists and antagonists affect VEGF production, especially VEGF production in regenerating tissues. Endometritis and endometriosis can occur during pregnancy, during the menstrual cycle, or during sex hormone therapy. Menstrual swelling and convulsions are associated with vascular hyperpermeability. Tamoxifen, a drug that reduces the risk of breast cancer, also increases uterine cell proliferation and tumor incidence. Like estradiol, this steroid analog causes uterine edema and cell proliferation that have been found to be partially increased in VEGF production. Ovarian hyperstimulation syndrome is a severe complication that affects ovulation induction. The most severe manifestations of these syndromes take the form of massive ovarian enlargement and cysts, ascites, hemoconcentration and third spatial accumulation of fluid. We believe that increased capillary permeability caused by the release of VEGF secreted by ovarian luteinized granulosa cells and others after stimulation with human chorionic gonadotropin plays a major role in these syndromes Have been. Hyperthecotic ovarian matrix of polycystic ovaries in Stein-Leventhal syndrome
Stroma) was shown to overexpress VEGF.

(10) VE in both neurons and pia after transient middle cerebral artery occlusion
Rapid specific induction of GF gene expression has been demonstrated in animal models of stroke. VEG
F may contribute to the recovery of brain cells from ischemic insults (strokes) such as stroke, head trauma or cerebral infarction by enhancing vascular neoplasia, but brain damage may be caused by the concomitant formation of cerebral edema It can get worse. Malaria can also induce edema as a result of VEGF-induced cerebral hypoxia. Because tumor capillaries lack normal blood-brain barrier function, brain tumor-related brain edema and fluid-filled bladders develop. There is a strong tendency for VEGF released by gliomas in vivo to kill the characteristic histologic changes and clinical features of glioblastoma tumors in patients with increased brain edema. Carpal tunnel syndrome is accompanied by improved nerve hydration and often with subsequent increased extracellular matrix deposition (entrapment neuropathy). Increased VE in the tissue surrounding the nerve
Levels of GF can cause nerve entrapment by inducing vascular permeability, outward flux of body fluids and matrix deposition in perineural tissue.

(11) VEGF production in tissues is dramatically up-regulated in response to hypoxia. Thus, in necrosis, ischemia, infarction, obstruction, anemia, circulatory dysfunction or other hypoxia,
VEGF levels are increased and vascular hyperpermeability, edema and extravasation are common. Hypoxia, which causes "altitude sickness," also causes rapid VEGF production, which is often the cause of life-threatening cerebral and pulmonary edema (HACE and HAPE) that can occur if humans do not adapt. It is.

(12) VEGF overproduction is similarly implicated in pericardial and pleural effusion resulting from vascular hyperpermeability. This vascular hyperpermeability can lead to radiation injury,
It is caused by rheumatic disease, lupus, myocardial infarction, trauma or drug reaction. Not surprisingly, VEGF overproduction associated with pericardial and pleural effusion is commonly observed when dissecting patients with lung or breast cancer, lymphoma and leukemia. VEG
The amount of F is significantly increased also in the synovial fluid of a swollen joint in an individual with rheumatoid arthritis. Although associated with certain swelling and vascular permeability, which is beneficial in promoting angiogenesis and healing, sprains and fractures can be painful and accompanied by excessive undesirable edema. Similarly, VEGF involvement is expected in conditions such as synovitis or meniscal injury with exudation (eg, water above the knee).

(13) Restriction of circulation (eg, lying posture (bed sores), sinking ulcer and varicose ulcer)
The ulceration associated with is often accompanied by edema and protein exudate. Diabetic complications occur as a result of elevated circulating glucose levels (hyperglycemia) and the formation of advanced glycation end products (AGEs), often with defective circulation. These conditions, either alone or in combination, are known to stimulate VEGF production and thus stimulate hyperpermeability, which can result in many diabetic complications.

(14) Because of the significant component production of endogenous VEGF by renal podocytes and the known vascular hyperpermeability of elevated VEGF levels, renal defects such as microalbuminuria, proteinuria Hypovolemia, electrolyte imbalance (often encountered as a diabetic complication) and nephrotic syndrome (particularly in the case of hypoxia caused by burns, shock or trauma) can be treated according to the present invention.

(15) Protein extravasation and extravasation commonly lead to edema and excessive matrix deposition and matrix (stromal) proliferation, leading to the progression of other diseases. These diseases include hyperviscosity syndrome, cirrhosis, fibrosis, keloids and unwanted scar tissue. Inhibitors of VEGF-mediated hyperpermeability interfere with the progression of these diseases.

(16) It is known that significant amounts of VEGF isoforms are stored in platelets, mast cells, etc. and extracellular matrix. In certain situations, these VEGs
F / VPF storage is released rapidly, which causes acute vascular hyperpermeability.

