WO2004006947A1 - A method for inhibiting vascular permeability and tissue edema - Google Patents

Abstract

A method for inhibiting or reducing increase in vascular permeability of blood vessels in a subject following exposure of the blood vessels to an elevated level of a factor, e.g. a vascular endothelial growth factor (VEGF), which increases vascular permeability comprises administering a Rho family member antagonist to the subject.

Description

A METHOD FOR INHIBITING VASCULAR PERMEABILITY AND TISSUE EDEMA

Field of the invention

The present invention relates to the regulation of vascular permeability and angiogenesis mediated by various factors, notably growth factors, such as growth factors of the vascular endothelial growth factor (VEGF) family or type.

Background of the invention

VEGF (more specifically VEGF-A) is a member of a family of homologous peptides that influence vascular permeability and angiogenesis. The family in question includes at least ten structurally related proteins, viz.: VEGF-A, VEGF-B, VEGF-C, VEGF-D and VEGF-E; placenta growth factor (PIGF); EG-VEGF and various platelet- derived growth factors (PDGF), viz. PDGF-A, PDGF-B, PDGF-C and PDGF-D (Nicosia, 1998); (Ogawa, Oku et al., 1998); (Veikkola and Alitalo, 1999); (LaRochelle, Jeffers et al., 2001); (LeCouter, Kowalski et al., 2001). At least three biological functions, viz. vasculogenesis, angiogenesis and vascular permeability, are mediated by the different isoforms of VEGF (Risau and Flamme, 1995). VEGF family members are key factors involved in initiation of angiogenesis under physiological as well as pathological conditions (Ferrara and Alitalo, 1999). They are responsible for angiogenesis in the female reproduction cycle, during wound healing, and in a variety of pathological conditions, such as tumor growth and metastasis, diabetic retinopathy, and rheumatoid arthritis (Folkman, 1995; Ferrara and Davissmyth, 1997).

Three structurally related high-affinity receptors for the members of the VEGF family have been identified, viz. VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and VEGFR- 3 (Flt-4) (Mustonen and Alitalo, 1995). VEGFR-3 is specifically expressed in lymphatic endothelial cells (Lymboussaki et al., 1998), whereas VEGFR-1 and VEGFR-2 are expressed in blood vessel endothelial cells (Millauer et al., 1993). Both VEGF-C and VEGF-D bind to VEGFR-2 and VEGFR-3 and induce angiogenic and lymphangiogenic responses (Achen et al., 1998; Cao et al., 1998; Marconcini et al., 1999), whilst VEGF-B and PIGF bind only to VEGFR-1 (Cao et al., 1996; Olofsson et al., 1998). The biological functions of these interactions remain to be characterized. VEGF-induced angiogenic responses are mediated by VEGFR-1 and VEGFR-2 (Waltenberger et al., 1994a), and the biological responses to VEGF isoforms via these two receptors appear to be different. For example, activation of VEGFR-2 leads to proliferation and migration of endothelial cells, whereas activation of VEGFR-1 does not. PDGF-A, PDGF-B, PDGF-C and PDGF-D may be important for endothelial cell responses during angiogenesis, and they are generally considered to be cell-survival factors and mitogens for vessel-wall cells such as smooth muscle cells, pericytes and mesangial cells (Abboud, 1995; Uutela et al., 2001). Members of the VEGF family can dimerise with each other in various combinations which may have implications for their receptor interactions and activity (Cao et al., 1996; Olofsson et al., 1998).

VEGF (more specifically VEGF-A) was initially characterized as a vascular permeability factor secreted by tumor cells (Senger et al., 1983). Owing to the fact that tumor vasculature is fenestrated, dilated, irregular, unstable and hemorrhagic, it has been suggested that VEGF is responsible for induction of vascular leakage in tumors. (Roberts and Palade, 1997); Dvorak et al., 1999). In addition to tumors, persistent expression of VEGF, VEGFR-1 and VEGFR-2 is also noted in organs and tissues with fenestrated endothelium, such as the choroid plexus and the kidney glomeruli (Breier et al., 1992). Moreover, interendothelial junctions are believed to be involved in VEGF-mediated endothelial leakage (Kevil et al., 1998). It has therefore been speculated that persistent production of VEGF family members is involved in the induction and maintenance of endothelial fenestrations. Indeed, VEGF is able to induce endothelial fenestrations in vivo in skeletal muscle, eye and skin after a short treatment (Roberts and Palade, 1995; Cao et al., 2001), as well as in vitro in cultured endothelial cells from adrenal cortex (Esser et al., 1998) and from the brain (Fischer et al., 2002).

The fact that VEGF increases vascular permeability is important not only for the spread of tumor cells but also for fluid accumulation in tissue. Transport of fluid across capillaries depends on the driving forces involved, i.e. the intracapillary hydrostatic pressure, the interstitial pressure and the colloid-osmotic pressure in the plasma and in the interstitial fluid, and on the permeability of the capillaries, i.e. on the capillary filtration coefficient (Guyton and Hall, 1996). As a rule there is an outward migration (filtration) of fluid in the arterial end of a capillary which is somewhat greater than the inward migration in the venous part of the capillary. The accumulating fluid is drained by the lymphatic system in most tissues, and via the cerebrospinal fluid in the brain. When the capillary permeability increases, e.g. by the creation of gaps (fenestra) between the endothelial cells, the resulting fluid accumulation causes tissue edema.

VEGF, also known as vascular permeability factor (VPF), has two major functions on blood vessels. While being a potent and specific angiogenic factor in vivo, it also increases vascular leakage. In fact, VEGF is the most potent vascular permeability factor identified so far. It has a more than 50,000-fold greater effect than histamine on blood vessels (Senger et al., 1990; Dvorak et al., 1999). Another unique biological feature of VEGF family members is that their expression can be dramatically upregulated by hypoxia (Makino et al., 2001; Simpson et al., 1999). Thus, conditions such as ischemic heart disease, atherosclerosis, circulatory insufficiency, stroke, diabetes, chronic inflammation, respiratory insufficiency and high-mountain disease can lead to a dramatic increase in VEGF production, thereby increasing vascular permeability (with attendant possible tissue edema) and instigating neovascularization.

