SK5052001A3 - Inhibition of the formation of vascular hyperpermeability - Google Patents

Abstract

Vascular hyperpermeability and the subsequent events such as macular edema, retinoblastoma, ocular ischemia, ocular inflammatory disease or infection, choroidal melanoma, edematous side-effects induced by iron chelation therapy, pulmonary edema, myocardial infarction, rheumatoid diseases, anaphylaxis, allergies, hypersensitive reactions, cerebral edema, brain tumor fluid-filled cysts, communicating hydrocephalus, carpal tunnel syndrome, organ damage resulting from a burn, irritation or infection, erythema multiforme, edematous macules and other disorders, brain tumors, tumor effusions, lung or breast carcinomas, ascites, pleural effusions, pericardial effusions, high altitude "sickness", radioanaphylaxis, radiodermatitis, glaucoma, conjunctivitis, choroidal melanoma, adult respiratory distress syndrome, asthma, bronchitis, ovarian hyperstimulation syndrome, polycystic ovary syndrome, menstrual swelling, menstrual cramps, stroke, head trauma, cerebral infarct or occlusion, hyotension, ulcerations, sprains, fractures, effusions associated with synovitis, diabetic complications, hyperviscosity syndrome, liver cirrhosis, microalbuminuria, proteinuria, oliguria, electrolyte imbalance, nephrotic syndrome, exudates, fibroses, keloid, 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. The preferred compound 4,5-dihydro-3-pyridin-4-yl-1(2)H-benzo[g]indazole selectively inhibits the function of KDR tyrosine kinase but do not block the activity of Flt-1 tyrosine kinase which is another VEGE tyrosine kinase receptor.

Description

The present invention relates to the use of a compound that inhibits the cellular signaling function of KDR tyrosine kinase for the preparation of a medicament for inhibiting vascular hyperpermeability. The invention further provides the use of a compound that inhibits the cellular signaling function of KDR tyrosine kinase, for the preparation of a medicament for inhibiting vascular hyperpermeability by employing agents that selectively inhibit the cellular signaling function of KDR.

BACKGROUND OF THE INVENTION

Edema can be described as an increase in interstitial fluid volume. This is usually an abnormal condition for which relief is typically sought. Quite often this condition arises because the fluid exits the blood vasculature due to increased endothelial permeability, often associated with macromolecular extravasation, and finds a new settlement in the interstitial spaces.

There are a number of physiological and biochemical mechanisms that are subject to edema and edema formation in individuals. An important mediator in one or more of these mechanisms is "vascular endothelial cell growth". This factor increasingly regulates transport in vascular endothelial cells and causes an increase in the permeability of a variety of vascular beds, including skin, subcutaneous tissues, peritoneal wall, mesentery, diaphragms, trachea, bronchi, duodenum and uterus. Significant diapedesis, endothelial exchange modifications, extravasation and deposition of macromolecules at these sites, and prolonged hypotension may accompany these increased permeability effects. These processes are believed to be an introduction facilitating neovascularization. VEGF is expressed by inflammatory T cells, macrophages, neutrophils and eosinophils, and the like, at sites of inflammation. This factor is increasingly regulated by hypoxia, certain vasopressive hormones, growth factors, reproductive hormones, and numerous inflammatory cytokines. VEGF-mediated vascular permeability is implicated in such diseases as tumor ascites, endometriosis, adult respiratory distress syndrome (distress syndrome respirators, ARDS), post-cardiopulmonary bypass-related hypotension and hyperpermeability pustules,

-Edematous responses to burns and trauma, endothelial dysfunction in diabetes, complications of ovarian hyperstimulation syndrome, and eye edema.

Thus, it is clear that inhibition of VEGF production or activity could be useful, particularly to block the manifestation of the above-mentioned diseases. In particular, agents that are able to block VEGF-mediated hyperpermeability and edema and concomitant syndromes could be useful in alleviating these diseases.

Protein tyrosine kinase. Protein tyrosine kinases (PTKs) include a large and diverse class of proteins exhibiting enzymatic activity. Protein tyrosine kinases play an important role in regulating cell growth and cell differentiation (for review see Schlessinger & Ullrich, 1992, Neuron 9, 383-391).

Aberrative stimulation, expression, or mutations in protein tyrosine kinases lead to either uncontrolled cell proliferation (e.g., malignant tumor growth) or defects in key developmental, regulatory or reparative processes. As a result, the biomedical community is investing significant resources to elucidate the specific biological role of protein tyrosine kinase family members, their function in different processes, their contribution to tumorigenesis and other diseases, the biochemical mechanisms subject to their signal transduction pathways activated after ligand stimulation and the development of new drugs.

Tyrosine kinases can be of the receptor type (containing extracellular, transmembrane and intracellular domains) or non-receptor type (which are exclusively intracellular).

Receptors of tyrosine kinases (RTKs). Tyrosine kinase receptors include a large family of transmembrane receptors with diverse biological activities. To date, at least nineteen (19) distinct subfamily of tyrosine kinase receptors have been identified. The tyrosine kinase (RTK) family of receptors includes receptors that are critical to 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 receptor tyrosine kinases is activated after ligand binding, which results in phosphorylation of the receptor and composite cellular substrates and

3 consequently diverse cellular responses (Ullrich & Schlessinger, 1990, Cell 61, 203 212). Thus, receptor tyrosine kinase-mediated signal transduction is initiated through extracellular interaction with a specific growth factor (ligand), typically followed by dimerization of the receptor, stimulation of intrinsic protein tyrosine kinase activity, and trans-phosphorylation of the receptor. Thus, binding sites form for intracellular signal transduction molecules and result in complexes with a spectrum of cytoplasmic signaling molecules, facilitating appropriate cellular responses (e.g., cell division, differentiation, metabolic effects, changes in the extracellular microenvironment), see Schlessinger and Ullrich, 1992, Neuron 9, 1 20.

Proteins with SH2 domains (src homology -2) or phosphotyrosine binding (PTB) bind activated tyrosine kinase receptors and their substrates with high affinity to transduce signals into cells. Both of the 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., 1993, Cell 72, 767 778; and Koch et al., 1991, Science 252, 668-678, Schoelson, Curr Opin, Chem Biol (1997), 1 (2), 227-234, Cowburn, Curr Opin, Struct Biol (1997) , 7 (6), 835-838). Some intercellular substrate proteins that are associated with receptor tyrosine kinases (RTKs) have been identified. These can be divided into two basic groups: (1) substrates having a catalytic domain; and (2) substrates which lack such a domain but serve as adapters and associate with catalytically active molecules (Songyang et al., 1993, Cell 72, 767-778). The specificity of the interactions between receptors or proteins and the SH2 or PTB domains of their substrates is determined by the amino acid residues immediately surrounding the phosphorylated tyrosine residue. For example, differences in binding affinities between SH2 domains and amino acid sequences flanking the phosphorothrosine residues at individual receptors are correlated with observed differences in their substrate phosphorylation profiles (Songyang et al., 1993, Cell 72, 767-778). Observations suggest that the function of each receptor tyrosine kinase is determined not only by its pattern of expression and ligand availability, but also by the alignment of progressive signal transduction pathways that are activated by the action of a specific receptor, as well as the synchronization and duration of these stimuli. Thus, phosphorylation provides a significant regulatory step that determines selectivity

-4 signal pathways generated by specific growth factor receptors as well as differentiation factor receptors.

It is believed that some receptor tyrosine kinases and the growth factors that bind to them play a role in angiogenesis, although some may promote angiogenesis indirectly (Mustonen and Alitalo, J. Cell Biol. 129, 895-898, 1995). One such receptor tyrosine kinase, known as "fetal liver kinase 1" (FLK-1), is a member of the type III subclass of receptor tyrosine kinases. An alternative term for human FLK-1 is "kinase insert domain-containing receptor (KDR)" (Terman et al., Oncogene 6, 1677-1683, 1991). Another alternative designation for FLK-1 / KDR is "vascular endothelial cell growth factor receptor 2" VEGFR-2 because it binds VEGF with high affinity. The murine version of FLK-1 / VEGFR-2 was also called NYK (Oelrichs et al., Oncogene 8, (1): 11-15, 1993). Acid DNAs encoding murine, rat and human FLK-1 have been isolated, and nucleotides and coding amino acid sequences have been published (Matthews et al., Proc. Natl. Acad. Sci. USA, 88, 9026-9030, 1991; Terman et al., 1991 as above; Terman et al., Biochem. Biophys. Res Comm. 187. 1579-1586, 1992; Sarzani et al., As above; and Millauer et al., Cell 72, 835-846 , 1993). Numerous studies, such as those published by Millauer et al., As mentioned above, suggest that VEGF and FLK-1 / KDR / VEGFR-2 are a ligand-receptor pair that plays an important role in vascular endothelial cell proliferation and formation and blood vessel growth, timed vasculogenesis, and angiogenesis.

Another type III RTK subclass referred to as "fms-like tyrosine kinase-1, Flt-1" refers to FLK-1 / KDR (DeVries 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" (VEGFR-1). To date, it has been found that members of the FLK-1 / KDR / VEGFR-2 and Flt-1 / VEGFR-1 subfamily are primarily expressed in endothelial cells. These members of the subclass are specifically stimulated by members of the vascular endothelial cell growth factor (VEGF) of the ligand family (Klagsburn and D'Amore, Cytokine & Growth Factor Reviews 7, 259, 270, 1996). Vascular endothelial cell growth factor (VEGF) binds to Flt-1

-5r r c o c

C rrs has a higher affinity than FLK-1 / KDR and is mitogenic to vascular endothelial cells (Terman et al., 1992 as above; Mustonen et al. As above; DeVries et al. As reported) higher). Flt-1 is believed to be essential for endothelial organization during vascular development. Expression of Flt-1 is associated with early vascular development in mouse embryos and with neovascularization during wound healing (Mustonen and Alitalo as above). Expression of Flt-1 in adult organs such as kidney glomeruli suggests an additional function for this receptor that is not associated with cell growth (Mustonen and Alitalo as above).