From these various examples, edema occurs under various physiological conditions and VEGF
/ VPF or related analogs have been strongly implicated in edema formation and extravasation. The compounds of the present invention minimize edema conditions associated with: I.e. macular edema, aphakic / membrane cataract alveolar spot edema, retinoblastoma, ocular ischemia, ocular inflammatory disease or ocular inflammatory infection, choroidal melanoma, edema side effects caused by iron chelation therapy, pulmonary edema Pleural effusion, pericardial effusion, myocardial infarction, rheumatic disease, tissue edema at sites of trauma and allergic inflammation, polyp edema at sites of chronic inflammation, cerebral edema, fluid-filled vesicles of brain tumors, traffic heads, burns Edema associated with organ damage and edema resulting from inhalation burn injury. The compounds of the present invention also minimize edema conditions associated with: Skin burns, blisters, erythema multiforme, edema and other skin disorders, head trauma, ascites and various effusions associated with cancer, carpal tunnel syndrome, altitude sickness, allergy and hypersensitivity reactions, radiation sensitivity Disease, radiation dermatitis, glaucoma, conjunctivitis, adult respiratory distress syndrome, asthma, bronchitis, ovarian hypersensitivity syndrome, polycystic ovary syndrome, menstrual swelling and menstrual paralysis (abdominal pain), stroke, head injury, cerebral infarction or obstruction , Ulcers,
Administration of sprains, fractures, synovitis effusion, diabetic complications, hyperviscosity syndrome, cirrhosis and growth factors. The compounds of the present invention may also include microalbuminuria, proteinuria, hypovolemia, electrolyte imbalance, nephrotic syndrome, hyperviscosity syndrome, exudate (symptoms),
It can be used to treat fibrosis, keloids, and the formation of unwanted scar tissue.

Compounds of the invention inhibit or prevent the production of VEGF, attenuate the intracellular response to VEGF, inhibit vascular hyperpermeability, reduce inflammation, or inhibit edema formation Or, it can be administered in combination with one or more additional agents, to prevent it. The compounds of the present invention can be administered sequentially or concomitantly prior to drug delivery, and any route is suitable. Additional agents include, but are not limited to, anti-specific steroids, NSAIDS, ras inhibitors, anti-TNF agents, anti-IL1 agents, antihistamines, PAF-antagonists, COX-1 inhibitors, COX-2
Inhibitors, NO synthase inhibitors, mention may be made of PKC inhibitors and PI _3-kinase inhibitors. The compounds of the invention and the additional agent may act additively or synergistically. Thus, administration of a combination of such substances that inhibits the formation of vascular permeability and / or edema has a greater reduction in the adverse effects of vascular permeability or edema as compared to administration of these substances alone. Can be done.

Since the formation of edema results from extravasation of fluid from the bloodstream, hypotension often occurs, as does extravasation. Hypotension can also occur as a result of VEGF or a VEGF activator binding to the VEGF receptor on vascular endothelial cells. The compounds of the present invention have a KD which is the result of VEGF (or other active ligand) binding to the receptor.
Inhibiting the cellular signaling function of R would minimize the occurrence of hypotension. The compounds of the present invention, when administered to an individual, inhibit hypotension in the individual.

Pharmaceutical Formulations The compounds of the present invention may be administered in a pharmaceutical composition alone or in combination with a suitable carrier or excipient (s) at a dose to treat or ameliorate vascular hyperpermeability, edema and related disorders. The composition can be administered to a human patient. Mixtures of these compounds may also be administered to the patient as a simple mixture or as a suitably formulated pharmaceutical composition. Further therapeutically effective dose means the dose of the compound, i.e., the dose of the compound that is sufficient to prevent the progression of edema, VEGF-related hyperpermeability and / or VEGF-associated hypotension. Techniques for formulating and administering the compounds of the present application are described in “Remin
gton's Pharmaceutical Sciences, "Mac Publishing Corporation, Easton, PA, found in the latest edition.

Routes of Administration Suitable routes of administration include, for example, oral, ophthalmic, rectal, transmucosal, topical or intestinal administration; intramuscular, subcutaneous, intramedullary injection and subarachnoid, direct intraventricular, intravenous, peritoneal cavity Parenteral administration, such as intra-, intra-nasal or intra-ocular injection may be used.

Alternatively, the compounds can be administered partially rather than systemically, for example, by injection of the compound directly into the site of edema, often in a depot or sustained release formulation.

Further, the drug can be administered in a targeted drug administration system, for example, in liposomes coated with an endothelial cell-specific antibody.

Composition / Formulation The pharmaceutical composition of the present invention can be prepared in a manner known per se, for example by means of conventional mixing, dissolving,
It can be manufactured by granulation, sugar-coated tablets, trituration, emulsification, encapsulation, entrapping or freeze-drying processes.

The pharmaceutical composition for use according to the invention is thus one or more physiologically acceptable excipients which facilitate the processing of the active compounds into pharmaceutically usable preparations. It can be formulated in a conventional manner using carriers such as agents and auxiliaries. Proper formulation is dependent upon the route of administration chosen.

For injection, the substances of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known to those skilled in the art. Such carriers enable the compounds of the present invention to be formulated as tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient to be treated. . Pharmaceutical preparations for oral use are prepared by combining the active compound with solid vehicles and, if desired, adding suitable auxiliaries to the core of the tablets or dragees, and grinding the resulting mixture to give a granulated mixture. It can be obtained by processing. Suitable excipients are, inter alia, fillers such as lactose, sucrose, mannitol or sorbitol; sugars such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose. And / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, a concentrated sugar solution may be used, optionally containing gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, a lacquer solution and a suitable organic solvent or solvent mixture. Can be. To identify or characterize different combinations of active compound doses,
Dyestuffs or pigments may be added to the tablets or dragee coatings.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Push-fit capsules can contain the active ingredient in admixture with fillers such as lactose, binders such as starch, 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. Further stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.

For buccal administration the compositions may take the form of tablets or dragees formulated in conventional manner.