An increase in the level of VEGF has important consequences in a number of organs. The resulting increase in capillary permeability leads to edema formation, subsequent tissue swelling and decreased oxygen transport, which in turn augments hypoxia. In the brain, the increased permeability of the blood-brain barrier (BBB), and hence development of edema, is of special importance. This is due to the fact that the cranial cavity in which the brain is positioned is essentially indistensible. Accordingly, brain swelling increases the intracranial pressure, thereby compromising the blood supply as well as causing incarceration/herniation and attendant brain damage. Edema accompanies and often complicates a number of diseases in the central nervous system, e.g. cancer, trauma, infections, and hemorrhagic and occlusive stroke, and has been suggested to be related to VEGF production. Current treatments for cerebral edema are very limited, and include osmotherapy and treatment with glucocorticoids (dexamethasone). The potentially beneficial effects of osmotherapy are often not achieved because osmotherapy shrinks healthy regions of the brain together with the damaged areas. A relationship between the effect of glucocorticoids and their ability to inhibit the expression of VEGF has been proposed (Criscuolo, 1993; Fischer, Renz et al., 2001). The disruption of the BBB and the occurrence of brain edema in meningitis has been suggested to be related to the production of VEGF (van der, Stockhammer et al., 2001), and significant relationships have been found between VEGF expression, Flk-1 expression and glioma malignancy grading, intratumoral vascularity and peritumoral brain edema in humans (Yao et al., 2001), suggesting a link between VEGF expression and development of edema. In human ischemic stroke, an upregulation of VEGF expression was demonstrated in the penumbral areas of the brain (Issa et al., 1999). In experimental focal cerebral ischemia, VEGF expression starts after 1-3 hours and peaks at 24-48 hours (Marti et al., 2000; Croll and Wiegand, 2001). Systemic administration of VEGF immediately following stroke significantly increases BBB leakage, hemorrhagic transformation, and ischemic lesions (Zhang et al., 2000). Expression of other growth factors is also upregulated in focal brain ischemia (Zhang and Chopp, 2002), but VEGF is of crucial importance for the adverse outcome. Treatment with a soluble VEGF receptor chimeric protein, which inactivates endogeneous VEGF, significantly decreased brain edema and the extent of ischemic brain lesions in mice (van Bruggen et al., 1999). However, VEGF may also exert beneficial effects in conjunction with ischemic conditions by stimulation of neovascularisation (vasculogenesis and angiogenesis). In this way, the brain tissue in the penumbral or other regions can establish a blood supply and thereby improve the extent of, and possibly rate of, neurological recovery (Zhang et al., 2000; Marti et al., 2000). In other organs, increased expression of VEGF is also of importance. While the growth of new blood vessels is beneficial in improving the condition of ischemic myocardium, the VEGF-induced edema will put an increased burden on heart function. A similar situation applies in ischemic diabetic retinopathy, in which VEGF-induced edema is responsible for virtually all blindness (wet type of macular degeneration) (Aiello, 1997). In addition, chronic inflammation is also accompanied by VEGF-induced vascular leakage and increases in tissue thickness. Attempts to alleviate ischemic conditions by inducing neovascularization by means of VEGF-based angiogenic therapy may produce serious side-effects due to the formation of highly permeable blood vessels. On the other hand, if the VEGF-induced increase in vascular permeability could be selectively blocked, it should be possible to greatly improve VEGF-based angiogenic therapy.

Despite the great efforts which have been made to understand the signaling pathways mediated by the VEGF receptors, no one has yet been able to separate the angiogenesis and vascular permeability signaling pathways induced by VEGF and related growth factors. One family of proteins that has been implicated in receptor-mediated signalling to the cytoskeleton is the small GTPases of the Rho family (Machesky and Hall, 1996; Ridley, 2001). Peptides in this family include Rho, Rac and Cdc42, and they affect adhesion, actin polymerization and the formation of lamellipodia and filopodia, all of which are processes important for endothelial cell function (Aspenstrδm, 1999).

The present inventor has investigated the therapeutic potential of antagonists of the Rho GTPases, and it has been found, for example, that the Rac antagonist denoted N17Rac (vide infra) selectively blocks the VEGF-induced vascular fenestration, but surprisingly not the VEGF-induced angiogenesis. It has thus proved possible to separate the angiogenesis-inducing effect of VEGF from the effect of VEGF on vascular permeability. Rho family antagonists of the type in question thus appear to have great therapeutic potential in the treatment of numerous diseases or conditions since they counteract the induction of increase in vascular permeability by VEGF substantially without interfering with the beneficial angiogenic effect of VEGF. In stroke, for example, the ischemic cerebral edema induced by VEGF is the main lethal complication in the acute phase (Bounds et al., 1981; Ropper and Shafran, 1984). In relation hereto it would thus be highly desirable to be able to block VEGF-induced edema while allowing new blood vessel growth (i.e. angiogenesis) to proceed in order to improve the ischemic condition. The same is true for ischemic heart disease; thus, while the growth of new blood vessels resulting from the heightened attendant VEGF expression is beneficial in improving the condition of ischemic myocardium, the associated edema will increase the burden on heart function. As a result, VEGF- based angiogenic therapy causes more harm than good. It is thus clear that VEGF-induced increase in vascular permeability must be blocked in clinical trials of VEGF's for the purpose of angiogenic therapy; no agent capable of separating these two biological effects has, however, hitherto been available.

Soga et al. (Soga et al., 2001) in studies of haptotaxis on type I collagen and VEGF-stimulated chemotaxis on type I collagen supports have shown, inter alia, that Rac is required and sufficient to stimulate the motility of human dermal (foreskin) microvascular endothelial cells, and that a Rac inhibitor, N17Rac, inhibited cell motility on type I collagen and totally prevented the stimulation of chemotaxis by VEGF. On the basis of Soga et al.'s results it might thus be expected that a Rac inhibitor of the type employed would also inhibit angiogenesis; however, no data relating to the effect on VEGF-induced angiogenesis were presented.

'I

The present inventor has now surprisingly found not only that a Rac inhibitor of the type in question (which is an example of a Rho family member antagonist or inhibitor) can inhibit the increase in vascular permeability which is normally induced by a growth factor or other factor of the VEGF type, but moreover that this inhibition can be achieved substantially without inhibiting angiogenesis which is normally induced by the factor in question. The present invention derives, inter alia, from the realization on the part of the inventor that VEGF- induced angiogenesis, i.e. blood vessel cell proliferation, and increase in blood vessel permeability are mediated by different intracellular pathways, despite the fact that they share the same cell membrane receptor (VEGFR-2).

Summary of the Invention

The invention relates overall to the use of Rho family member antagonists or inhibitors for therapeutic, diagnostic and research purposes. A first aspect of the present invention thus relates to a method for inhibiting or reducing increase in vascular permeability of blood vessels in a subject (animal or human subject) following exposure of the blood vessels in question to an elevated level of a factor which increases vascular permeability, the method comprising administering to the subject a Rho family member antagonist. As discussed above, increase in vascular permeability of blood vessels is a major cause of tissue edema, and a further aspect of the invention accordingly relates to a method for treating a subject so as to prevent or reduce edema in tissues in the subject which contain blood vessels which have been exposed to an elevated level of a factor which increases vascular permeability, the method comprising administering to the subject a Rho family member antagonist, i.e. a substance which is an antagonist or inhibitor of peptides or proteins belonging to the Rho family of GTPases (vide infra) or which is an inhibitor or antagonist of Rho regulatory pathways.

As also discussed above, vascular permeability-inducing factors of the type in question in the context of the latter methods of the invention will typically be factors which increase vascular permeability via the agency of a Rho family member, such as Rho, Rac, Cdc42 and isoforms thereof (vide infra).