As mentioned above, recent evidence suggests that VEGF plays a role in stimulating both normal and pathological 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 regulation and improvement of vascular permeability (Connolly et al., J. Biol. Chem. 264, 20017-20024, 1989; Brown et al., Regulation of Angiogenesis (ed. LD Goldberg and EM Rosen), 233 269, 1997).

Various forms of VEGF resulting from alternative mRNA splicing, including the four species described by Ferrara et al. (J. Cell. Biochem. 47, 211-218, 1991). Ferrara et al. (see above), both species, both secreted and predominantly cell-associated VEGF species, have been identified and the protein is known to exist as disulfide-linked dimers.

Recently, several related VEGF homologues have been identified. However, their role in normal physiological and disease processes has not yet been explained. In addition, members of the VEGF family are often co-expressed with VEGF in a variety of tissues and are generally capable of forming heterodimers with VEGF. This property is likely to alter receptor specificity and biological effects of heterodimers and further complicate the elucidation of their specific functions as explained below (Korpelainen and Alitalo, Curr. Opin. Cell Biol., 159, 164, 1998 and references cited therein).

The placenta growth factor (PIGF) contains an amino acid sequence that exhibits significant homology to the VEGF sequence (Park et al., J. Biol. Chem. 269, 25646-25654, 1994; Maglione et al. Oncogene 8, 925) 931 (1993). As with VEGF, a variety of PIGF species are derived from alternative mRNA splicing, and the protein exists in the dimeric form (Park et al., Supra). PIGF-1 and PIGF-2 bind to Flt-1 with high affinity, and PIGF-2 also readily binds to neuropilin-1 (Migdal et al., J. Biol. Chem. 273 (35): 22272 22278) but does not bind FLK-1 / KDR at all (Park et al., supra). PIGF has been reported to potentiate both the vascular permeability and the mitogenic effect of VEGF in endothelial cells when VEGF is present at low concentrations (allegedly due to heterodimer formation) (Park et al., Supra).

VEGF-B is produced as two isoforms (167 and 185 residues), which also appear to bind to Flt-1 / VEGFR-1. This may play a role in regulating extracellular matrix degradation, cell adhesion and migration through modulation of urokinase-type plasminogen activator expression and activity and plasminogen activator inhibitor 1 (Pepper et al., Proc. Natl. Acad. Sci. USA (1998), 95, (20). ): 11709-11714).

VEGF-C was originally cloned as a ligand for VEGFR-3 / Flt-4, which is primarily expressed by lymphatic endothelial cells. This is a fully mature form, VEGF-C can also bind KDR / VEGFR-2 and stimulate endothelial cell proliferation and migration in vitro and angiogenesis in in vivo models (Lymboussaki et al., Am. 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 widening of only lymphatic vessels and blood vessels are unaffected. In contrast to VEGF, VEGF-C expression is not induced by hypoxia (Ristimaki et al., J. Biol. Chem. (1998), 273, (14), 8413-8418).

The recently discovered VEGF-D is structurally very similar to VEGF-C. VEGF-D has been reported to bind and activate at least two VEGFRs, VEGFR-3 / Flt-4 and KDRA / EGFR-2. It was originally cloned as a c-fos-inducible mitogen for fibroblasts and is most prominent in mesenchymal lung cells and

Skin (Achen et al., Proc. Natl. Acad. Sci. U.S.A. (1998), 95, (2), 548-553 and references cited therein).

VEGF-C and VEGF-D have been reported to induce an increase in vascular permeability in vivo in the Miles assay when injected into skin tissue (PCT / US97 / 14696; WO98 / 07832, Witzenbichler et al., Supra).

I

The physiological role and significance of these ligands in modulating vascular hyperpermeability and endothelial responses in the tissues in which they are expressed remains unclear.

Based on the discovery of other VEGF and VEGFR homologues and precedents for ligand and receptor heterodimerization, the effect of such VEGF homologs may include the formation of VEGF ligand heterodimers and / or receptor heterodimerization, or binding to undiscovered VEGFR (Witzenbichler et al., Supra), as discussed above. Also, recent studies suggest the possible involvement of neuropilin-1 (Migdal et al., As above) or VEGFR-3 / Flt4 (Wtzenbichler et al., As above), and also that other receptors such as KDR / VEGFR- 2 are responsible for inducing vascular permeability (Stacker, SA, Vitali, A., Domagala, T., Nice, E. and Wks, AF, Angiogenesis and Cancer Conference, Amer. Assoc. Cancer Res., Jan. 1998, Orlando, FL; Williams, Diabetelogia 40, S118-120 (1997)).

Development of compounds for modulating protein tyrosine kinases. In view of the supposed importance of protein tyrosine kinases in the control, regulation and modulation of cell proliferation as well as diseases and disorders associated with abnormal cell proliferation, many attempts have been made to identify inhibitors of receptor and non-receptor tyrosine kinases, using a variety of approaches, including mutant ligands. No. 4,966,849), soluble receptors and antibodies (International Application No. WO 94/10202; Kendall & Thomas, 1994, Proc. Natl. Acad. Sci 90. 10705-10709; Kim et al., 1993, Nature 362, 841 844), RNA ligands (Jellinek et al., Biochemistry 33, 10450-10456; 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, 448-457) and tyrosine kinase inhibitors (International Documents WO 94/03427; WO 92/21660; WO

-891/15495; WO 94/14808; U.S. Pat. No. 5,330,992; Mariani et al., 1994, Proc. Am. Assoc. Cancer Res. 35, 2268).

More recently, attempts have been made to identify small molecules that act as tyrosine kinase inhibitors. For example, bismonocyclic, bicyclic or heterocyclic aryl compounds (PCT International Patent Publication WO 92/20642) and vinylene-azaindole derivatives (PCT International Patent Publication WO 94/14808) have generally been described as tyrosine kinase inhibitors. Styryl compounds (U.S. Patent No. 5,217,999), styryl substituted pyridyl compounds (U.S. Patent No. 5,302,606), certain quinazoline derivatives (European Patent Application EP 0 566 266 A1); Expert Opin. Ther. Pat. (1998), 8 (4): 475-478, seleoindoles and selenides (PCT International Patent Publication WO 94/03427), tricyclic polyhydroxyl compounds (PCT International Patent Publication WO 92/21660) and benzylphosphonic acid compounds (PCT International Patent Publication WO 91/15495) have been described as being useful as tyrosine kinase inhibitors for use in the treatment of cancer. Anilinocinolines (PCT International Patent Publication WO 97/34876) and quinazoline derivative compounds (PCT International Patent Publication 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 serine / threonine kinase inhibitors. In particular, bis (indolylmaleinimide) compounds have been described as inhibiting, in particular, the PKC serine / threonine kinase isoforms whose dysfunction is associated with altered vascular permeability in VEGF-related diseases (International Patent Documents PCT WO97 / 40830; PCT WO97 / 40831).

Therefore, it is appropriate to identify active macromolecules and small organic compounds that specifically inhibit tyrosine signal transduction by modulating receptor and non-receptor tyrosine kinase activity to regulate and modulate abnormal or inappropriate cellular function, cell proliferation or differentiation. In particular, it would be useful to identify methods and compounds that specifically inhibit the function of tyrosine kinases that are essential for the formation of vascular hyperpermeability leading to edema,

-9fusions, exudates and macromolecular extravasation and deposition as well as related diseases.

SUMMARY OF THE INVENTION

The present invention is directed to inhibiting vascular hyperpermeability by inhibiting the cellular signaling function of KDR tyrosine kinase. The present invention also provides the use of a compound that inhibits the cellular signaling function of KDR for the preparation of a medicament for inhibiting vascular hyperpermeability by selectively disrupting the catalytic kinase response of KDR / VEGFR-2 without significantly affecting Flt-1 / VEGFR1 activity or other tyrosine kinases. Agents that act in accordance with this use have a clear pharmacological advantage over prior therapeutic approaches involving materials, such as steroids, that are prone to cause numerous adverse effects. The use of the present invention is also advantageous compared to the use of less specific kinase inhibitors, including those that inhibit composite VEGF receptors, as this use will not directly interfere with the significant normal physiological function of other kinases. As a result of inhibition of vascular endothelial hyperpermeability, subsequent formation of edema, associated diapedesis, trans-endothelial molecular exchange changes, extravasation, exudates and effusion will also be inhibited by suppression of KDR tyrosine kinase activity. Since this latter case often leads to excessive matrix deposition, abnormal tree proliferation and organ dysfunction, inhibition of KDR tyrosine kinase is also useful in the treatment of the numerous non-cancerous diseases involved in these etiological characteristics. In addition, vascular hypotension, which may be due to VEGF-related activation of ligand binding to the KDR receptor tyrosine kinase, is also minimized by inhibiting KDR tyrosine kinase activity.