When administered by inhalation, the compounds for use according to the invention may be in the form of an aerosol spray, a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It is conveniently administered from a pressurized pack or nebulizer, which is used with the drug. In the case of a pressurized aerosol the dosage unit will be determined by providing a valve to administer the measured amount. For use in an inhaler or insufflator, capsules and cartridges of, for example, gelatin may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds can be formulated for parenteral administration by injection, eg, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, eg, in ampoules 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 may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0067] 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, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated in a depot preparation. Such long acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly or by intramuscular injection). Thus, for example, the compound can be formulated with a suitable polymeric or hydrophobic material (eg, as an emulsion of an acceptable oil) or an ion exchange resin, or as a sparingly soluble derivative, eg, as a sparingly soluble salt.

Examples of pharmaceutical carriers for the hydrophobic compounds of the present invention include benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer and a co-soluble system comprising an aqueous phase. The co-solvent system may be a VPD co-solvent system. VPD is 3% w / v benzyl alcohol,
8% w / v non-polar surfactant polysorbate 80 and 65% w / v
Solution of polyethylene glycol 300, made up with absolute ethanol.
The VPD compatibilizing system (VPD: 5W) is a 1: 1 mixture of VPD and 5% dextrose solution
Consists of one diluted with This compatibilizing system dissolves hydrophobic compounds well and as such has low toxicity upon systemic administration. Of course, the proportions of a compatibilizing system can be varied considerably without destroying its solubility and toxicity properties. In addition, the components of the compatibilizing system can themselves be varied: for example, less toxic non-polar surfactants can be used instead of polysorbate 80; the size of the polyethylene glycol fraction can be varied; Other biocompatible polymers can be substituted for polyethylene glycol, eg, polyvinylpyrrolidone; and other sugars or polysaccharides can substitute for dextrose.

Alternatively, other distribution systems for hydrophobic pharmaceutical compounds can be used. Liposomes and emulsions are well known as examples of hydrophobic drug distribution vehicles or carriers. Certain organic solvents, such as dimethyl sulfoxide, can also be used, but at the cost of usually greater toxicity. In addition, the compounds can be distributed using a sustained release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic substance. Various sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may release the compound for several weeks up to over 100 days depending on its chemical properties. Depending on the chemical properties and biological stability of the therapeutic, additional strategies for protein stabilization can be used.

The pharmaceutical compositions may also include stable solid or gel phase 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 molecular compounds of the present invention can be provided as salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts can be formed with many acids, such as, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, maleic acid, succinic acid, and the like. Salts tend to be more soluble in water or other protic solvents than in the corresponding free base form.

Effective Amount Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent or ameliorate the existing symptoms of the subject being treated. Determination of an effective amount can be well within the capability of those skilled in the art.

An effective amount of the compound inhibits the cell signaling function of KDR and sufficiently suppresses vascular hyperpermeability without causing significant adverse reactions by inhibiting Flt-1 or other tyrosine kinase functions. Certain compounds having such activity are identified as KDR
It can be identified by in vitro assays that determine dose-dependent inhibition of tyrosine kinase. The IC ₅₀ for KDR is [ATP] / K _m (A
TP) and significantly lower compound than _{IC 50} for Flt-1 or other PTK as determined under conditions similar substrate is preferred (ideally 100 times KD
R tyrosine kinase).

For any compound used in the method of the invention, the therapeutically effective amount can be estimated initially from cellular assays. A circulating concentration range that includes the IC ₅₀ as determined by cellular assays, eg, in cell and animal models (ie, the concentration of test compound that achieves 50% of maximal inhibition of cell signaling function KDR, typically in response to VEGF or other activation stimuli) Can be formulated to achieve Determination of cellular IC ₅₀ of the presence of 3-5% serum albumin, will be close to the combined effect of plasma proteins on the compound. Such information can be used to more accurately determine useful doses in humans. In addition, the most preferred compounds for systemic administration effectively inhibit cell signaling function KDR in intact cells at levels that can safely be reached in plasma.

A therapeutically effective amount refers to that amount of the compound that results in amelioration of symptoms in the patient. The toxic and therapeutic effects of such compounds can be measured by standard pharmaceutical methods in cell culture or experimental animals, such as maximum tolerated dose (MTD) and ED ₅₀ (50% of maximal response).
Effective amount that causes The dose ratio between efficacy on toxicity and treatment is the therapeutic index and it can be expressed as the ratio between MTD and ED _50.
Compounds that exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage in humans.
The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. Dosages may vary within this range depending upon the dosage form employed and the route of administration utilized. Individual physicians will be able to select the exact formulation, route of administration and dosage in light of the patient's symptoms (eg, Fin
gl et al., "The Pharmacological Basis of Th.
erapetics ”, Ch. 1, p. 1 (1975)). In emergency treatment, a rapid bolus or infusion dose near the MTD may be required to obtain a rapid response.

The dose and interval are adjusted separately to achieve a KDR modulating effect or the lowest effective concentration (ME
Plasma levels of the active part sufficient to maintain C) are obtained. The MEC will vary for each compound but can be estimated from in vitro data, for example, the concentration required to achieve 50-90% inhibition of KDR tyrosine kinase using the assays described herein. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, plasma concentrations can be determined using HPLC assays or bioassays.

Dosage intervals can also be determined using MEC value. Compounds should be administered using a regimen that maintains plasma levels above the MEC in 10-90%, preferably 30-90%, and most preferably 50-90% of the time until the desired symptom improvement is achieved. It is. In the case of local administration or selective uptake, the local concentration of the active drug may not correlate with plasma concentration.

The amount of composition administered will, of course, depend on the subject being treated, the subject's weight, the severity of the affliction,
It depends on the method of administration and the judgment of the prescribing physician.