As already indicated to some extent above, in a further aspect of the invention the factor in question in the context of a method according to the invention is one which additionally stimulates angiogenesis in tissues containing the blood vessels, and the method causes substantially no inhibition or reduction of this factor-induced angiogenesis. Among further aspects of the invention are: pharmaceutical compositions comprising, as an active ingredient, a Rho family member antagonist together with a pharmaceutically acceptable carrier or diluent; and the use of a Rho family member antagonist for the preparation of a medicament for the treatment or prophylaxis of a condition arising from an increase in vascular permeability of blood vessels following exposure of the blood vessels to an elevated level of a factor which increases vascular permeability.

Detailed description of the invention

The present invention is based on the fact that members of the Rho family of small GTPases, that includes the Rho, Rac and Cdc42 subfamilies, are key molecules in the regulation of the increase of vascular permeability by factors such as VEGF and related growth factors. However, the angiogenic response induced by VEGF and related growth factors involves another pathway. It is thus possible to selectively counteract the increase in permeability of blood vessels induced by VEGF and related growth factors by interfering with the function of Rho GTPases.

In the practice of the invention, increased vascular permeability and tissue edema mediated by VEGF or other causative factors, e.g local or systemic hypoxia, is counteracted without compromising any ensuing angiogenesis by administering an effective amount of at least one Rho family member antagonist to a patient suffering from a condition for which such treatment is indicated, e.g. a condition such as stroke, traumatic brain injury or other central nervous system injury, myocardial ischemia, hypoxic lung edema or edema in another organ to which there is insufficient blood supply, retinopathy, chronic inflammation, neoplastic diseases (cancer), or patients undergoing angiogenic therapy in which VEGF activity is involved. An aspect of the invention pertains to the use of the Rho regulatory pathway as a target for Rho antagonists. This pathway involves the GDP/GTP exchange proteins (GEP's). Rho proteins exist in two forms, viz. GDP- bound inactive and GTP-bound active forms, that are interconvertible. The GEP's facilitate the exhange of GDP/GTP and thereby constitute a target for regulating Rho activity. In other aspects, GDP dissociation inhibitors (GDI's), that prevent dissociation of GDP from Rho, and hence the activation via GTP binding, are therefore targets for agents to regulate Rho activity. The active, GTP-bound Rho is converted into the inactive GDP-Rho by a GTPase reaction that is facilitated by specific GTPase activating proteins (GAP's). Thus another aspect pertains to the use of GAP's as targets for Rho activity regulation. The fact that Rho is found in the cytoplasma complexed with a GDI, and that Rho becomes active when it binds GTP followed by translocation to the cell membrane, render the use of agents that promote the binding of Rho to GDI and block Rho binding to the cell membrane another aspect. Also, the fact that C3 transferase, a bacterial ADP ribosyltransferase, ribosylates Rho to inactivate the protein renders the use of C3 transferase to inactivate Rho another aspect of the invention. In addition, the use of other bacterial toxins, such as toxin A and B, that also have Rho-inhibitory activity constitutes another aspect. Various mutations of the Rho protein may function as dominant-negative Rho and hence function as inhibitors of the biological activity of endogenous Rho activity in vascular cells; their use to inhibit the activity of Rho thus constitutes another aspect of the invention. Mixtures of inhibitors or antagonists can also be employed, as well as inhibitors/antagonists of Rho protein synthesis or stability. Rho protein inhibitors/antagonists include any inhibitor/antagonist of RhoA, Rad or Cdc42, as well as other members of the Rho GTP-binding family.

The term "vascular permeability" as employed in the context of the present invention refers to the ease with which transport of fluid (liquid) and solutes present therein from the blood vessel lumen into the tissue, and Wee versa, takes place.

The term "angiogenesis" as employed in the present context refers to the generation of new blood supply, i.e. generation of new blood vessels (capillaries, veins and/or arteries), from existing blood vessel tissue. The process of angiogenesis can involve a number of tissue cell types, including, for example, endothelial cells which line all blood vessels in the form of a single layer of cells and are involved in regulating exchange of fluid, solutes, cells, blood gases etc. between the bloodstream and the surrounding tissues. Development of new blood vessels (angiogenesis) can take place from the walls of existing small vessels by the outgrowth of endothelial cells. Angiogenesis is important not only in relation to normal physiological processes in healthy individuals, but also in relation to numerous pathological conditions; thus, for example, the process of angiogenesis ensures that hypoxic tissues have an adequate supply of oxygen and nutrients. In neoplastic tissues on the other hand, angiogenesis is important in providing the tissue with a supply of blood sufficient for tumor cell survival and proliferation (growth, metastasis). In this connection, one aspect of methods of the invention relates to the administration of an anti-angiogenic agent and/or an anti-cancer agent (e.g. a cytostatic agent) in addition to the Rho family member antagonist.

The term "Rho family member" as employed in the context of the present invention refers to any member of the so-called Rho family of small GTPases (Rho GTPases), including: RhoA, RhoB, RhoC, RhoD, RhoE, RhoG and RhoH; Rad, Rac2 and Rac3; Cdc42; TC10; and Rnd1 and Rnd2. These have been reported to regulate the polymerization of actin filaments to produce stress fibers, lamellipodia and filopodia (Ridley et al., 1992; Nobes and Hall, 1995; Aspenstrom, 1999). Additionally, Rho-associated protein kinase is included within the scope of this term in the context of the present invention.

A Rho family member antagonist in the present context is any substance which functions as an antagonist or inhibitor with respect to a Rho family member as defined in the context of the invention. Such antagonists - which may not only be peptides or proteins (including, e.g., analogues of Rho family members that bind to receptors, antibodies against a Rho family member protein or fragment thereof), but also non-peptidic substances (typically small organic molecules) - include, for example: the small molecule designated Y-27632 (see, e.g., US 4,997,834), that inhibits Rho-associated protein kinase; Clostridium botulinum exoenzyme C3, that inhibits RhoA GTPase; and Clostridium difficile toxin B, that non-specifically inhibits most Rho GTPases, such as isoforms of Rho and Rac, as well as Cdc42 (Aktories et al., 2000).

Blood vessels of particular interest in relation to the present invention will include, for example, blood vessels contained in cardiac muscle tissue, brain tissue, lung tissue, skeletal muscle tissue, kidney tissue, liver tissue or skin, and vascular permeability-inducing or -increasing factors of particular causative importance in relation to the invention will include - as already indicated to some extent above - growth factors of the VEGF family (vide supra); certain members of the VEGF family have also been denoted "vascular permeability factor" (VPF).

In an interesting further aspect of methods according to the present invention, a factor that promotes angiogenesis may be administered in addition to the Rho family member(s) in question in order to induce or enhance angiogenesis; thus, for example, in the case of a causative factor (i.e. vascular permeability-inducing or -increasing factor) which itself promotes angiogenesis, such as a growth factor of the VEGFΛ PF family or type, the additionally administered angiogenesis- promoting factor in question may be identical to, or of the same type or family as, the causative factor in question.

In relation to the invention, the cause of an elevated level of the vascular permeability-inducing or permeability-increasing factor in question will normally be a chronic or acute disease condition in the subject or patient. Frequently encountered chronic or acute conditions of relevance will include: ischemic heart disease (e.g. atherosclerosis); ischemic stroke; hemorrhagic stroke; diabetes mellitus (Type 1 and Type 2); inflammation (including rheumatoid arthritis); and hypoxia-induced conditions such as so-called "high-mountain disease".