The present invention also provides a therapeutic approach to inhibiting vascular hyperpermeability and edema formation in individuals by administering a compound that specifically inhibits KDR tyrosine kinase activity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes the use of a compound that inhibits the cellular signaling function of KDR for the preparation of a medicament for inhibiting vascular hyperpermeability. The present invention also describes the use of a compound that inhibits cellular signaling

KDR tyrosine kinase function, for the preparation of a medicament for inhibiting vascular hyperpermeability by employing agents that selectively inhibit the cellular signaling function of KDR. By identifying and utilizing highly selective KDR inhibitors that effectively block KDR cellular signaling and consequently vascular hyperpermeability, in accordance with the use of the present invention, the essential role of KDR in mediating the vascular permeability response to VEGF has been established. Such highly selective KDR inhibitors have demonstrated efficacy in modulating vascular permeability without the need to inhibit the function of the high affinity receptor, VEGFR-1 / Flt-1. This property should provide better treatment tolerance than prior therapies or treatments 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 the KDR tyrosine kinase receptor, which lies on the surface of vascular endothelial cells. Although naturally occurring kinase-activating mutations and truncations have not yet been identified for KDR, they have been described for EGFR and Tie-2 receptor kinases. Consequently, cases of constitutive activation of DRC are also envisaged. 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 also induces hyperpermeability of blood vessels. As a result, the fluid moves from the bloodstream around the walls of the blood vessels to the interstitial spaces, thereby creating an edema area. This response is also often accompanied by diapedesis. Similarly, excessive vascular hyperpermeability may disrupt normal molecular exchange through the endothelium in critical tissues and organs (e.g., lung and kidney), causing organ dysfunction, macromolecular extravasation, and matrix deposition, often with the support of stromal proliferation. Upon appearance in confined compartments, edema (for example, cerebral edema) can lead to impaired organ function and damage.

Inhibition of KDR cellular signaling function may include: either blocking the production of activating ligand, blocking activating ligand binding to the receptor

-11 KDR tyrosine kinase, prevention of receptor dimerization and transphosphorylation, inhibition of KDR tyrosine kinase enzyme activity (by inhibiting the phosphorylation capacity of the enzyme), or some other mechanism that interrupts its output signaling (D. Mukhopedhyay et al., Cancer Res. 58, 1278-1284 (1998) ) and the references therein). In accordance with the use described herein, such procedures that are selective for disrupting the KDR cellular signaling function will reduce vascular hyperpermeability as well as associated extravasation, subsequent edema formation, and matrix deposition.

There are a number of compounds that exhibit the desired inhibitory property of KDR tyrosine kinase. Among these compounds are antibodies (hereinafter meaning to include single chain antibody constructs) that bind the extracellular KDR receptor domain or part of a cellular kinase enzyme or, alternatively, that bind VEGF alone. These antibodies interfere with the binding of VEGF to the KDR receptor tyrosine kinase and / or, significantly, the cellular signaling function of KDR tyrosine kinase. Antibodies that bind to KDR tyrosine kinase can act as VEGF antagonists or, more generally, VEGF activator antagonists. Alternatively, these antibodies may block functional receptor dimerization or may be KDR tyrosine kinase inhibitors. Antibodies that bind to VEGF or an activating ligand are neutralizing antibodies of VEGF or an activating ligand. It should be noted that such VEGF neutralizing antibodies can block VEGF responses through both KDR receptors and Flt-1 receptors and, typically, are specific for a single activating ligand. In most cases, blocking VEGF responses via Flt-1 receptors is neither necessary nor required. Since these VEGFRs are reported to recognize different epitopes on VEGF, the desired specific KDR activation can be achieved through the use of antibodies that specifically bind to and "mask" the KDR-binding epitope of VEGF or another activating ligand.

Other compounds that can inhibit KDR tyrosine kinase activity and consequently minimize vascular hyperpermeability and edema formation include peptides and organic molecules. Among the peptides are soluble extracellular domains of KDR and KDR binding fragments. Other useful peptides are VEGF mutants or VEGF-related growth factors (e.g., VEGF-C, VEGF-D or VEGF-D).

(12HIV Tat protein and fusion proteins thereof) that bind and block another ligand binding to this receptor but do not stimulate KDR tyrosine kinase dimerization, activation or transphosphorylation. Such mutants may act as monomers or non-functional heterodimers, thereby blocking binding of native dimemic VEGF or activating ligand. Similarly, other peptides or small molecules that block receptor dimerization and / or activation can be successfully used. These compounds also act as antagonists of activating ligands or are inhibitors of KDR tyrosine kinase activity. Preferred compounds are small organic molecules.

In addition, molecules such as KDR-specific ribozymes, antisense polynucleotides (such as antisense mRNA) or single-chain intracellular antibodies (S _C F _V ) that inhibit biosynthesis or correct presentation of active, functional KDR tyrosine kinase will effectively block KDR-mediated responses. against VEGF. These molecules may be introduced into cells preformed or their production induced intracellularly (for example, by use of suitable adenoviral, retroviral or baculoviral vectors).

Preferred compounds of the invention exhibit the ability to inhibit the cellular signaling function of KDR without significantly inhibiting the cellular signaling function of Flt-1 (Flt-1 tyrosine kinase is also referred to as VEGFR-1 tyrosine kinase). Both KDR tyrosine kinase and Flt-1 tyrosine kinase are activated by VEGF binding to KDR tyrosine kinase receptors and Flt-1 tyrosine kinase receptors. Since Flt-1 tyrosine kinase activity may mediate significant events in endothelial maintenance and vascular function, inhibition of this enzymatic activity or associated transduced signals may lead to toxic or inverse effects. At the very least, such inhibition is not necessary to block the induction of vascular hyperpermeability and to produce edema, so that it is unnecessary and of no value to the individual. Preferred compounds of the invention are unique in that they inhibit the activity of one VEGF receptor tyrosine kinase (KDR), which 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 the invention are therefore selective with respect to their tyrosine kinase inhibitory activity.

13a <·

VEGF is known to contribute to vascular hyperpermeability and edema formation. VEGF is expressed by inflammatory T cells, macrophages, neutrophils and eosinophils, and the like, at inflammatory sites. The production of this factor is rapidly regulated by hypoxia, certain vasopressing hormones, growth factors, reproductive hormones, and numerous inflammatory cytokines. In fact, vascular hyperpermeability and edema that is associated with the expression or administration of many other growth factors appears to be mediated through the production of VEGF. Vascular hyperpermeability, associated edema, altered transendothelial exchange, and macromolecular extravasation, which is often accompanied by diapedesis, may result in excessive matrix deposition, abnormal stromal proliferation, fibrosis, and the like. Therefore, VEGF-mediated hyperpermeability may significantly contribute to disorders in these etiological characteristics. For example:

(1) VEGF significantly increases in the epidermis of injured psoriatic skin. This factor effectively stimulates dermal endothelial cell proliferation and microvascular hyperpermeability associated with psoriasis.

(2) Major organs are often damaged after burns and severe scalds. This appears to be manifested by an uncontrollable "mediator disease" resulting from ischemic-reperfusion injury, swelling and visceral edema, i. endothelial cell damage. For burn victims, inhalation burn injury is one of the primary causes of death. The tracheobronchial epithelium forms rattles and associates with protein-rich exudate, producing airway fingerprints, which can lead to obstruction of these airways. The combination of inhalation burns and hypoxia followed by exposure to high oxygen (in an attempt to help the individual) may aggravate the situation by causing progressive changes in the lungs, such as a diffuse exudative formation, tracheal bleeding and edematous changes in the blood vessel wall. Circulating serum VEGF levels increase dramatically (up to 20 times) in victims due to burns and multiple trauma, and may be the primary mediator of these complications (Grád et al., Clin. Chem. Lab. Med. 36, 379-383, 1998) .

-14 (3) Sunburn is also associated with edema. It is also known that VEGF production is increasingly regulated upon exposure to UV radiation. Other skin diseases in which edema is formed include blistering symptomatic erythema (acrrodinium), persistent acrodema, and vesicular diseases such as multiform erythema, bullous pemphigoid, and herpetiform dermatitis (i.e., acute or chronic inflammatory conditions). Edematous maculars and rosacea, conditions associated with teleangiectase, are other diseases in which edema is manifested.

(4) Increased microvascular permeability and edema are common characteristics of inflammatory and neoplastic diseases. Brain tumors, such as gliomas where tumor and peritumoral brain edema and liquid-filled cysts are formed, and meningiomas, with concomitant massive cerebral edema, are examples of such diseases. Locally high levels of VEGF are associated with these diseases. The induction of malignant ascites fluid and tumor effusions (especially malignant and pericardial effusions) are further examples of such edema-producing diseases and are known to cause VEGF production. In addition, edema resulting from head trauma can create concussions and impaired brain function. Similarly, communicating hydrocephalus has been shown to cause cytokines such as IGF-1 and TGF-βΙ, which are known to modulate VEGF production.

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

(6) Cytokine-activated eosinophils may be an important source of VEGF, thereby contributing to the formation of tissue edema at sites of allergic inflammation. Edema and exudates are common complications that occur during allergic reactions and delayed-type hypersensitivity reactions; also often include anaphylaxis. VEGF is primarily involved in reactions that are not susceptible to antihistamines

-15 ° C C C Γ Γ asp asp asp asp asp asp asp asp asp asp asp asp asp asp asp asp asp asp asp asp asp asp asp asp asp asp asp ín asp zvýš zvýš regul asp asp sa regul regul asp asp asp asp asp or aspirin, In addition, examples of such allergic reactions and delayed-type hypersensitivity reactions in which VEGF may also be involved are tuberculosis, certain viral infections, angioedema, urticaria (urticaria) and anaphylaxis induced by physical exertion. Edema also often arises as a result of drug sensitivity or as a result of hypersensitivity reactions, or in response to the administration of growth factor with increased regulation of VEGF or cytokines (e.g. IGF-1, FGF-2, or IL-2). Radioanaphylaxis and radiodermatitis are associated with vascular hyperpermeability.