Packaging The composition may, if desired, be obtained in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, for example a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Compositions containing a compound of the invention formulated in a compatible pharmaceutical carrier can be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. Suitable symptoms indicated on the label include treatment of edema, inhibition of vascular hyperpermeability and extravasation, matrix deposition, minimization of VEGF-related hypotension, and the like.

Example I. In Vitro PTK Assays The following in vitro assays can be used to determine the activity levels and effects of various compounds of the invention on one or more PTKs. Similar assays can be designed along the same procedure for other tyrosine kinases using techniques well known to those skilled in the art.

A. KDR tyrosine kinase production using baculovirus system In human KDR cells by PCR using cDNA isolated from HUVEC cells
A coding sequence for the domain (amino acids 789-1354) was created. Likewise
Poly-His at the N-terminus of this protein₆The sequence was introduced. This fragment to X
The transfection vector pVL1393 at the ba1 and Not1 sites
Cloned. Baculogold Transfection Reagent (Pharmin
Recombinant Baculo by co-transfection using PharMingen
Irs (BV) was produced. Plaque purification of recombinant BV for Western analysis
More confirmed. For protein production, 2x10 in SF-900-II medium ⁶ Per gram of SF-9 cells at 0.5 plaque forming units (MO
Infected in I). Cells were harvested 48 hours after infection.

B. KDR purification Triton X-100 lysis buffer (20 mM Tris, pH 8.0, 1
37 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 10 μg / ml aprotinin, 1 μg / ml leupeptin) 50
ml to the cell pellet obtained from 1 L of cell culture and add (His) ₆ KDR (
SF-9 cells expressing amino acids 789-1354) were lysed. Solval S
The lysate was centrifuged at 19000 rpm at 4 ° C. for 30 minutes in an S-34 rotor. 5 equilibrated with 50 mM HEPES, pH 7.5, 0.3 M NaCl
The cell lysate was applied to a ml NiCl ₂ chelating Sepharose column. 0.
KDR was eluted using the same buffer containing 25 M imidazole. S
Column fraction analysis was performed using DS-PAGE and an ELISA assay measuring kinase activity (below). Purified KDR was added to 25 mM HEPES, pH 7
. It was changed to 5, 25 mM NaCl and 5 mM DTT buffer and stored at -80 ° C.

C. Human Tie-2 Kinase Generation and Purification Human Tie-2 kinase was generated by PCR using cDNA isolated from human placenta as a template.
A coding sequence for two intracellular domains (amino acids 775 to 1124) was created. A poly-His ₆ sequence was introduced at the N-terminus and this construct was constructed with Xba 1 and Not.
One site was cloned into the transfection vector pVL1939. Recombinant baculovirus (BV) was produced by co-transfection using baculogold transfection reagent (Pharmingen). Recombinant B
V was plaque purified and confirmed by Western analysis. For protein production, SF-9 insect cells were grown at 2 × 10 ⁶ / ml in SF-900-II medium and infected at an MOI of 0.5. Purification of the His-tagged kinase used in the screen was performed as described for KDR.

D. Human Flt-1 tyrosine kinase generation and purification The baculovirus expression vector pVL1393 (Pharmingen, Los Angeles, CA) was used. The nucleotide sequence encoding poly-His ₆ was located 5 'of the nucleotide sequence (amino acids 786-1338) encoding the entire intracellular kinase domain of human Flt-1. The nucleotide sequence encoding the kinase domain was generated by PCR using a cDNA library isolated from HUVEC cells. KDR (Part B) and ZAP70 (Part F)
Affinity purification of proteins with histidine residues is possible in a manner similar to that described above. SF-9 insect cells were infected at a multiplicity of 0.5 and harvested 48 hours post infection.

E. Lck and EGFR tyrosine kinase sources Lck or truncated forms of Lck are commercially available (
For example, Upstate Biotechnology, Inc., Saranac Lake, New York, or Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.), Known natural or natural forms using conventional methods. Purified from a replacement source. Sigma to EGFR
(Cat # E-3641; ~ 500 units / 50 μl), and
EGF ligand was obtained from Research Products / Calbiochem (Cat
# PF011-100).

F. ZAP70 tyrosine kinase production The baculovirus expression vector pVL1393 (Fal Mingen, Los Angeles, CA) was used. The nucleotide sequence encoding poly-His ₆ was placed 5 'of the nucleotide region encoding the entire ZAP70 (amino acids 1 to 619). A nucleotide sequence encoding the ZAP70 coding region was generated by PCR using a cDNA library isolated from Jurkat immortalized T cells. Histidine residues allow for affinity purification of proteins (see Part B). The LVPRGS bridge constitutes the recognition sequence for proteolytic cleavage by thrombin, thereby removing the affinity tag from the enzyme. SF-9 insect cells were infected at a multiplicity of 0.5 and harvested 48 hours post infection.

G. Purification of ZAP70 20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 1 μg / ml leupeptin, 10 μg / ml aprotinin and 1 mM sodium orthovanadate SF-9 cells were lysed with the buffer contained. 50 mM HEPES, p
The soluble lysate was applied to a chelating Sepharose high trap column (Pharmacia) equilibrated with H7.5, 0.3 M NaCl. The fusion protein was eluted with 250 mM imidazole. The recovered enzyme was added to 50 mM HEPES, p
Stored in a buffer containing H7.5, 50 mM NaCl and 5 mM DTT.

H. Enzyme-linked immunosorbent assay (ELISA) for RTK An enzyme-linked immunosorbent assay (ELISA) was used to detect and measure the presence of tyrosine kinase activity. See, for example, Rose and Frieman, M.
annual of Clinical Immunology, 2nd edition, Am.
Sock of Microbiology, Washington DC, 359-3
71 (1980), Voller et al., "Enzyme-linked immunosorbent assay".
ELISA was performed according to the well-known protocol described in.