As already mentioned (vide supra), a Rho family member antagonist as employed in the context of the invention may be a protein (peptide) or a non-peptidic substance. In the case of antagonists of the peptide/protein type, one useful group of such antagonists consists of dominant negative forms of Rho family members. An example of such a peptide/protein is N17Rac (sometimes denoted RacN17) and peptides/proteins comprising the amino acid sequence thereof, i.e. comprising the amino acid sequence of Rac in which a threonine residue (T) at position 17 in native Rac has been replaced with an asparagine (N) residue. Other relevant groups of Rho family member antagonists include, for example, Cdc42 antagonists and GDP dissociation inhibitors (GDI's).

It will be clear that it will, in general, be desirable that the Rho family member antagonist(s) employed in accordance with the invention display(s) adequate cell- permeability towards (i.e. adequate ability to enter or be transported into) the cells which are involved in connection with the increase in vascular permeability induced by the causative factor (e.g. a growth factor of the VEGF/VPF type) in question. One way of enhancing the cell permeability of a potentially useful type of antagonist which, a priori, displays inadequate, or undesirably low, cell- permeability is to incorporate a moiety (i.e. a group, substituent, amino acid sequence or the like) which in itself has been found to be associated with enhanced ability to be transported into cells, and which may thus be expected to enhance the cell-permeability of the Rho family member antagonist(s) in question. With regard to peptide amino acid sequences conferring this property (see, e.g., Gariepy and Kawamura, 2001), a number of relevant candidates may be envisaged, including the following:

the N-terminal hydrophobic region of K-FGF (Kaposi fibroblast growth factor), residues 1 -16 (Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro);

Src homoloαv 2 (SH2) domain of Grb2. residues 1-12 (Ala-Ala-Val-Leu-Leu-Pro-Val- Leu-Leu-Ala-Ala-Pro);

Integrin fo (Val-Thr-Val-Leu-Ala-Leu-Gly-Ala-Leu-Ala-Gly-Val-Gly-Val-Gly);

HIV-1 αp41 (1-23) (Glv-Ala-Leu-Phe-Leu-Glv-Phe-Leu-Glv-Ala-Ala-Gly-Ser-Thr-Met- Gly-Ala. Caiiman crocodylus immunoglobin (v) light chain (Met-Gly-Leu-Gly-Leu-His-Leu- Leu-Val-Leu-Ala-Ala-Ala-Leu-Gln-Gly-Ala-Met-Gly-Leu-Gly-Leu-His-Leu-Leu-Leu- Ala-Ala-Ala-Leu-Gln-Gly-Ala);

Synthetic analogues of the fusion peptide of influenza hemagluttinin, such as

KALA: Trp-Glu-Ala-Lys-Leu-Ala-Lys-Ala-Leu-Ala-Lys-Ala-Leu-Ala-Lys-His-Leu-Ala-

Lys-Ala-Leu-Ala-Lys-Ala-Leu-Lys-Ala-Cys-Glu-Ala;

GALA: Trp-Glu-Ala-Ala-Leu-Ala-Glu-Ala-Leu-Ala-Glu-Ala-Leu-Ala-Glu-His-Leu-Ala-

Glu-Ala-Leu-Ala-Glu-Ala-Leu-Glu-Ala-Leu-Ala-Ala;

Aβ. Leu-Ala-Arg-Leu-Leu-Ala-Arg-Leu-Leu-Ala-Arg-Leu-Leu-Arg-Ala-Leu-Leu-Arg-

Ala-Leu-Leu-Arg-Ala-Leu;

Hel 11-7: Lys-Leu-Leu-Lys-Leu-Leu-Leu-Lys-Leu-Trp-Lys-Leu-Leu-Leu-Lys-Leu-Leu-

Lys;

Penetratin from Antennapedia third helix (43-58): Arg-Gln-lle-Lys-lle-Trp-Phe-Gln- Arg-Arg-Met-Lys-Lys-Trp-Lys;

Transportan resulting from the fusion of galanin to mastoparan: Gly-Trp-Thr-Leu-

Asn-Ser-Ala-Gly-Tyr-Leu-Leu-Gly-Lys-lle-Asn-Leu-Lys-Ala-Leu-Ala-Ala-Leu-Ala-Lys-

Lys-lle-Leu;

Basic residues from HIV-1 Tat protein (47-57): Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln- Arg-Arg-Arg; and

VP22 from HSV transcription factor (267-300): Asp-Ala-Ala-Tyr-Ala-Tyr-Arg-Gly-

Arg-Ser-Ala-Ala-Ser-Arg-Pro-Tyr-Glu-Arg-Pro-Arg-Ala-Pro-Ala-Arg-Ser-Ala-Ser-

Arg-Pro-Arg-Arg-Pro-Val-Glu.

Other ways of achieving/enhancing transport of Rho family member antagonists into the cells in question are to encapsulate them in a cell-membrane-permeable vehicle, such as liposomes or lipofectin, or to attach them to antibodies, viral protein carriers or other "homing" carrier molecules that target the relevant vessels. Delivery may also be accomplished by injection of recombinant viruses expressing the antagonist. A working example herein (vide infra) illustrates the use of a so-called TAT protein sequence fused to N17Rac (Soga et al., 2001) as a carrier protein to enhance the transport of N17Rac into cells.

Administration of a Rho family member antagonist (together with any other therapeutically active substances, relevant auxiliary substances, vehicles, carriers, excipients or the like) in the manner of the invention may take place by any suitable route of administration, including topical, parenteral, oral and rectal routes. Owing to the nature of the chronic or acute underlying conditions (vide supra) which are often associated with the increase in vascular permeability which is to be counteracted (inhibited or reduced) in the manner of the invention, parenteral routes of administration will often be particularly well suited, and a parenteral administration route of choice will then normally be one of the following: intraarterial administration; intravenous administration; intracranial administration; intracutaneous administration; subcutaneous administration; intramuscular administration; pulmonary administration; nasal administration; and vaginal administration. In certain cases, e.g. during surgical procedures, direct administration into or onto affected tissues (by injection, infusion or other means) may be an effective parenteral route of administration.

As already mentioned above, further aspects of the present invention relate to pharmaceutical compositions comprising, as an active ingredient, a Rho family member antagonist together with a pharmaceutically acceptable carrier or diluent. Compositions of this type will thus be applicable, for example, to the treatment or prophylaxis of a condition arising from an increase in vascular permeability of blood vessels following exposure of the blood vessels to an elevated level of a factor [e.g. a growth factor of one of the types described earlier above, such as a VEGF (VPF), PDGF or PIGF] which increases vascular permeability; edema in tissues containing the blood vessels - particularly in tissues of one of the types already discussed above - is an important example of such a condition.

Yet another aspect of the invention relates to the use of a Rho family member antagonist for the preparation of a medicament for the treatment or prophylaxis of a condition (such as one of those already mentioned above) arising from an increase in vascular permeability of blood vessels following exposure of the blood vessels to an elevated level of a factor (such as one of those discussed above) which increases vascular permeability.