(7) VEGF has been implicated in ocular neovascularization leading to diabetic retinopathy and microangiopathy, age-related macular degeneration and neonatal blindness resulting from hyperoxic exposure. In many cases, this condition is preceded by macular or other ocular edema. VEGF has been identified as the primary mediator of the neovascularization of the iris, cornea and retina in cases of ocular ischemia and vascular edema. VEGF-induced vascular hyperpermeability contributes to the breakdown of the blood-retinal barrier in a number of extravasational eye diseases, and matrix deposition provides the basis for subsequent angiogenesis. Corneal neovascularization is a major consequence of chemical burns, corneal inflammation and edema. Recent evidence has shown the involvement of VEGF in processes following such eye trauma. Iron chelating agent, deferoxamine, has been used in the clinical treatment of cancer patients. However, this treatment often causes macular edema. The concentrations of this iron chelating agent achieved in patients induce a 3- to 5-fold increase in VEGF mRNA expression in all normal and tumor cell lines examined, suggesting that VEGF is likely to be a mediator of edema formation Increased intraocular pressures due to overproduction of VEGF and edema can lead to inappropriate matrix deposition, eye deformation, optical disc changes, visual field defects, and can result in giaucoma. Vascular hyperpermeability is also often associated with conjunctivitis.

(8) Chronic pulmonary disease in neonates and adults results from both lung injury and inappropriate healing processes. VEGF production is reported in some animal models with lung injury. The destruction of pulmonary endothelial cells is also characteristic of hyperoxic lung damage. During recovery from hyperoxia, VEGF is over-regulated by type II alveolar cells and subsequently causes proliferation and regeneration of pulmonary endothelial cells. However, this result may cause disruption of the exchange between pulmonary endothelium and pulmonary edema. Asthma and bronchitis often include bronchial vascular dilatation, vascular congestion, bronchial wall edema, and exudates that result in thinning of the airway mucosa and narrowing of the bronchial lumen. Protein exudate edema and faulty tree growth are typically associated with this phenomenon. In related processes, pulmonary edema is formed during adult respiratory distress syndrome. Cases of adult respiratory distress syndrome typically include pneumonia, inhalation of noxious substances, lung contusion, a thaw-like condition, and inhalation of stomach contents.

(9) Corticosteroids such as cortisone, hydrocortisone, dexamethasone or prednisolone are among the most widely used therapeutic agents for oedematous conditions. They are potent inhibitors of VEGF expression. It is currently believed that this property contributes significantly to the well-known anti-oedematous efficacy of such steroids. However, their pluripotent biological activities are also responsible for their undesirable side effects. Steroid hormones and their agonists and antagonists also dramatically affect VEGF production, particularly in reproductive tissues. Endometritis and endometriosis may occur during pregnancy, menstrual cycle or sex hormone treatment. Swelling and convulsions during menstruation are associated with vascular hyperpermeability. Tamoxifen, an agent that reduces the risk of breast cancer, also increases uterine cell proliferation and tumor incidence. This steroid analog, as well as estradiol, cause uterine edema and cell proliferation, which has been shown to be involved in a local increase in VEGF production. Ovarian hyperstimulation syndrome is severe

-17 • * r r c r * r e ... e e r e e r r, r i r <

r r <· r f a complication that affects the induction of ovulation. The most serious manifestation of these syndromes is massive ovarian enlargement and multiple cysts, ascites, haemoconcentration and fluid accumulation in tertiary spaces. Increased capillary permeability, triggered by the release of VEGF secreted by luteinizing granulosa cells, and the like, is believed to play a key role in these syndromes upon stimulation with human chorionic gonadotropin. VEGF has been shown to be overexpressed in the hypertekotic ovarian tree of polycystic ovaries in Stein-Leventhal syndrome.

(10) Rapidly differentiated induction of VEGF expression gene in both neuronal and choroid cells after transient mild cerebral arterial occlusion has been demonstrated in animal stroke models. VEGF may contribute to the recovery of brain cells from ischemic injury, such as stroke head injury or cerebral infarction, by enhancing neovascularization, but brain injury may also be exacerbated by concomitant cerebral edema. Malaria can also induce edema as a result of VEGF-induced cerebral hypoxia. Brain-associated cerebral edema and fluid-filled cysts arise because tumor capillaries do not have a normal barrier function with respect to blood flow in the brain. VEGF released by glioma cells in situ is most likely responsible for the pathognomonic histopathology and clinical features of glioblastoma tumors in patients, including increased cerebral edema. Carpal channel syndrome is accompanied by increased nerve hydration and also often by subsequent increases in extracellular matrix deposition (neuropathy captured). Elevated levels of VEGF in tissues that surround the nerve can cause nerve entrapment by inducing vascular permeability, fluid efflux, and stromal deposition into the perineural tissues.

(11) VEGF production in tissues is dramatically over-regulated in response to hypoxia. Hence, there are observations that in areas of necrosis, ischemia, heart attack, occlusion, anemia, circulatory impairment or other

-18e r r p r r-r p rp ^ ppc r -r r

P r f. <C ~ r r> c Í r.

In the absence of oxygen deficiency, VEGF levels are elevated and vascular hyperpermeability, edema and extravasation are common. Reducing oxygen pressure, which is responsible for “altitude sickness”, also causes rapid production of VEGF, which is likely to cause imminent cerebral and pulmonary edema (HACE and HAPE), which may occur if the person is not acclimatized.

(12) Overproduction of VEGF is similarly demonstrated in pericardial and pleural effusions caused by vascular hyperpermeability resulting from radiation damage, rheumatoid diseases, skin tuberculosis, myocardial infarction, trauma or drug reactions. Not surprisingly, overproduction of VEGF in conjunction with pericardial and pleural effusions is usually observed in autopsy in patients with lung or breast cancer, lymphoma and leukemia. Amounts of VEGF also increase significantly in synovial fluid of swollen joints in individuals with rheumatoid arthritis. Sprains and fractures, although associated with some swelling and vascular hyperpermeability that is beneficial in promoting angiogenesis and healing, may be accompanied by pain and excessive, unwanted edema. Similarly, VEGF is believed to be involved in conditions such as synovitis or meniscus with effusion (for example, "knee water").

(13) Ulceration associated with reduced circulation (eg pressure sores, gravitational and varicose ulcers) is also often accompanied by protein edema and exudates. Diabetic complications often arise as a result of elevated blood glucose levels (hyperglycemia) and the formation of higher glycemic end products (AGEs), often accompanied by impaired circulation. It is known that these conditions, either alone or in combination, stimulate VEGF production and hence vascular hyperpermeability, which can lead to numerous diabetic complications.

(14) Because of the significant constitutive production of endogenous VEGF by the kidney podocytes and the known effects of vascular hyperpermeability of elevated VEGF levels, kidney disease such as microalbuminuria,

Proteinuria, oliguria, electrolytic imbalance (often encountered as diabetic complications) and nephrotic syndrome (particularly when hypoxia is induced by burns, shock or trauma) can be treated using the present invention.

(15) Protein extravasation and diapedesis, which usually accompanies edema and leads to excessive matrix deposition and stromal proliferation, contribute to the progression of other diseases. These diseases include hyperviscosity syndrome, liver cirrhosis, fibrosis, keloid and formation of unwanted tissue scarring. Inhibition of VEGF-mediated hyperpermeability will delay the progression of such diseases.

(16) Significant amounts of VEGF isoforms are known to be deposited in platelets, mast cells and the like, and in extracellular matrices. In certain situations, these stocks of VEGF / VPF can be rapidly released and thereby contribute to acute vascular hyperpermeability.

From these diverse examples, it is readily apparent that edema occurs in various physiological conditions and VEGF / VPF or a related analogue is strongly involved in edema formation and extravasation. The compounds of the present invention minimize the edematous condition associated with macular edema, aphakic / pseudoaphacic cystoid macular edema, retinoblastoma, ocular ischemia, ocular inflammatory disease or infection, choroidal melanoma, edematous side effects, pleural pain, pulmonary edema, myocardial infarction, rheumatoid disease, tissue edema at sites of injury or allergic inflammation, polyp edema at sites of chronic inflammation, cerebral edema, fluid-filled brain tumor Cysts, communicative hydrocephalus, edema associated with organ damage resulting from burn burns and edema. The compounds of the invention also minimize edematous conditions associated with skin burns, blisters, erythema multiforme, edematous macula and other skin diseases, brain tumors, ascites and various cancers associated with cancer, carpal duct syndrome, "hypersensitivity reactions", allergic and hypersensitivity reactions , radioanaphylaxis,

-20radiodermatitis, glaucoma, conjunctivitis, adult respiratory distress syndrome, asthma, bronchitis, ovarian hyperstimulation syndrome, polycystic ovarian syndrome, menstrual swelling and convulsions, stroke, head trauma, conjunctivitis, ulceration, cerebral infarction, cerebral infarction , diabetic complications, liver cirrhosis and administration of growth factors. The compounds of the present invention can be used to treat microalbuminuria, proteinuria, oliguria, electrolytic imbalance, nephrotic syndrome, hyperviscosity syndrome, exudates, fibrosis, keloid and formation of unwanted tissue scarring.