The disclosed protocol was adapted to determine activity for a specific RTK. A preferred protocol for performing an ELISA experiment for, for example, KDR is shown below. Adaptation of this protocol to determine the activity of compounds against other RTK family members and non-receptor tyrosine kinases is well within the ability of those skilled in the art. All PTs for the purpose of determining inhibitor selectivity
K substrate (for example, random copolymer of poly (Glu ₄ Tyr), molecular weight 2000)
0-50000) was used with a concentration of ATP (typically 5 μM) that nearly doubled the apparent Km of the assay.

KDR in In Vitro ELISA The inhibitory effect of compounds of the present invention on KDR tyrosine kinase activity was assayed using the following method.

Buffers and Solutions Store the PGT: Poly (Glu, Tyr) 4: 1 powder at -20 ° C. Dissolve the powder in phosphate buffered saline (PBS) to make a 50 mg / ml solution. Store 1 ml aliquots at -20 <0> C. Dilute to 250 μg / ml with Gibco PBS when making plates.

Reaction buffer: 100 mM Hepes, 20 mM MgCl ₂ , 4 mM MnCl ₂ , 5 mM DTT, 0.02% BSA, 200 μM NaVO ₄ , pH 7.10 ATP: Store 100 mM aliquots at −20 ° C. Dilute to 20 μM with water.

Wash buffer: PBS containing 0.1% Tween 20.

Antibody dilution buffer: 0.1% bovine serum albumin (BSA) in PBS.

TMB Substrate: Mix TMB substrate and peroxide solution 9: 1 immediately before use or use Kay Blue substrate of neogen.

Stop solution: 1 M phosphoric acid.

Procedure 1. Plate preparation Dilute the PGT stock (50 mg / ml, frozen) to 250 μg / ml with PBS. Corning Modified Flat Bottom High Affinity ELISA Plate (Corning #
Add 125 μl per well (25805-96). Add 125 μl of PBS to blank wells. Cover with sealing tape and incubate at 37 ° C overnight. Wash once with 250 μl of wash buffer and dry in a 37 ° C. dry incubator for about 2 hours. Store the coated plate in a sealed bag at 4 ° C. until use.

[0100] 2. Tyrosine kinase reaction:-Prepare a 4x concentration of inhibitor solution in 20% DMSO in water. -Prepare the reaction buffer. Prepare the enzyme solution and make the desired units in 50 μl, for example for KDR, 1 ng / μl in the reaction solution to a total volume of 50 ng per well. Store on ice. -Bring 4x ATP solution to 20 [mu] M from a 100 mM stock solution in water. Store on ice. Add 50 μl enzyme solution per well (typically 5-50 ng enzyme / well depending on the specific activity of the kinase) -Add 25 [mu] l of 4x inhibitor. -Add 25 [mu] l of 4x ATP for the inhibitor assay. -Incubate for 10 minutes at room temperature. Stop the reaction by adding 50 μl of 0.05 N HCl per well. -Wash the plate. ** Final concentration for reaction ATP: 5 μM 5% DMSO

3. Antibody binding-Dilute a 1 mg / ml aliquot of PY20-HRP (Pierce) antibody (phosphotyrosine antibody) to 50 ng / ml with 0.1% BSA in PBS by two-step dilution (100x then 200x). -Add 100 [mu] l antibody per well. Incubate for 1 hour at room temperature. Incubate at 4 ° C for 1 hour. -Wash the plate 4 times.

[0102] 4. Color reaction-Prepare TMB substrate and add 100 μl per well. -Monitor and monitor the OD at 650 nm until 0.6. -Stop with 1M phosphoric acid. Shake on plate reader. -Read the OD at 450 nm immediately.

The optimal incubation times and enzyme reaction conditions will vary slightly for the enzyme preparation, but will be determined empirically for each lot.

Similar assay conditions are used for Flt-1, Tie-2, EGFR and ZAP70. For Lck, the reaction buffer used was 100 mM MOPSO, pH 6.5, 4 mM MnCl ₂ , 20 mM MgCl ₂ , 5 mM DTT, 0.2% BSA, 200 mM NaVO ₄ under similar assay conditions. .

PKC Kinase Source The catalytic subunit of PKC was obtained commercially (Calbiochem).

PKC Kinase Assay According to established methods (Yasuda, I., Kirshimoto, A .;
Tanaka, S .; , Tominaga, M .; Sakurai, A .; , Ni
Shizuka, Y .; , Biochemical and Biophysic
al Research Communication, 3 (166): 122.
0-1227 (1990)) radioactive kinase assay. Briefly, all reactions were performed with 50 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ ,
mM DTT, 1 mM EGTA, 100 μM ATP, 8 μM peptide, 5 mM
% DMSO and ³³ P ATP (8 Ci / mM) in a kinase buffer. The compound and enzyme were mixed in the reaction vessel, and ATP and the substrate mixture were added to start the reaction. Subsequently, 10 μl of stop buffer (5 mM ATP in 75 mM phosphoric acid) was added to terminate the reaction, and a part of the mixture was spotted on a phosphocellulose filter. The spotted sample was washed three times in 75 mM phosphoric acid at room temperature for 5 to 15 minutes. Radiolabel incorporation was quantified by liquid scintillation counting.