To summarize, the present invention can be used in vitro or ex vivo to inhibit vessel permeability increases. Methods of the invention can be used in vivo to treat a variety of diseases or conditions involving, typically, growth-factor- induced (e.g. VEGF-induced) vessel permeability increases in subjects, including animals and humans. Such diseases or conditions include, for example, stroke, head and brain trauma, various forms of cancer, diabetic retinopathy, chronic inflammation and autoimmune diseases, such as rheumatoid arthritis, and diseases related to high-altitudes and low tissue oxygenation. Moreover, when a growth factor such as VEGF is used in angiogenic therapy to remedy insufficient blood flow in organs (e.g. the heart or limbs), using the present invention it will be possible to counteract the unwanted effect of increase in blood vessel permeability.

Brief Description of Figures

Fig. 1 shows that VEGF induces endothelial actin reorganization and cell-shape changes in PAE endothelial cells transfected with VEGFR-2, but not those cells transfected with VEGFR-1. Addition of 100 ng/ml VEGFι₆₅ to PAE/VEGFR-2 cells for 6-12h resulted in reorganization of actin filaments and adoption of an elongated cell shape as determined by fixation and staining with TRITC-labelled phalloidin (b), while (a) shows the cells without stimulation by VEGF. Under identical conditions VEGF did not induce any cell-shape changes in VEGFR-1 expressing cells (f) and without VEGF (e). The dominant negative mutant Rad was introduced into PAE/VEGFR-2 cells by microinjection of the protein (g and h). Overexpression of the mutant Rad protein completely blocked the VEGF- stimulated cell-shape changes. In the presence (d, g and h) and absence (c), respectively, of VEGF, the PAE/VEGFR-2 cells microinjected with dominant negative mutant Rad (g and h) were stained with a FITC-conjugated goat anti- rabbit antibody (g). The same cells as in panel g were visualized with TRITC- phalloidin in panel h.

Fig. 2 shows the effect of TAT-N17Rac on the neovascularization pattern induced by VEGF. Corneal neovascularization was examined and quantitated on day 5 after pellet implantation into the corneas of C57BL6/J mice. Large arrows in panels a, b and c point to the implanted pellet. With VEGF alone (a) there was a high density of capillary sprouts, and the vessel appeared leaky and tended to form capillary blobs [fusion of capillaries at the growing edge (small arrows)]. The presence of both VEGF and TAT-N17Rac in the slow-release polymer (b) provoked neovascularization, but now there were no capillary sprouts/blobs. Small arrows in b point to well-defined microvessels. In c, it is demonstrated that TAT-N17Rac alone in the slow-release polymer does not induce neovascularization. Quantification of areas of corneal neovascularization is shown in d. Data represent mean determinants for 10-12 eyes (±SEM).

Fiα. 3 demonstrates, using immunohistochemistry, that TAT-N17Rac is taken up by microvessels. Histological sections of VEGF/TAT-N17Rac-implanted corneas at day 5 were incubated with an anti-CD31 antibody and stained with a Cy3-conjugated secondary antibody (e and h; seen in red colour in the original images from which Fig. 3 was prepared). The same corneal sections (f and i) were stained with an anti-HA tag antibody (for TAT-N17Rac; seen in green colour in the original images from which Fig. 3 was prepared). The CD31 positive signals (red colour in the original images of e and h) were superimposed with HA signals (green colour in the original images of f and i) using a digital imaging program to create double staining signals. Overlapping signals (yellow colour in the original images of g and j) represent microvessels which have taken up TAT-N17Rac.

Fig. 4 shows that TAT-N17Rac blocked VEGF-induced vascular fenestrations. Thin parts of the walls of microvessels growing into the mouse cornea were examined under the electron microscope. VEGF-induced vessels consist of a thin layer of endothelium with high number of fenestrations (i). In contrast, microvessels induced by VEGF/TAT-N17Rac completely lacked endothelial fenestrations (j), suggesting that TAT-N17Rac effectively attenuated VEGF-induced vascular fenestrations. Arrowheads indicate endothelial fenestrations, L indicates capillary lumen, and M indicates collagenous matrix of the cornea. Quantification analysis showed that barely detectable fenestrations were found in the VEGF/TAT- N17Rac-stimulation (k). Interestingly, TAT-N17Rac did not reduce the number of endothelial caveolae. Instead, a significant increase in endothelial caveolae was found in the VEGF TAT-N17Rac-induced vessels (I) as compared with those induced by VEGF alone (m). In agreement with this finding, endothelium induced by VEGF/TAT-N17Rac was significantly thicker than that induced by VEGF alone. Data represent means of at least 15 fields (±SEM).

Fig. 5 shows the results of a modified Miles^' assay in mice in order to detect the . vascular permeability effect of VEGF/VPF. Penetration of intravenous Evans blue into dermal tissues was an indicator in monitoring of the VEGF-induced permeability. Evans blue was injected intravenously into the tail vein of Balb/c mice. After 5 min, 2 μg of TAT-N17Rac in 20 μl was injected intradermally. After 30 min, 50 ng of VEGF in 20 μl of phosphate-buffered saline (PBS) were administered intradermally in the same TAT-N17Rac-injected locations. The same amount of TAT-N17Rac alone and bovine serum albumin (BSA) were used as negative controls. The extravasation of Evans blue was recorded at intervals using a digital camera system. As shown in the upper and lower panel, intradermal injections of VEGF, but not BSA, induced a rapid vascular permeability response as measured by the extravasation of Evans blue dye. The VEGF-induced permeability was detectable after 3 min of injection, and maximal effect was achieved after approximately 135 min. However, this potent effect was completely prevented when TAT-N17Rac was pre-injected in the same locations, and injection of TAT-N17Rac itself did not affect extravasation of Evans blue dye. These results demonstrate that Rac is an essential component in mediating VEGF-induced vascular permeability.

Fig. 6 demonstrates that Rac promotes, but is not strictly required for, VEGF- induced angiogenesis, but makes up a necessary part of the signal transduction system mediating the stimulatory effect of VEGF on formation of endothelial fenestrations and vascular permeability. Earlier studies have demonstrated that VEGF acts primarily via VEGFR-2 to induce angiogenesis and vascular permeability. To further dissect the VEGF-Rac signaling pathways and to understand the underlying mechanisms, the VEGFR-2-transduced signaling pathways in PAE/VEGFR-2 cells were studied in connection with the present invention. Upon VEGF stimulation (10 ng/ml), VEGFR-2 became phosphorylated, which led to phosphorylation of PLCγ, Akt, eNOS and Erk1/2 in a time-dependent manner (see panels 2-6 in Fig. 6). Maximum phosphorylation was observed after 10-15 minutes for PLCγ, Akt and eNOS, while maximum phosphorylation for Erk1/2 was observed after 30-60 minutes. To characterize to what extent the phosphatidylinositol-3-OH kinase (PI3K) was involved in activating both Rac and the intracellular signaling cascades, we conducted experiments with the specific PI3K inhibitor known as wortmannin. This agent specifically blocked the phosphorylation of Akt and eNOS over a timespan of 30 minutes, consistent with Akt being an important activator of eNOS in endothelial cells. Wortmannin reduced the phosphorylation of Erk1/2 to only a small extent (data not shown). To elucidate the functional relationship between VEGFR-2 and Rac, we carried out a pull-down assay with GST-PAK bound to glutathione-coupled agarose beads and using subsequent blotting with a Rac antibody. This assay allowed us to specifically detect the activated fraction of Rac. After 10 minutes of VEGF stimulation an at least two-fold increase in GTP-Rac was observed, and wortmannin prevented this effect (see the figure). These results show that activation of Rac is dependent on PI3K, and that PI3K also leads to activation of Akt and eNOS. Thus, during VEGF stimulation both signaling pathways share PI3K as an essential activating factor.