The compounds of the present invention may be administered in combination with one or more additional pharmaceutical agents that inhibit or prevent the formation of VEGF, attenuate the intracellular response to VEFG, inhibit vascular hyperpermeability, reduce inflammation, or inhibit or prevent the formation of edema. The compounds of the present invention may be administered prior to, sequentially or concurrently with an additional pharmaceutical agent, any route of administration being suitable. Additional pharmaceutical agents include, but are not limited to, anti-edemic steroids, NSAIDS, Ras inhibitors, anti-TNF agents, anti-IL1 agents, antihistamines, PAF-antagonists, COX-1 inhibitors, COX-2 inhibitors, NO synthase inhibitors , PKC inhibitors and PI ₃ kinase inhibitors. The compounds of the present invention and the additional pharmaceutical agents act either additively or synergistically. Thus, the administration of such combinations of agents that inhibit vascular hyperpermeability and / or inhibit edema formation may provide greater relief from the deleterious effects of vascular hyperpermeability or edema than administration of either agent alone.

Because edema often results from extravasation of the fluid from the bloodstream, hypotension occurs frequently when extravasation occurs. Hypotension may also occur as a result of the activator of VEGF or VEGF binding to VEGF receptors on vascular endothelial cells. The compounds of the invention minimize the development of hypotension by, apparently, inhibiting the cellular signaling function of KDR resulting from the binding of VEGF (or other activating ligands) to this receptor. The compounds of the present invention inhibit hypotension in individuals when administered to those individuals.

-21 Pharmaceutical preparations

The compounds of the invention may be administered to a human patient alone or in pharmaceutical compositions in which they are mixed with suitable carriers or excipient (s) at dosages to treat or ameliorate vascular hyperpermeability, edema, and associated diseases. Mixtures of these compounds may also be administered to the patient in the form of a simple mixture or in suitably formulated pharmaceutical compositions. The therapeutically effective dose further relates to an amount of the compound or compounds that is sufficient to prevent edema, VEGF-associated hyperpermeability, and / or the progression of VEGF-related hypotension. Techniques for formulating and administering instant application compounds can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, last edition.

Routes of administration

Suitable routes of administration may include, for example, oral administration, eye drops, rectal, transmucosal, topical or intestinal administration; parenteral administration, including intramuscular, subcutaneous administration, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal or intraocular injection.

Alternatively, the compound may be administered in a local rather than systemic manner, for example by injecting the compound directly into the oedematous site, often as a sustained-release preparation or a sustained release preparation.

In addition, the drug may be administered as a targeted drug delivery system, for example, as a liposome coated with an endothelial cell-specific antibody.

Compositions / Preparations

The pharmaceutical compositions of the present invention can be produced by a method known in the art, for example, by means of conventional mixing, dissolving, granulating, dragee-making, fine-crushing, emulsifying, encapsulating, entrapping or lyophilizing processes.

-22r r

C r

Thus, pharmaceutical compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers containing excipients and excipients which facilitate the incorporation of the active compounds into preparations which can be used pharmaceutically. The suitable formulation depends on the route of administration chosen.

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

For oral administration, the compounds may be formulated simply by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers allow the compounds of the present invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by the patient to be treated. Pharmaceutical preparations for oral use can be obtained by combining the active compound with a solid excipient, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable excipients, if necessary, to obtain tablet cores or dragees. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulosic preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP). If desired, disintegrating agents such as cross-linked polyvinylpyrrolidone, agar or alginic acid or a salt thereof, such as sodium alginate, may be added.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopolgel, polyethylene glycol, and / or titanium dioxide, coating solutions and suitable organic solvents or solvent mixtures. To

- 23 r f yy r, r- »1 c yy y ry ·.

Dyestuffs or pigments may be added to identify or characterize different combinations of active ingredient doses, or tablets, or dragees may be added to the tablet coatings or dragees.

Pharmaceutical compositions which can be used orally include extrusion capsules prepared from gelatin, as well as soft, hermetically sealed capsules prepared from gelatin and a plasticizer such as glycerol or sorbitol. The extrusion capsules may contain the active ingredients in admixture with a filler such as lactose, a binder such as starches, and / or lubricants, such as talc or magnesium stearate, and optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, 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 lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention are conventionally administered in the form of an aerosol spray from a pressurized pack or nebulizer, using a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases. In the case of pressurized aerosols, the dosage unit may be determined by means of a metered delivery valve. Capsules and cartridges, for example gelatin for use in an inhaler or insufflator, may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, for example, as ampoules or multidose containers, with the addition of a preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating 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 in the form of suitable oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or esters of synthetic fatty acids such as ethyl oleic acid or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspensions may also contain suitable stabilizers or agents which increase the solubility of the compounds, thereby allowing the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for use with a suitable vehicle, for example sterile, pyrogen-free water, before use.

The compounds may also be formulated as 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 above, the compounds may also be formulated as sustained-release formulations. Such sustained-release preparations may be administered by implantation (for example, subcutaneously or intramuscularly or by intramuscular injection). Thus, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or as ion exchange resins, or as poorly soluble derivatives, for example, as a poorly soluble salt.

An example of a pharmaceutical carrier for the hydrophobic compounds of the present invention is a cosolvent system comprising benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. The co-solvent system may be a VPD co-solvent system. VPD is a 3% w / v solution. benzyl alcohol, 8% w / v non-polar polysorbate 80 surfactant and 65% w / v; polyethylene glycol 300, made up to volume in absolute ethanol. The VPD cosolvent system (VPD: 5W) consists of VPD diluted 1: 1 with 5% dextrose in aqueous

-25r r. rr '· r. · _Γ p _{r Γ} f, _r r, - r r r solution. This cosolvent system dissolves hydrophobic compounds well and produces low toxicity upon systemic administration alone. Of course, the proportions of the cosolvent system can vary considerably without compromising its solubility and toxicity characteristics. In addition, the identity of the cosolvent components may vary, for example, other non-polar surfactants may be used.

low toxicity substances instead of polysorbate 80; the fraction size of the polyethylene glycol may vary; polyethylene glycol may be replaced by other biocompatible polymers such as polyvinylpyrrolidone; and other sugars or polysaccharides may be used instead of dextrose.

Alternatively, other delivery systems may be used for the hydrophobic pharmaceutical compounds. Liposomes and emulsions are well known examples of delivery aids or carriers for hydrophobic drugs. Certain organic solvents, such as dimethyl sulfoxide, may also be used, although usually at the cost of greater toxicity. In addition, the compounds can be delivered using a sustained release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained release materials have been introduced and are well known to those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for several weeks up to more than 100 days. Depending on the chemical nature and biological stability of the therapeutic reagents, additional strategies for protein stabilization may be used.

. The pharmaceutical compositions may also contain suitable 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 organic molecular compounds of the present invention can be used in the form of salts with pharmaceutically compatible counterions. Pharmaceutically compatible salts can be formed with many acids including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, and the like.

-26podobne. The salts tend to have higher solubility in aqueous or other proton solvents than the corresponding free base forms.

Effective dose

Pharmaceutical compositions suitable for use in the present invention include compositions in which the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent the development or amelioration of the existing symptoms of the subject being treated. Determination of effective amounts is within the skill of those skilled in the art.

An effective dose of the compound inhibits the cellular signaling function of KDR in a sufficient manner to suppress vascular hyperpermeability without causing significant adverse effects due to inhibition of Flt-1 or other tyrosine kinase functions. Certain compounds that exhibit such activity can be identified using in vitro assays that determine dose-dependent inhibition of KDR tyrosine kinase. Preferred compounds exhibit an IC ₅₀ versus KDR that is significantly lower than the IC 50 over Flt-1 or other PTKs determined under similar conditions [ATP] / K _m (ATP) and substrate (ideally, ~ 100x selective for KDR tyrosine kinase).

For any compound used in the present invention, the therapeutically effective dose can be initially determined from cellular samples. For example, a dose can be formulated in cellular and animal models to achieve a circulating concentration range that includes IC ₅₀ , as determined in cellular samples (ie, the concentration of test compound that achieves half maximal inhibition of cellular signaling function of KDR, usually in response to VEGF or other activation prompt). Determination of cellular IC ₅₀ in the presence of 3-5% serum albumin may approximate the binding effects of plasma protein on the compound. Such information may be used to more accurately determine useful doses in humans. In addition, most preferred compounds for systemic administration effectively inhibit the cellular signaling function of KDR in intact cells at levels that can be safely achieved in plasma.

The therapeutically effective dose is an amount of a compound that results in an improvement in the patient's symptoms. Toxicity and therapeutic efficacy of such compounds can be determined using standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the maximum tolerated dose (MTD) and ED ₅₀ ((effective dose at 50% maximum response). The dose ratio between toxic and therapeutic effects is the therapeutic index, and this can be expressed as the ratio between MTD and ED ₅₀ . Compounds that exhibit high therapeutic endpoints are preferred. Data obtained from these cell culture samples and animal studies can be used in formulating a dosage range for human use. The dose of such compounds lies preferably within a range of circulating concentrations that contain the ED 50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dose can be selected by the attending physician taking into account the patient's condition. (See, for example, Fingl et al., 1975, The Pharmacological Basis of Therapeutics, Chapter 1, p. 1). Acute bolus or MTD infusion may be required for emergency response to achieve rapid response.