Estrogen Receptor Binding Assay 1 for MCF-7 Mammalian Cancer Cell Cytosolic Human Estrogen Receptor
Binding of nM radiolabeled 17β-estradiol was determined by Shein et al., Cance.
r Res. , 45: 4192 (1985) (incorporated herein by reference) and determined after incubation for 20 hours at 4 ° C. After incubation, the cytosolic fraction was mixed with a dextran-coated charcoal suspension at 4 ° C for 10 minutes, centrifuged and the supernatant was collected. The bound radioactivity remaining in the charcoal supernatant was measured with a scintillation counter (LS6000, Beckman) using a liquid scintillation cocktail (Formula 989 Packard). The compounds were tested simultaneously at 8 concentrations in duplicate and a competition curve was obtained to quantify the inhibitory activity. Specific radioligand binding to the estrogen receptor was defined as the difference between total binding and non-specific binding determined in the presence of excess unlabeled 17β-estradiol (6 μM).

Results Inhibitory concentrations of representative compounds having the following structural formula were obtained:

[0109]

Embedded image

[Table 1]

These results indicate that the compounds of the present invention and shown in the examples herein have significant KDR tyrosine kinase inhibitory activity and are particularly selective as KDR tyrosine kinase inhibitors.

II. Cellular RTK Assay The following cellular assays were used to determine the activity levels and effects on KDR of various compounds of the invention. Similar assays can be designed along the same procedure for another tyrosine kinase using appropriate antibodies, reagents and techniques such as immunoprecipitation and western blotting, which are well known to those skilled in the art.

A. Human umbilical vein endothelial cells (H
VEGF-induced KDR phosphorylation in UVEC) HUVEC cells (pooled from donor) were purchased from Clontech (San Diego, CA).
(California) and cultured according to the manufacturer's instructions. Early passage (
Only 3 to 8) were used in this assay. Cells were cultured in 100 mm culture dishes (Falcon for tissue culture, Becton Dickinson, Primos, UK) using complete EBM medium (Clontech).

[0113] 2. To evaluate the inhibitory activity of the compounds, cells were trypsinized and seeded at 0.5-1.0 × 10 ⁵ cells / well in each well of a 6-well cluster plate (Costar, Cambridge, Mass.).

[0114] 3. Plates were grown 90-100% confluent 3-4 days after seeding. Media was removed from all wells, cells were rinsed with 5-10 ml of PBS, and incubated for 18-24 hours with 5 ml of EBM basal medium without supplements (ie, serum depleted).

[0115] 4. Serial dilutions of the inhibitor in 1 ml of EBM medium are added to the cells (final concentration 2
5 μM, 5 μM or 1 μM) and incubated at 37 ° C. for 1 hour. Next, human recombinant VEGF ₍₁₆₅₎ (R & G Systems) was added to EBM2.
A final concentration of 50 ng / ml was added to all wells containing ml and incubated at 37 ° C for 10 minutes. Phosphorylation background and phosphorylation induction by VEGF were evaluated using control cells untreated or treated with VEGF alone.

[0116] 5. Then cold P containing 1 mM sodium orthovanadate (Sigma)
Rinse all the wells with 5-10 ml of BS, lyse the cells, and remove the protease inhibitor (1 mM PMSF, 1 μg / ml aprotinin, 1 μg / ml pepstatin, 1 μg / ml leupeptin, 1 mM sodium vanadate, 1 mM
RIPA buffer (50 mM Tris-HCl, pH 7, 150 mM NaCl, 1% NP) containing 1 μg / ml DNase
-40, 0.25% sodium deoxycholate, 1 mM EDTA (all chemicals obtained from Sigma Chemical Company, St. Louis, Mo.). The lysate was centrifuged at 14000 rpm for 30 minutes to remove nuclei.

[0117] 6. Equivalent protein was then precipitated by addition of cold (−20 ° C.) ethanol (2 volumes) for a minimum of 1 hour or a maximum of overnight. The pellet was reconstituted in Laemli sample buffer containing 5% β-mercaptoethanol (Bio-Rad, Hercules, Calif.) And boiled for 5 minutes. Proteins were analyzed by polyacrylamide gel electrophoresis (6%, 1.5 mm Novex, San Diego, CA) and transferred to nitrocellulose membranes using the Novex system. After blocking with bovine serum albumin (3%), anti-KDR polyclonal antibody (C20, Santa Cruz Biotechnology, Santa Cruz, Calif.) Or anti-phosphotyrosine monoclonal antibody (4G10, Upstate Biotechnology, Lake Placid, 4 ° C using New York State)
For overnight. After washing and incubation with HRP-conjugated F (ab) 2 of goat anti-rabbit or goat anti-mouse IgG for 1 hour, bands were visualized using a chemiluminescent system (ESL) (Amersham Life Sciences, Arlington Heights, Ill.). did.

Results The inhibitory concentrations of a representative compound I having the following structural formula are as follows:

[0119]

Embedded image

[Table 2]

This compound also exhibits KDR tyrosine kinase selectivity (see Section I)
.

These results indicate that suitable compounds of the invention have significant inhibitory activity on VEGF-induced tyrosine phosphorylation of KDR tyrosine kinase in endothelial cells.

III. Uterine edema model This assay measures the ability of compounds to block the rapid increase in uterine weight in mice that occurs during the first few hours after estrogen stimulation. It has been found that the initial manifestation of this increase in uterine weight is due to edema caused by increased permeability of the uterine vasculature. Cullinan-Bove and Koss (Endocr
Inology, 133: 829-837 (1993)) has shown that there is a close transient relationship between estrogen-stimulated uterine edema and increased expression of VEGF mRNA in the uterus. These results have been confirmed by using a neutralizing monoclonal antibody against VEGF that significantly reduces the sudden increase in uterine weight following estrogen stimulation (WO 97/42187). Thus, this system can be presented as an in vivo model for inhibiting VEGF-mediated hyperosmosis and edema.