Fig. 7 shows that the infarct volume in rat brain 24h after a 2h transient occlusion of the right middle cerebral artery is decreased by treatment with TAT-N17Rac. In the control group that received vehicle/the infarct volume was 20.1 ± 2.7%, while the infarct volume in the rats given TAT-N17Rac (indicated in the figure simply as "N17Rac") was 12.3 ± 2.7 % (P = 0.05). This is thus statistically significant.

EXAMPLES Example 1

The experiments described below provide evidence that N17Rac inhibits VEGF- mediated permeability of blood vessels substantially without affecting the VEGF- mediated angiogenic response. In vitro, VEGF induced actin reorganization and provoked an elongated, spindle-like cell-shape change in endothelial cells expressing VEGFR-2, but not in cells expressing VEGFR-1. When N17Rac was introduced into VEGFR-2-expressing cells, which may be achieved either by cDNA transfection or by microinjection of the mutant protein, VEGF was unable to induce actin reorganization and the spindle-like cell-shape change. Further, the cell-membrane-permeable Rac antagonist (Rho antagonist) TAT-N17Rac alters the leaky angiogenic pattern and essentially completely blocks the VEGF-induced vascular fenestrations, but not angiogenesis. Finally, VEGF-induced vascular permeability increase is also prevented by the Rac antagonist. In vivo, VEGF induces the formation of fenestrated new capillaries in the mouse corneal angiogenesis model, whereas no fenestrations are found in new blood vessels induced by fibroblast growth factor 2 (FGF-2). VEGF further increased the number of caveolae which might mediate endothelial cell transcytosis. The VEGF- induced vascular fenestrations correlated with increased permeability of newly formed microvessels. Example 2

As described below, upregulation of VEGF has important consequences in tissue hypoxia, e.g. stroke. The increased permeability of the blood-brain barrier promotes edema formation that may increase tissue pressure and compromise blood supply. In serious cases this may not only cause incarceration/herniation, but also decrease local blood supply in and around the affected brain region so that the volume of infarction expands with time. In a rat model of stroke we observed that when N17Rac was administered following 2h of middle cerebral artery occlusion (mcao), the infarcted volume was smaller than in rats given vehicle only.

Materials and methods for Example 1

Reagents, cells and animals: Recombinant human VEGF₁₆₅ was prepared as previously described (Cao, Chen et al., 1996). Recombinant human FGF-2 was obtained from Scios Nova Inc., Mountainview, California. Plasmid constructs containing dominant negative Rac cDNA (pGEX N17Rac1 and pEXV myc tag V12N17Rac1) were kindly provided by Dr. Alan Hall (University College, London, UK). Stable porcine aortic endothelial (PAE) cell lines expressing VEGFR-1 and VEGFR-2 were established as previously reported (Cao et al.,1998; Waltenberger et al., 1994b) and maintained in Ham's F12 medium supplemented with penicillin/streptomycin and 10% foetal calf serum (FCS). Male, 5-6-week-old C57BI6/J mice were acclimatized and caged in groups of four or less. The animals were anaesthetized in a methoxyflurane chamber before all procedures, and were subsequently killed using a lethal dose of methoxyflurane. All animal studies were reviewed and approved by the animal care and use committee of the Stockholm Animal Board, Stockholm, Sweden.

Mouse corneal angiogenesis assay: The mouse corneal assay was performed according to procedures described previously (Cao et al., 1999). Corneal micropockets were created with a modified von Graefe cataract knife in both eyes of male, 5-6-week-old C57BI6/J mice. A micropellet (0.35 X 0.35 mm) of sucrose and aluminium sulfate (Bukh Meditec, Copenhagen, Denmark) coated with hydron polymer type NCC (IFN Sciences, New Brunswick, NJ, USA) containing 160 ng of VEGF or 80 ng of FGF-2 with or without a cell-membrane- permeable Rac antagonist (TAT-N17Rac) was implanted into each pocket. The pellet was positioned 0.6-0.8 mm from the corneal limbus. After implantation, erythromycin ophthalmic ointment was applied to each eye. The eyes were examined by a slit-lamp biomicroscope on day 5 or day 6 after pellet implantation. Vessel length and clock hours of circumferential neovascularization were measured.

Electron microscopy: Six days after implantation of VEGF or FGF-2 pellets into corneas, the animals were sacrificed and the eyes removed and immersed and fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate-HCI buffer (pH 7.3) with 0.05 M sucrose. A few hours later, the parts of the corneas containing ingrowing blood vessels were dissected out, cut into small pieces, and placed in fresh fixative. After rinsing in buffer, the specimens were post-fixed in 1.5% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.3) with 0.7% potassium ferrocyanate for 2 h at 4°C, dehydrated in ethanol (70%, 95%, 100%), stained with 2% uranyl acetate in ethanol, and embedded in Spurr low-viscosity epoxy resin. Thin sections were cut perpendicular to the surface of the cornea with diamond knifes on an LKB Ultrotome IV, picked up on carbon-coated Formvar films, stained with alkaline lead citrate, and examined in a Philips CM120Twin electron microscope at 80 kV.

Permeability assay: Six week-old female Balb/c white mice were shaved. Four days later, the mice were anaesthetized with a mixture of hypnorm and dormicum (1:1) in a H₂O solution and 150 μl of 1 % Evans blue dye solution was injected in the tail vein of each mouse. After 5 minutes, 50 ng VEGF with and without 2 μg of TAT-N17Rac in a volume of 20 μl PBS was injected at adjacent locations intradermally in the mid dorsum of the same animal. The extravasation of Evans blue dye was recorded with a digital camera for up to 4 hours. As controls, 2 μg of BSA or TAT-N17Rac were injected in the same positions in animals in a separate group. Five animals were used in each of the treated and control groups.