Dosage amounts and intervals may be adjusted individually to provide plasma levels of the effective fraction that are sufficient to maintain the modulating effects of KDR or minimum effective concentration (MEC). The MEC will vary for each compound, but can be determined for in vitro data, for example, the concentration required to achieve 50-90% inhibition of KDR tyrosine kinase using the assays described herein. The dosages required to achieve the MEC will depend on the individual characteristics and route of administration. However, HPLC samples and bio-samples may be used to determine plasma concentrations.

Dosage intervals can also be determined using the MEC value. Compounds should be administered using a regimen that plasma levels above MEC for 10 to 90% of the period, preferably between 30 and 90%, and particularly preferably between 50 and 90%, until the desired symptom improvement is achieved. In cases of local administration or selective absorption, the effective local concentration of the drug should not be related to the plasma concentration.

-28r c

The amount of composition administered will, of course, depend upon the subject being treated, the subject's weight, the severity of the disease, the mode of administration, and the judgment of the attending physician.

packing

The compositions may be provided, as appropriate, in a pack or dispenser device, which may contain yen or more unit dosage forms containing the active ingredient. For example, the pack may comprise a metal or plastic foil, such as a blister pack. Administration instructions may be attached to the package or dispensing device. Compositions comprising a compound of the present invention adapted on a compatible pharmaceutical carrier, placed in a suitable container and labeled with a treatment for the indicated condition, may also be prepared. Suitable conditions indicated on the label may include treatment of edema, inhibition of vascular hyperpermeability and extravasation of stromal deposition, minimization of VEGF-related hypotension, and the like.

DETAILED DESCRIPTION OF THE INVENTION

I. In vitro PTK assay

The following in vitro assays may be used to determine the level of activity and effect of the various compounds of the present invention on one or more PTKs. Similar assays can be designed in this manner for other tyrosine kinases using procedures well known in the art.

A. Preparation of KDR tyrosine kinase using baculovirus system:

The coding sequences for the human KDR intracellular domain (aa789-1354) were generated by PCR using cDNA isolated from HUVEC cells. The poly-His6 sequence was also introduced at the N-terminus of this protein. This fragment was cloned into the pVL1393 transfection vector at the Xba 1 and Not 1 sites. Recombinant baculovirus (BV) was generated by co-transfection using BaculoGold transfection reagent (PharMingen). Recombinant BV was plaque purified and verified by Western analysis. For preparation

-29e e e e

PP-P r 'p C r C Γ rer-rpprr protein SF-9 cells were grown in SF-900-II medium at 2 x 10 ⁶ / ml, and infected at 0.5 plaque-forming units per cell (MOI) ). Cells were harvested 48 hours after infection.

B. Purification of the DRC

SF-9 cells expressing (His) ₆ KDR (aa789-1354) were lysed by adding 50 ml of Triton X-100 lysis buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol, 1% Triton X-100 , 1 mM PMSF, 10 µg / ml aprotin, 1 µg / ml leupeptin) per cell pellet from 1 L 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 used in a 5 ml NiCb chelating sepharose column, equilibrated with 50 mM HEPES, pH 7.5, 0.3 M NaCl. KDR was eluted using the same buffer containing 0.25 M imidazole. Column fractions were analyzed using SDS-PAGE and an ELISA assay (below) to measure kinase activity. The purified KDR was exchanged into 25 mM HEPES, pH 7.5, 25 mM NaCl, 5 mM DTT buffer and stored at 80 ° C.

C. Preparation and purification of human Tie-2 kinase

The coding sequence for the human Tie-2 intracellular domain (aa775-1124) was generated by PCR using cDNAs isolated from the human placenta matrix. A poly-His6 sequence was introduced at the N-terminal and this construct was cloned into the pVL 1939 transfection vector at the Xba 1 and Not 1 sites. Recombinant BV was generated by co-transfection using BaculoGold Transfection reagent (PharMingen). Recombinant BV was plaque purified and verified by Western analysis. For protein preparation, SF-9 insect cells were grown in SF-900-II medium at 2 x 10β / β, and infected at an MOI of 0.5. Purification of His-recipient kinase used in screening was analogous to that described for KDR.

D. Preparation and Purification of Human Flt-1 Tyrosine Kinase

The baculovirus expression vector pVL1393 (Phar Mingen, Los Angeles, CA) was used. The nucleotide sequence encoding poly-His ₆ was located 5 'to the nucleotide region encoding the total intracellular kinase domain of human Flt-1.

(Amino acids 786-1338). The nucleotide sequence encoding the kinase domain was generated by PCR using cDNA standards isolated from HUVEC cells. Histidine residues allowed affinity purification of the protein in a manner analogous to that for KDR (Part B.) and ZAP70 (Part F.) SF-9 insect cells were infected at 0.5 abundance and harvested 48 hours post infection.

E. Sources of Lck and EGFR tyrosine kinases

Lck or truncated forms of Lck were obtained commercially (e.g., from Upstate Biotechnology Inc., Saranac Lake, NY or Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or purified from known natural or recombinant sources using conventional procedures. EGFR was purchased from Sigma (Cat # E-3641; 500 units / 50 μΙ) and EGF ligands were purchased from Oncogene Research Products / Calbiochem (Cat # PF011-100).

F. Preparation of ZAP70 Tyrosine Kinase

Baculovirus expression of the pVL 1393 vector (Phar Migen, Los Angeles, CA) was used. The nucleotide sequence encoding poly-His6 was located 5 'to the nucleotide region of total ZAP70 (amino acids 1 to 619). The nucleotide sequence encoding the ZAP70 coding region was generated by PCR using cDNA standards isolated from Jurkat immortalized T cells. The histidine residues allowed affinity purification of the protein (see Part B.). The LVPRGS bridge constituted the recognition of the sequence for proteolytic cleavage by thrombin, allowing the removal of affinity labeling from the enzyme. SF-9 insect cells were infected at a frequency of 0.5 and harvested 48 hours after infection.

G. ZAP70 Purification

SF-9 cells were lysed in a buffer containing 20 nM Tris, pH 8.0, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 1 µg / ml leupeptin, 10 µg / ml aprotin and 1 mM sodium ortovanadate. The soluble lysate was applied to a chelating sepharose Hi Trap column (Pharmacia) equilibrated in 50 mM HEPES, pH 7.5, 0.3 M NaCl. The fusion protein was eluted with 250 mM imidazole. trapped

-31 e r c f * e r Γ r Z9 r c r i - <·, ·.

The enzyme was stored in a buffer containing 50 mM HEPES, pH 7.5, 50 mM NaCl, and 5 mM DTT.

H. Enzyme-linked Immunosorbent Assay (ELISA) for RTK

Enzyme-linked immunosorbent assays (ELISA) were used to detect and measure the presence of tyrosine kinase activity. ELISAs were performed according to known protocols as described, for example, in Aviary, et al., 1980, Enzyme-Linked Immunosorbent Assay, V: Manual of Clinical Immunology, 2d ed.

Ed. Rose and Friedman, p. 359-371 Am. Soc. of Microbiology, Washington, D.C.

The published protocol was adapted to determine activity relative to a specific RTK. For example, a preferred protocol for performing ELISA experiments for DRC is shown below. Adaptation of this protocol to determine the activity of the compound for other members of the RTK family as well as non-receptor tyrosine kinases are within the scope of their ability in the art. For the determination of inhibitor selectivity, a universal substrate (for example, a random copolymer of poly (Glu ₄ Tyr), with a molecular weight of 20,000 to 50,000) was used together with ATP (typically 5 μΜ) at concentrations approximately approximately twice the apparent Km in the sample.

KDR in vitro ELISA

The following procedures were used to evaluate the inhibitory effect of the compounds of the present invention on KDR tyrosine kinase activity:

Buffers and solutions

PGT: Poly (Glu, Tyr) 4: 1

Store powder at -20 ° C. Dissolve the powder in phosphate buffered saline (PBS) to a 50 mg / ml solution. Store 1 ml aliquot at -20 ° C. Dilute to 250 µg / ml in Gibco PBS to form plates.

Reaction Buffer:

100 mM Hepes, 20 mM MgCl 2, 4 mM MnCl 3, 5 mM DTT, 0.02% BSA, 200 μΜ NaVO ₄ , pH 7.10.

-32ATP:

Store aliquots of 100 mM at -20 ° C. Dilute to 20 μΜ in water. Wash Buffer:

PBS with 0.1% Tween 20

Antibody dilution buffer:

0.1% bovine serum albumin (BSA) in PBS

TMB Substrate:

Mix 9: 1 TMB substrate and peroxide solutions immediately before use or use Neogen K-Blue substrate

Stop solution

M phosphoric acid

Workflow

1. Preparation of plates

Dilute PGT stock solution (50 mg / ml, frozen) in PBS to 250 µg / ml. Add 125 μΙ per well of Corning modified high-affinity flat bottom ELISA plates (Corning # 25805-96). Add 125 μΙ PBS to empty wells. Cover with sealing tape and incubate overnight at 37 ° C. Wash 1x with 250 μΙ wash solution and dry for approximately 2 hours in a dry incubator at 37 ° C.

Store coated plates in sealed bags at 4 ° C until use.

2. Tyrosine kinase reaction:

- Prepare inhibitor solution at 4x concentration in 20% DMSO in water.