Materials: All hormones were purchased from Sigma (St. Louis, Mo.) or Cal Biochem (La Jolla, Calif.) As lyophilized powders and prepared according to the supplier's instructions.

The vehicle components (DMSO, Cremaphor EL) were purchased from Sigma (St. Louis, Mo.).

Mice (Bulb / c, 8-12 weeks old) were purchased from Taconic (Germantown, NY) and housed in sterile veterinary facilities according to institutional animal care and use committee guidelines.

Methods: 1 day: Pregnant mare serum gonadotropin (PMSG) 12.5 in bulb / c mice.
Units were injected intraperitoneally (ip).

Day 3: Mice received 15 units of human chorionic gonadotropin (hCG).

Day 4: Mice were randomized and divided into groups of 5-10. Dosage range 1-200m
g / kg depending on solubility and vehicle i. p. , I. v. Or p. o.
Test compounds were administered by the route. The vehicle control group received only vehicle and two groups were left untreated.

Typically, 30 minutes later, one of the experimental, vehicle and untreated groups received 17β-estradiol (500 μg / kg) i. p. Injected. After 2-3 hours, the animals were sacrificed by CO ₂ inhalation. Following a midline incision, each uterus was isolated and cut out just below the cervix and at the junction of the uterus and fallopian tubes. Fat and connective tissue were removed with care not to compromise uterine integrity before weighing. The average weight of the treated groups was compared to the untreated or vehicle treated groups. Significance was determined by student test. Estradiol responsiveness was monitored using a non-stimulated control group.

Results% inhibition of uterine edema after estradiol stimulation was obtained for a representative compound having the structural formula at a dose of 100 mg / kg for three routes of administration:

[0131]

Embedded image

[Table 3]

[0132] Compound I was shown herein to selectively inhibit KDR in vitro kinase activity and effectively block cellular autophosphorylation of KDR in response to VEGF stimulation.

These results indicate that suitable compounds of the present invention, eg, Compound I, which selectively inhibits KDR function, effectively block edema formation. The results also indicate that i.p. v. And i. p. It also shows that the administration is particularly effective. Importantly, the results of similar anti-edema effects also show many structurally distinct KDRs
Obtained by selective inhibitors of function.

Equivalents While the invention has been particularly shown and described with respect to preferred embodiments thereof, various changes may be made in form and detail without departing from the spirit and scope of the invention as defined by the appended claims. It will be appreciated by those skilled in the art that 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 considered to be within the scope of the claims.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] December 22, 2000 (200.12.22)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

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Claims (30)