Microinjection: PAE/VEGFR-2 cells were grown on coverslips to about 70% confluency in Ham's F12 medium containing 10% FCS. Before microinjection, the medium was changed to serum-free Ham's F12. Microinjection of recombinant, dominant negative Rac protein (purified from E. coli using a GST-tag purification method) into the cytoplasm of growing cells was carried out using a Zeiss automated injection system and glass capillaries from Eppendorf. The concentration of injected Rac protein was 0.5 μg/μl in microinjection buffer (50 mM Tris, 50 mM NaCI, 5 mM MgCl₂, 0.1 mM DTT) containing 0.5 μg/μl of rabbit IgG. Microinjection buffer containing 0.5 μg/μl of rabbit IgG was injected into cells as controls. Shortly after the injections, the cells were given 100 ng/ml of VEGF in Ham's F12 medium containing 2% FCS and incubated for 12 h. The cells were fixed in 3% formaldehyde in PBS (pH 7.5) for 30 min, rinsed three times with PBS, and permeabilized in 0.5% Triton X-100 in PBS for 30 min. They were then washed three times with PBS, exposed to FITC-conjugated goat anti-rabbit IgG for 2 h, washed three times with PBS, and stained with 1 μg/ml of TRITC- phalloidin (Sigma) in PBS for 30 min. After washing five times with PBS, the coverslips were mounted in a mixture of glycerol and PBS (90:10) containing 0.1 % p-phenylenediamine, and the cells were finally examined in a fluorescence microscope.

Transfection: Monolayers of PAE/VEGFR-2 cells are grown on coverslips in six- well-plates to 70% confluency in Ham's F12 medium containing 10% FCS. The medium is replaced with 1 ml of Opti-MEM (Gibco BRL) and the cells transfected with 10 μg V12N17Rac DNA containing a c-myc tag using 20 μg of Lipofectin (Gibco Life Technologies) according to the protocol recommended by the manufacturer. After 8 h, 100 ng/ml of VEGF in Ham's F12 medium containing 10% FCS is added and the incubation continued for 12 h. The cells are fixed in 3% formaldehyde in PBS (pH 7.5) for 30 min. After rinsing three times with PBS, the cells are permeabilized with 0.5% Triton X-100 in PBS for 30 min and washed again with PBS. They are then incubated with a monoclonal antibody against the myc-tag (9E10) for 1 h, washed several times with PBS, stained with FITC- conjugated rabbit anti-mouse IgG for 1 h, and washed again with PBS. The cells are finally stained with TRITC-phalloidin and further processed for fluorescence microscopy as described above.

Immunohistochemistry: The growth-factor-implanted mouse eyes were enucleated at day 5 after implantation, and immediately frozen on dry ice and stored at -80°C before use. Frozen sections of 10 μm were cut using a cryomicrotome. Sections were air-dried for 10 min, fixed with acetone and blocked with 30% non-immune goat serum. Endogenous biotin was blocked by using an avidin-biotin reagent (Vector laboratories, Burlingame, USA). A mixture of primary antibodies consisting of rat anti-mouse CD31 (1 :100, Pharmingen, San Diego, USA) and mouse anti-human desmin (1:50 NOVO Castra, Newcastle upon Tyne, UK) were added and incubated for 1 h at room temperature. After repeated washing, secondary antibodies of rabbit anti-rat-FITC (Dako A/S, Glostrup, Denmark) and biotinylated goat anti-mouse IgG (Southern Biotechnology Associates Inc., Birmingham, USA) were added to the tissue sections before incubation for 30 min. Following rigorous rinsing, streptavidin- conjugated Cy3 (1:2500, Jackson ImmunoResearch, West Grove, USA) was added to samples and incubated for 30 min. After washing in PBS, slides were mounted in 90% glycerol and examined under a fluorescent microscope (Nikon) at 20x magnification. Images were collected with a digital camera system, and further analyzed using the Adobe Photoshop 6.0 software program

Cell-shape assay and actin staining: VEGFR-1/PAE and VEGFR-2/PAE cells were grown on coverslips in 12-well plates to about 40-60% confluency in Ham's F12 medium supplemented with 10% FCS. The medium was removed and replaced with fresh Ham's F12 medium containing 2% FCS with and without 100 ng/ml of VEGF, PLGF, PLGF/VEGF, or 25% of conditioned media. After 16 h, cells were fixed with 3% paraformaldehyde in PBS (pH 7.5) for 30 min, rinsed three times with PBS, and permeabilized with 0.5% Triton X-100 in PBS for 15 min. The cells were then washed three times with PBS and stained for 30 min with 1 ug/ml of TRITC-phalloidin (Sigma) in PBS. After washing 3 times with PBS, the coverslips were mounted in a mixture of glycerol and PBS (9:1) and the cells were examined in a combined light and fluorescence microscope.

Detection of GTP-Rac: VEGFR-2/PAE cells (5 x 10⁶) were starved in serum-free Ham's F12 medium for 19 hours, pre-treated with or without 100 nmol/l wortmannin for 90 minutes, and stimulated with 50 ng/ml VEGF for 10 minutes. Cells were lysed in a GST-Fish buffer (50 mmol/l Tris pH 7.2, 1 % Triton X-100, 0.5% sodium deoxycholate, 0.1 % SDS, 500 mmol/l NaCI, 10 mmol/l MgCI₂, 10 μg/μl aprotinin/leupeptin, 1 mmol/l PMSF). After centrifugation of cell lysates, supernatants were used for binding to GST-PAK-CD-Sepharose as previously described [Fischer et al., 2002 (ref. 21)]. Briefly, supernatants of bacterial lysates (1.2 ml) containing GST-PAK were incubated in end-to-end rotation with 300 μl of 50% GSH-Sepharose in PBS for 1 hour at 4 °C. The bound Sepharose beads were then washed 3 times with a bacterial lysis buffer and resuspended in 0.5 ml of GST-Fish buffer. GST-PAK Sepharose in 100 μl was incubated with cell lysates for 1 hour. Following extensive washing, bound material was released with by treatment with LDS loading buffer and analyzed by Western blotting (ECL) using a mouse anti-human Rad monoclonal antibody (1 :1000, BD Science).

Signal Transduction Assays: VEGFR-2/PAE cells were grown to 90% confluency in 60 mm dishes, washed, and incubated for 30 minutes in serum-free medium (RPMI 1640). Cellular activity was stopped by adding 500 μl LDS lysis buffer (Invitrogen) containing 1.2 μg/ml aprotinin, pepstatin and leupeptin and 1.25 mmol/l NaF, PMSF and sodium orthovanadate. Samples were mixed for 30 seconds and then centrifuged for 10 minutes at 14,000 rpm. DNA was removed, and equal amounts of protein samples were separated by SDS-PAGE using a 10% BIS-Tris gel (Invitrogen). Proteins were transferred to nitrocellulose membranes, and non-specific sites were blocked with 5% bovine albumin (Sigma) in PBS containing 0.1 % Tween. The membranes were probed overnight at 4°C with antibodies solubilized in PBS containing 5% bovine serum albumin (BSA) and 0.1 % Tween for detecting P-Akt (Ser473) and P-eNOS (Ser1177) (Cell Signalling Technology, Beverly, MA), P-Erk 1/2 (Tyr204) (Santa Cruz Biotechnology, Santa Cruz, CA), P-KDR (Oncogene Research Products, Boston, MA). This was followed by incubation for 1 hour in PBS containing 1 % BSA and 0.1 % Tween. The peroxidase-conjugated rabbit immunoglobulin was diluted 1 :1000 for P-Akt, P- eNOS and P-PLCγ, and diluted 1:5000 for P-Erk1/2 (Dako, Taastrup, Denmark). For the P-VEGFR-2, a peroxidase-conjugated rabbit immunoglobulin was diluted 1:40000 (Santa Cruz Biotechnology). Protein bands were visualized by enhanced chemiluminiscence (Pierce, Rockford, IL).