- Prepare reaction buffer

-33e o r r p e <·, r 'i' r r e e r <.- / · <»

C λ r r r

- Prepare the enzyme solution so that the required units are at 50 μΙ, for example for KDR n adjust to 1 ng / μΙ for a total of 50 ng per well in reactions. Store on ice.

- Prepare 4 x ATP solution at 20 μΜ from 100 mM stock solution in water. Store on ice.

- Add 50 μΙ of enzyme solution per well (typically 5 to 50 ng enzyme / well depending on specific kinase activity)

- Add 25 μΙ 4 x inhibitor

Add 25 μΙ 4 x ATP per inhibitor sample

- Incubate for 10 minutes at room temperature

- Stop the reaction by adding 50 μΙ 0.05 N HCl per well

- Wash plate ** Final concentrations for reaction:

ATP: 5 μΜ% DMSO

3. Antibody binding

- Dilute 1 mg / ml aliquot of PY20-HPR (Pierce) antibody (phosphotyrosine antibody) to 50 ng / ml in 0.1% BSA in PBS by dilution in 2 steps (100 x then 200 x)

- Add 100 μΙ Ab per well. Incubate for one hour at room temperature. Incubate for one hour at 4 ° C.

- Wash 4 plates

4. Color reaction

- Prepare the TMB substrate and add 100 μΙ per well

- Monitor OD at 650 nm until 0.6.

- Stop with 1 M phosphoric acid, Shake on a plate reader.

- Immediately read OD at 450 nm.

-34 * f

C C C Γ: * r r c r ť * β C <'' '

Q n f r r · r '

Optimal incubation times and enzymatic reaction conditions slightly vary according to enzyme preparations and are determined empirically for each group.

Analogous experimental conditions were used for Flt-1, Tie-2, EGFR and ZAP70. For Lck, a reaction buffer consisting of 100 mM MOPSO, pH 6.5, 4 mM MnCl ₂ , 20 mM MgCl ₂ , 5 mM DTT, 0.2% BSA, 200 mM NaVO _{4 was} used.

analogous experimental conditions.

Source of PKO kinase

The catalytic PKC subunit can be obtained commercially (Calbiochem).

PKC Kinase Assay

A radioactive kinase assay was performed using the following 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 kinase buffer consisting of 50 mM Tris-HCl pH7.5, 10 mM MgCl ₂ , 2 mM DIT, 1 mM EGTA, 100 μΜ ATP, 8 μΜ peptide, 5% DMSO and ³³ P ATP ( 8 (Ci / mM). Compounds and enzyme were mixed in a reaction vessel and the reaction was initiated by addition of ATP and substrate mixture. After completion of the reaction by adding 10 μΙ of stop buffer (5 mM ATP in 75 mM phosphoric acid), a portion of the mixture was applied to phosphocellulose filters. The loaded samples were washed three times in 75 mM phosphoric acid at room temperature for 5 to 15 minutes. Incorporation of radiolabeled isotopes was quantified using a liquid scintillation counter.

Estrogen receptor binding assays

Binding of 1 nM radiolabeled 17β-estradiol to the human estrogen receptor in the cytosol of mammalian cancer tumor MCF-7 cells was determined after incubation for 20 hours at 4 ° C using reaction conditions as described by Shein et al., Cancer Res. 45, 4192 (1985) (incorporated herein by reference). After incubation, the cytosolic fractions were mixed with a suspension of dextran coated activated carbon for 10 minutes at 4 ° C, centrifuged and the supernatant collected. Bound radioactivity remaining in the activated carbon supernatant was added

I

- 35 t rr rr. Rr rrr r was measured using a scintillation counter (LS 6000, Beckman) using a liquid sensitization cocktail (Formula 989 Packard). Compounds were tested simultaneously at eight concentrations in duplicate to obtain a competitive curve to quantify inhibitor activity. Specific binding of the radioligand to the estrogen receptor was defined as the difference between total binding and non-specific binding determined in the presence of an excess of unlabeled 17β-estradiof (6 μΜ). .

The results

The following inhibitory concentrations of representative compounds of the structural formula were obtained: θ ° = _s = Q

sample IC ₅₀ for compound DRC 0.20 μΜ Flt-1 > 50.0 μΜ lck > 50.0 μΜ TIE2 > 50.0 μΜ ZAP70 > 50.0 μΜ EGFR > 50.0 μΜ PKC > 50.0 μΜ Estrogen receptor > 10.0 μΜ (<10% inh @ 10 μΜ)

These results demonstrate that the compounds of the present invention and exemplified herein exhibit significant KDR tyrosine kinase inhibitory activity and are particularly selective as KDR tyrosine kinase inhibitors.

-36II. Cellular RTK tests

The following cellular assays were used to determine the level of activity and effect of the various compounds of the present invention on KDR. Similar assays can be designed in the same series for other tyrosine kinases using appropriate antibody reagents and methodologies such as immunoprecipitation and Western blotting, which are well known in the art.

A. VEGF-Induced KDR Phosphorylation in Human Umbilical Vein Endothelial Cell (HUVEC) Endothelial Cells as Measured by Western Blot Assays.

1. HUVEC cells (from pooled donors) were purchased from Clonetics (San Diego, CA) and cultured according to the manufacturer's instructions. Only the primary flow rates (3 to 8) were used in this test. Cells were cultured in 100 mm dishes (Falcon for tissue culture; Becton Dickinson; Plymouth, England) using complex EBM medium (Clonetics).

2. To evaluate the inhibitory activity of the compound, cells were trypsinized and seeded at 0.5 to 1.0 x 10 ⁵ cells / well in each of the 6-well cluster plates (Costar; Cambridge, MA).

3. Three to four days after seeding, the plates were run down to 90-100%. Medium was removed from all wells, cells were washed with 5-10 ml PBS and incubated for 18-24 hours with 5 ml EBM basic medium without the addition of additives (i.e. serum fasting).

4. Sequential dilution of inhibitors was added in 1ml EBM medium (25 μΜ, 5 μΜ or 1 μΜ final concentration) to the cells and incubated for one hour at 37 ° C. Human recombinant VEGF (165) (R&D systems) was then added to all wells in 2 ml EBM medium at a final concentration of 50 ng / ml and incubated at 37 ° C for 10 minutes. Control cells untreated or treated with VEGF were used only to assess background phosphorylation and VEGF-induced phosphorylation.

-37e r ·

5. All wells were then rinsed with 5-10 ml cold PBS containing 1 mM sodium orthovanadate (Sigma) and cells were lysed and scraped into 200 μΙ RIPA buffer (50 mM Tris-HCl) pH 7, 150 mM NaCl, 1% NP- 40, 0.25% sodium deoxycholate, 1 mM EDTA) containing protease inhibitors (PMSF 1 mM, aprotin 1 µg / ml, pepstatin 1 µg / ml, leupeptin 1 µg / ml, sodium vanadate 1 mM, sodium fluoride 1 mM) and 1 pg / ml Dnase (all chemicals from Sigma Chemical Company, St Louis, MO). The lysate was centrifuged at 14,000 rpm for 30 minutes to eliminate the core.

6. Equal amounts of proteins were then precipitated by addition of cold (20 ° C) ethanol (2 volumes) for at least one hour or maximum overnight. The granules were reconstituted in Laemli sample buffer containing 5% β-mercaptoethanol (BioRad; Hercules, CA) and boiled for 5 minutes. Proteins were resolved by polyacrylamide gel electrophoresis (6%, 1.5 mm Novex, San Diego, CA) and transferred to a nitrocellulose membrane using a Novex system. After blocking with bovine serum albumin (3%), proteins were tested overnight with anti-KDR polyclonal 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 incubating for one hour with HRP-conjugated F (ab) 2 goat anti-rabbit or goat anti-mouse IgG, the bands were visualized using an emission chemiluminescence (ECL) system (Amersham Life Sciences, Arlington Heights, IL).

The results

Inhibitory concentrations of a representative compound of formula I: o ‘

O = _s = 0

-38e r r e r

C yy yy were:

HUVEC Cellular IC ₅₀ for compound KDR phosphorylation 5 to 10 μΜ

This compound also demonstrated selectivity of KDR tyrosine kinase (see para

I) ·

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

III. Model of uterine edema

This assay determined the capacity of the compounds to inhibit the acute increase in uterine weight in mice in the first few hours after estrogen stimulation. It is known that this early onset of uterine weight increase is due to edema due to the increased permeability of the uterine vasculature. Cullinan-Bove and Koss (Endocrinology (1993), 133, 829-837) demonstrated a close temporal relationship of estrogen-stimulated uterine edema with increased VEGF mRNA expression in the uterus. These results were confirmed by the use of a neutralizing monoclonal antibody to VEGF, which significantly reduced the acute increase in uterine weight after estrogen stimulation (WO 97/42187). Therefore, 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) in the form of lyophilized powders and prepared according to the supplier's instructions.

Excipient ingredients (DMSO, Cremaphor EL) were obtained from Sigma (St. Louis, MO).

Mice (Balb / c, 8-12 weeks of age) were obtained from Taconic (Germantown, NY) and housed in pathogen-free animal husbandry facilities, in accordance with the institutional guidelines of the Committee for Animal Breeding and Use.

-39Metódy:

Day 1: Balb / c mice were injected intraperitoneally with 12.5 units of pregnant mares serum gonadotropin (PMSG).

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

Day 4: Mice were randomized into groups of 5 to 10. Test compounds were administered by the intraperitoneal, intravenous or oral route, depending on solubility and excipient at doses ranging from 1 to 200 mg / kg. The excipient control group received only the excipient and the two groups were left untreated.