[Claims] 1. A method for inhibiting vascular hyperpermeability in an individual, comprising inhibiting the cell signal function of KDR. 2. The method according to claim 1, wherein the inhibition of the cell signal function of KDR is selectivity of KDR signal function. 3. The cell signaling function of KDR, wherein the activating ligand K
2. The method of claim 1, wherein the method is stimulated by binding to the receptor portion of DR. 4. The method according to claim 3, wherein the inhibition of the cell signal function of KDR is KDR signal function selectivity. 5. The method of claim 1, wherein the inhibition of the cell signaling function of KDR comprises blocking the production of an activating ligand, regulating the binding of the activating ligand to the KDR tyrosine kinase receptor, preventing dimerization of the receptor, A method selected from the group consisting of blocking the KDR phosphoryl transfer reaction, inhibiting the activity of KDR tyrosine kinase, impairing the recruitment of the intracellular substrate of KDR, and inhibiting the downstream signal initiated by the phosphorylation activity of KDR tyrosine kinase. The method of claim 1, wherein 6. The method of claim 5, wherein said inhibition of KDR cell signaling function is selective for KDR signaling function. 7. The method of claim 1, wherein said inhibiting occurs upon administration of the compound to an individual.
The method described in. 8. The method of claim 7, wherein said compound inhibits the catalytic kinase activity of said KDR. 9. The method of claim 7, wherein said compound is an antagonist of KDR tyrosine kinase activation. 10. The method of claim 7, wherein said compound selectively inhibits phosphorylation of a KDR kinase substrate. 11. The method of claim 7, wherein said compound is selective for said KDR tyrosine kinase. 12. The method according to claim 11, wherein said compound is selected from a peptide, an antibody and an organic molecule, and said compound binds to said KDR tyrosine kinase. 13. The method of claim 1, wherein the administration of the compound comprises macular edema, aphakia / alveolar edematous cataract, retinoblastoma, ocular ischemia, ocular inflammatory disease or ocular inflammatory infection, choroidal melanoma, iron Edema side effects caused by chelation therapy, pulmonary edema, myocardial infarction,
Rheumatic disease, hypersensitivity, tissue edema at the site of trauma and allergic inflammation, allergy, hypersensitivity reaction, polyp edema at the site of chronic inflammation, cerebral edema, fluid filling of brain tumor, traffichead, carpal tunnel syndrome, burn Organ damage, skin burns, tanning, irritating or infectious blisters, erythema multiforme, edema spots and other skin disorders, brain species, tumor effusion, lung or breast cancer, ascites, pleural effusion, pericardium Exudation, altitude sickness, radiation sensitivity, radiation dermatitis, glaucoma, conjunctivitis, choroidal melanoma, adult respiratory distress syndrome, asthma, bronchitis, ovarian hypersensitivity syndrome, polycystic ovary syndrome, menstrual swelling, menstrual paralysis, stroke , Head trauma, cerebral infarction or obstruction, hypotension, ulcer, sprain, fracture,
Synovitis effusion, diabetic complications, hyperviscosity syndrome, cirrhosis, microalbuminuria, proteinuria, hypovolemia, electrolyte imbalance, nephrotic syndrome, effusion, fibrosis, keloids, and growth factors 13. The method of claim 12, wherein the method inhibits the formation of a condition selected from: administering. 14. The method of claim 11, wherein the adverse effects associated with alterations in cell signaling function of a tyrosine kinase other than KDR are avoided by administering said compound. 15. The compound is selected from a single chain antibody, a KDR-specific ribozyme and an antisense polynucleotide, and wherein the compound is introduced or produced intracellularly, thereby providing a functional 8. The method according to claim 7, which inhibits the proper appearance of KDR tyrosine kinase. 16. The method according to claim 16, wherein the compound is an anti-specific steroid, a Ras inhibitor, an anti-
TNF agent, anti-IL1 agent, antihistamine, PAF-antagonist, COX-1 inhibitor, COX-2 inhibitor, NO synthase inhibitor, nonsteroidal anti-inflammatory agent (NSA
ID), in combination with an agent selected from a PKC inhibitor and a PI _3-kinase inhibitor, the method according to claim 7 to be administered. 17. A method of inhibiting a physiological process or condition in an individual, said physiological process or condition comprising edema formation, extravasation, blood bleeding, exudation from blood vessels or lymphatic vessels, tissue or capillaries. A method selected from the group consisting of exudation, ascites formation, matrix deposition and vascular blood pressure lowering, wherein the inhibition is achieved by administering a compound that inhibits the cell signaling function of KDR. 18. The method of claim 17, wherein said compound is KDR tyrosine kinase selective. 19. The method of claim 18, wherein said compound is selected from a peptide, an antibody and an organic molecule, said compound binding to said KDR tyrosine kinase. 20. The method of claim 19, wherein the administration of the compound is macular edema, aphakia / alveolar edematous cataract, retinoblastoma, ocular ischemia, ocular inflammatory disease or ocular inflammatory infection, choroidal melanoma, iron Edema side effects caused by chelation therapy, pulmonary edema, myocardial infarction,
Rheumatic disease, hypersensitivity, tissue edema at the site of trauma and allergic inflammation, allergy, hypersensitivity reaction, polyp edema at the site of chronic inflammation, cerebral edema, fluid filling of brain tumor, traffichead, carpal tunnel syndrome, burn Organ damage, skin burns, tanning, irritating or infectious blisters, erythema multiforme, edema spots and other skin disorders, brain species, tumor effusion, lung or breast cancer, ascites, pleural effusion, pericardium Exudation, altitude sickness, radiation sensitivity, radiation dermatitis, glaucoma, conjunctivitis, choroidal melanoma, adult respiratory distress syndrome, asthma, bronchitis, ovarian hypersensitivity syndrome, polycystic ovary syndrome, menstrual swelling, menstrual paralysis, stroke , Head trauma, cerebral infarction or obstruction, hypotension, ulcer, sprain, fracture,
Synovitis effusion, diabetic complications, hyperviscosity syndrome, cirrhosis, microalbuminuria, proteinuria, hypovolemia, electrolyte imbalance, nephrotic syndrome, effusion, fibrosis, keloids, and growth factors 20. The method of claim 19, wherein the method inhibits the formation of a condition selected from: administering. 21. The method of claim 17, wherein said compound inhibits the catalytic kinase activity of said KDR. 22. The method of claim 17, wherein said compound is an antagonist of KDR tyrosine kinase activation. 23. The method of claim 17, wherein said compound selectively inhibits phosphorylation of a KDR kinase substrate. 24. The method of claim 17, wherein said compound is said KDR tyrosine kinase selective. 25. The method of claim 17, wherein the cell signaling function of KDR is stimulated by binding its activating ligand to the receptor portion of KDR. 26. The method of claim 25, wherein said compound is selective for said KDR tyrosine kinase. 27. The compound is selected from a single chain antibody, a KDR-specific ribozyme and an antisense polynucleotide, and the compound is introduced or produced intracellularly, thereby producing a functional KDR tyrosine. 18. The method of claim 17, which inhibits the proper appearance of the kinase. 28. The method of claim 26, wherein the inhibition of KDR cell signaling functions includes blocking activation ligand production, regulating the binding of the activation ligand to the KDR tyrosine kinase receptor, disrupting receptor dimerization, and KDR phosphorylation. A process selected from the group consisting of blocking a reaction, inhibiting KDR tyrosine kinase activity, inhibiting KDR intracellular substrate recruitment, and inhibiting downstream signals initiated by phosphorylation activity of KDR tyrosine kinase. Item 18. The method according to Item 17. 29. The method of claim 17, wherein adverse effects associated with alterations in cell signaling function of a tyrosine kinase other than KDR are avoided by administering said compound. 30. The method according to claim 17, wherein the compound is an anti-specific steroid, a Ras inhibitor, an anti-
TNF agent, anti-IL1 agent, antihistamine, PAF-antagonist, COX-1 inhibitor, COX-2 inhibitor, NO synthase inhibitor, nonsteroidal anti-inflammatory agent (NSA
ID), in combination with an agent selected from a PKC inhibitor and a PI _3-kinase inhibitor, the method according to claim 17 to be administered.