In VEGFR-2/PAE cells, VEGF-induced (10 ng/ml) phosphorylation of VEGFR-2, PLCγ, Erk1/2, Akt and e-NOS and activation of Rac were detected by Western blotting. Equal amounts of cell lysates were used in each sample. VEGF-induced autophosphorylation of VEGFR-2 was measured after 10 minutes of incubation with 10 ng/ml VEGF and the other phosphoenzymes at the indicated times. The formation of GTP-Rac was measured after 10 minutes of incubation. Cells were preincubated for 10 minutes with wortmannin (100 nM) to block PI3K activity.

Materials and methods for Example 2

Rat model of stroke: Adult male Sprague-Dawley rats weighing 275-300 g were fasted overnight with free access to water. The rats were anaesthetised using isoflurane in a mixture of 70% nitrous oxide and 30% oxygen. The right middle cerebral artery was occluded using the intraluminal suture technique (Longa, Weinstein et al., 1989 29 /id). Following a mid-line cervical incision the common and the external carotid was ligated with a 3-0 suture. A filament of nylon (0.25mm) whose tip was rounded (0.30-0.32 mm) by flame polishing and coated with poly-L-lysin was inserted from the carotid bifurcation into the internal carotid artery until an increase in resistance was felt - at the start of the middle cerebral artery (usually after 19 mm of the filament was introduced). After insertion of the filament, the anaesthesia was discontinued and the rats were allowed to regain consciousness. After two hours the rats were neurologically evaluated (according to Bederson, Pitts et al., 1986 30 /id). Only rats demonstrating circling behavior to the right were included in the study. Under anaesthesia, circulation in the middle cerebral artery was reestablished by withdrawal of the filament. Thereafter a catheter was inserted in the common carotid artery and the peptide TAT-N17Rac (15μg) was administered to the rats (n=7) in a volume of 0.3 ml over a period of 10 seconds. Control rats (n=5) received only vehicle. The artery was then ligated, the skin sutured and the rats allowed to regain consciousness.

After 24 h the rats were again anaesthetized (pentobarbital, 50mg/kg). They were decapitated, and the brain cut into coronal slices (2mm) which were exposed to 2% 2,3,5-triphenyltetrasolium chloride solution to mark the living (red) and infarcted (white) regions. The extent of infarction was measured using an image analysis system (SigmaScan Pro 5.0). The measured infarct volume was adjusted for edema by division of "edema index", i.e. volume of right brain divided by left brain volume for each slice. The infarct volume is expressed as percentage of the total brain volume.

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Claims

1. A method for inhibiting or reducing increase in vascular permeability of blood vessels in a subject following exposure of said blood vessels to an elevated level of a factor which increases vascular permeability, said method comprising administering to said subject a Rho family member antagonist.

2. A method for treating a subject so as to prevent or reduce edema in tissues in said subject which contain blood vessels which have been exposed to an elevated level of a factor which increases vascular permeability, said method comprising administering to said subject a Rho family member antagonist.

3. A method according to claim 1 or 2, wherein said factor additionally stimulates angiogenesis in tissues containing said blood vessels, said method causing substantially no inhibition or reduction of said angiogenesis.

4. The method according to any one of claims 1-3, wherein said blood vessels are blood vessels contained in tissues selected from the group consisting of: cardiac muscle tissue; brain tissue; lung tissue; skeletal muscle tissue; kidney tissue; liver tissue; and skin.

5. The method according to any one of claims 1-4, wherein said factor is a vascular endothelial growth factor (VEGF) or vascular permeability factor (VPF).

6. The method according to any one of the preceding claims, said method further comprising administering a factor that promotes angiogenesis.

7. The method according to claim 6, wherein said angiogenesis-promoting factor is identical with said permeability-increasing factor.

8. The method according to any one of claims 1-7, wherein said exposure to an elevated level of said factor is a consequence of a chronic or acute disease condition in said subject.

9. The method according to claim 8, wherein said chronic or acute disease condition is selected from the group consisting of: ι ischemic heart disease, including atherosclerosis; ischemic stroke; hemorrhagic stroke; diabetes; inflammation, including rheumatoid arthritis; high-mountain disease;

10. The method according to any one of claims 1-9, wherein said Rho family member is selected from the group consisting of RhoA, RhoB, RhoC, Rad, Rac2, Rac3, CDC-42 and Rho-associated protein kinase.

11. The method according to any one of claims 1-10, wherein said Rho family member antagonist is a peptide or protein.

12. The method according to any one of claims 1-10, wherein said Rho family member antagonist is a non-peptide small molecule.

13. The method according to claim 11, wherein said Rho family member antagonist comprises the amino acid sequence of N17Rac.

14. The method according to claim 11, wherein said Rho family member antagonist is a Cdc42 antagonist.

15. The method according to any one of the preceding claims, wherein said Rho family member antagonist comprises a moiety which enhances cell-permeability of said antagonist.

16. The method according to any one of the preceding claims, wherein said moiety is a peptide amino acid sequence.

17. The method according to claim 16, said amino acid sequence being identical or partially identical to the amino acid sequence of a peptide selected from the group consisting of TAT, Kaposi fibroblast growth factor (K-FGF), Grb2 (SH2 domain) and integrin /33.

18. A method according to any one of the preceding claims, wherein said administration takes place by a route selected from the group consisting of: parenteral administration; oral administration; and rectal administration.

19. A method according to any one of the preceding claims, wherein said administration takes place parenterally by a route selected from the group consisting of: intraarterial administration; intravenous administration; intracranial administration; intracutaneous administration; subcutaneous administration; intramuscular administration; nasal administration; and pulmonary administration.

20. A pharmaceutical composition comprising, as an active ingredient, a Rho family member antagonist together with a pharmaceutically acceptable carrier or diluent.

21. A pharmaceutical composition for treatment or prophylaxis of a condition arising from an increase in vascular permeability of blood vessels following exposure of said blood vessels to an elevated level of a factor which increases vascular permeability, said composition comprising, as an active ingredient, a Rho family member antagonist together with a pharmaceutically acceptable carrier or diluent.

22. A pharmaceutical composition according to claim 20 or 21 for treatment or prophylaxis of a condition arising from an increase in vascular permeability of blood vessels following exposure of said blood vessels to an elevated level of a vascular endothelial growth factor (VEGF) or vascular permeability factor (VPF).

23. A pharmaceutical composition according to any one of claims 20-23 for treatment or prophylaxis of edema in tissues containing said blood vessels.

24. A pharmaceutical composition according to claim 24 for treatment or prophylaxis of edema in tissue selected from the group consisting of: cardiac muscle tissue; brain tissue; lung tissue; skeletal muscle tissue; kidney tissue; liver tissue; and skin.

25. Use of a Rho family member antagonist for the preparation of a medicament for the treatment or prophylaxis of a condition arising from an increase in vascular permeability of blood vessels following exposure of said blood vessels to an elevated level of a factor which increases vascular permeability.