. Typically thirty minutes later, experimental groups, excipient groups, and one of the untreated groups were injected intraperitoneally with 17β-estradiol (500 µg / kg). After two to three hours, the animals were sacrificed by CO2 inhalation. After cutting the midline, each uterus was isolated and removed by cutting just below the cervix and the uterine junction and the fallopian tubes. The fat and connective tissue were carefully removed so as not to compromise the integrity of the uterus before weighing. Mean weights in the treatment groups were compared to the untreated or excipient treated groups. Significance was determined using the Student's test. Unstimulated control groups were used to monitor the estradiol response.

The results

Percent inhibition of uterine edema following estradiol stimulation for a representative compound of the structural formula:

⁰ - s ⁼ °

-40 was obtained for three routes of administration at doses of 100 mg / kg.

Method of administration % inhibition Value p oral 17 nonsignificant intravenous 57 0.01 intraperitoneally 52 0.04

Compound I has been shown herein to be KDR-selective for inhibiting kinase activity in vitro and effective in blocking cellular autophosphorylation of KDR in response to VEGF-stimulus.

These results demonstrate that suitable compounds of the present invention, such as a compound of formula I, that selectively inhibit KDR function effectively block edema. The results also demonstrate that intravenous and intraperitoneal administration are particularly effective for this compound. Importantly, similar anti-edema efficacy results were also obtained with numerous structurally different selective inhibitors of KDR function.

equivalents

Since the present invention has been illustrated and described in particular with reference to preferred embodiments thereof, those skilled in the art will appreciate that various changes in form and details may be made without departing from the spirit and scope of the invention as defined in the appended claims. . Those skilled in the art will find, or will 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 fall within the scope of the claims.

Claims (28)

Use of a compound that inhibits the cellular signaling function of KDR for the preparation of a medicament for inhibiting vascular hyperpermeability in an individual. The use of claim 1, wherein said inhibition of the cellular signaling function of KDR is selective for the KDR signaling function. The use of claim 1, wherein said cellular signaling function of KDR is stimulated by binding of an activating ligand to the receptor portion of KDR. The use of claim 3, wherein said inhibition of the cellular signaling function of KDR is selective for the signaling function of KDR. The use of claim 1, wherein said inhibition of the cellular signaling function of KDR comprises blocking the production of an activating ligand, modulating the binding of an activating ligand to the KDR receptor tyrosine kinase, disrupting receptor dimerization, blocking KDR trans-phosphorylation, inhibiting KDR tyrosine kinase activity, decreasing uptake of intracellular KDR substrates. disruption of the signaling flow initiated through the phosphorylation activity of KDR tyrosine kinase. The use of claim 5, wherein said inhibition of the cellular signaling function of KDR is selective for the KDR signaling function. The use of claim 1, wherein said compound inhibits the kinase catalytic activity of said KDR. The use of claim 1, wherein said compound is an antagonist of KDR tyrosine kinase activation. The use of claim 1, wherein said compound selectively inhibits phosphorylation of KDR kinase substrates. The use of claim 1, wherein said compound is selective for said KDR tyrosine kinase. -4211. The use of claim 10, wherein said compound is selected from the group consisting of peptides, antibodies, and organic molecules, wherein said compound binds to said KDR tyrosine kinase. Use according to claim 11, wherein the disease condition is selected from the group consisting of macular edema, aphakic / pseudoaphakic cystoid macular edema, retinoblastoma, ocular ischemia, ocular inflammatory disease or infection, choroidal melanoma, edematous side effects induced by chelating pulmonary edema, iron chelation therapy, myocardial infarction, rheumatoid diseases, anaphylaxis, tissue edema at sites of injury or allergic inflammation, allergy, hypersensitivity reactions, polyp edema at sites of chronic inflammation, cerebral edema, fluid-filled cerebral tumor cysts, communicative hydrocephalus, carpal canal dysfunction syndrome, , inhalation burn injury, skin burns, sunburn blisters, irritation or infection, erythema multiforme, edematous macula and other skin diseases, brain tumors, tumor effusions, lung or breast cancers, ascites γ, pleural effusions, pericardial effusions, "altitude disease", radioanaphylaxis, radiodermatitis, glaucoma, conjunctivitis, choroidal melanoma, adult respiratory distress syndrome, asthma, bronchitis, ovarian ovarian hyperstimulation syndrome, , stroke, head trauma, cerebral infarction or occlusion, hypotension, ulceration, sprains, fractures, synovitis-related effusions, diabetic complications, hyperviscosity syndrome, liver cirrhosis, microalbuminuria, proteinuria, oliguria, fibrosis, electrolytic nephrosis, electrolytic nephrosis and administration of growth factors. The use of claim 10, wherein the inverse effects associated with a change in the cellular signaling function of tyrosine kinases other than KDR are excluded if the dosage form comprises said compound. The use of claim 1, wherein said compound is selected from the group consisting of single chain antibodies, KDR-specific ribozymes, and antisense polynucleotides, wherein said compound is introduced or is administered. It produces intracellularly, thereby inhibiting self-presentation of functional KDR tyrosine kinase. The use of claim 1, wherein the dosage form comprises said compound in combination with a pharmaceutical agent selected from the group consisting of an antiedemic steroid, Ras inhibitor, anti-TNF agents, anti-IL1 agents, antihistamine, PAF antagonist, COX-1 inhibitor, inhibitor COX-2, an inhibitor of NO synthesis, nonsteroidal anti-inflammatory agent (NSAID), a PKC inhibitor and PI ₃ kinase. Use of a compound that inhibits the cellular signaling function of KDR for the preparation of a medicament for inhibiting a physiological process or condition in an individual, wherein said physiological process or condition is selected from the group consisting of edema, diapedesis, extravasation, effusion, exudation, ascites formation, matrix deposition and vascular hypotension. The use of claim 16, wherein said compound is selective for said KDR tyrosine kinase. The use of claim 17, wherein said compound is selected from the group consisting of peptides, antibodies, and organic molecules, wherein said compound binds to said KDR tyrosine kinase. The use according to claim 18, wherein the disease state is selected from the group consisting of macular edema, aphakic / pseudoaphakic cystoid macular edema, retinoblastoma, ocular ischemia of ocular inflammatory disease or infection, choroidal melanoma, edematous side effects induced by chelating pulmonary edema, chelating pulmonary therapy, myocardium, rheumatoid diseases, anaphylaxis, tissue edema at the site of injury or allergic inflammation, allergy, hypersensitivity reactions, polyp edema at sites of chronic inflammation, cerebral edema, brain tumor cysts filled with fluid, communicative hydrocephalus, carpal canal syndrome, organ damage resulting from organ damage inhalation burn injury, skin burns, sunburn blisters, irritation or infection, erythema multiforme, edematous macula and other skin diseases, brain tumors, tumor effusions, lung or breast cancers, ascites y, pleural effusions, pericardial effusions, "altitude sickness" radioanaphylaxis, radiodermatitis, -44glaucoma, conjunctivitis, choroidal melanoma, adult respiratory distress syndrome, asthma, bronchitis, ovarian overstimulation syndrome, polycystic ovarian syndrome, menstrual swelling, menstrual cramps, stroke, head trauma, ulcer, brain trauma, cerebral palsy, ulcer, hypotension, ulcer fractures, synovitis-associated effusions, diabetic complications, hyperviscosity syndrome, liver cirrhosis, microalbuminuria, proteinuria, oliguria, electrolytic imbalance, nephrotic syndrome, exudates, fibrosis, keloid, and administration of growth factors. The use of claim 16, wherein said compound inhibits the catalytic activity of the kinase of said KDR. The use of claim 16, wherein said compound is an antagonist of KDR tyrosine kinase activation. The use of claim 16, wherein said compound selectively inhibits phosphorylation of KDR kinase substrates. The use of claim 16, wherein said compound is selective for said KDR tyrosine kinase. The use of claim 16, wherein said cellular signaling function of KDR is stimulated by binding of an activating ligand to the receptor portion of KDR. The use of claim 24, wherein said compound is selective for said KDR tyrosine kinase. The use of claim 16, wherein said compound is selected from the group consisting of single chain antibodies, KDR-specific ribozymes and anti-sensitive polynucleotides, wherein said compound is introduced or produced intracellularly, thereby inhibiting self-presentation of a functional KDR tyrosine kinase. The use of claim 16, wherein said inhibition of the cellular signaling function of KDR comprises blocking the production of an activating ligand, modulating the binding of an activating ligand to a KDR receptor tyrosine kinase, disrupting receptor dimerization, blocking trans-phosphorylation of KDR, inhibiting KDR activity -45 tyrosine kinase, decreased uptake of intracellular KDR substrates, and disruption of signaling flow initiated through the phosphorylation activity of KDR tyrosine kinase. Use according to claim 16, wherein the inverse effects associated with a change in the cellular signaling function of tyrosine kinases other than KDR are excluded if the dosage form comprises said compound. The use of claim 16, wherein the dosage form comprises said compound in combination with a pharmaceutical agent selected from the group consisting of an anti-dermal steroid, an Ras inhibitor, an anti-TNF agent, an anti-IL1 agent, an antihistamine, a PAF antagonist, a COX-1 inhibitor, COX-2, an inhibitor of NO synthesis, nonsteroidal anti-inflammatory agent '(NSAID), a PKC inhibitor and PI ₃ kinase.