MXPA98009159A - Therapeutic treatment of diseases related to the vascular endothelial growth factor (ve - Google Patents

Abstract

Disclosed is a method for inhibiting eyelid endothelial cell growth (VEGF), such as that associated with neoplasia, and stimulated capillary permeability (VEGF), such as that associated with pulmonary adema, using particularly the beta-isozyme-selective inhibitor PKC. , to the hydrochloride of (S) 3,4-. { N, N'-1,1 '- ((2' '- ethoxy) -3' '' (O) -4 '' '- (N, N-dimatylamino) -butane) -bis-3,3'- indolil)} -1- (H) -pyrrole-2,5-dio

Description

THERAPEUTIC TREATMENT OF DISEASES RELATED TO THE VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) This application claims the priority benefit of Serial Serial US Application No. 60 / 016,658 filed May 1, 1996.

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is broadly directed to a method for inhibiting endothelial cell growth and capillary permeability associated with vascular endothelial growth factor (VEGF), eg, increased cell growth and permeability. induced by (BEGF) using a β isozyme inhibitor of Protein C Kinase (PKC). This induced VEGF condition is closely associated with neoplasia in mammals and other disorders include pulmonary edema The present invention is particularly directed to the use of an inhibitor of protein C kinase (PKC) ß isozyme to treat neoplastic diseases including capillary hemangioblastoma, breast cancer, Kaposi's sarcoma, glioblastoma, angiomatous disorders, colorectal cancer, medulloblastoma, Gastric carcinoma, adenocarcinomas of the gastrointestinal tract, malignant melanoma, ovarian cancer, non-small cell lung cancer, prostate cancer, malignant effusions, pre-cumulative edema, for example, intracerebral edema and cysts associated with brain tumor, bladder carcinoma, Syndrome by von Hlppel Lindau, renal cell carcinoma, skin cancer, thyroid malignancies, cervical cancer, hepatocellular carcinoma, rhabdomyosarcoma, and leiomisarcoma and some other disorders related to VEGF as described herein. 2. Description of the Related Art VPF / VEGF is a glycosylated, multifunctional cytokine. The over-expression of VPF / VEGF is associated with neoplasia, and various other disease conditions. VPF / VEGF induces endothelial cell proliferation, excessive permeability via vesicle-vacuolar organelle-mediated transport activation, migration and reorganization of actin with shape and shirring changes. Alters the expression of endothelial cells, inducing increased production of the tissue and several proteases, including interstitial collagenase and both tissue plasminogen activators and urokinase-like. Most of these same genes are induced by the stimulated activation of phorbol myristate acetate (PMA) of PKC. VPF / VEGF is abundantly expressed and secreted by most of the human and animal tumors examined so far. VPF / VEGF can directly affect tumor cells, for example, glioblastoppa tumor cells, as well as play an important role in the induction of tumor angiogenesis (Claffey, et al., Cancer Research 56, 172-181 ( 1996) and the references cited therein) The angiogenic potential of VEGF probably TS intensified by the synergistic activity of fibroblast growth factor released by cell rupture or death. (Pepper, et al., Biochßm Biophys Res. Commun, 189: 824-831 (1992), Muthukrishnan, et al., J. Cell Physiol., 148: 1-16 (1991)). The growth of tumor and metastasis are closely related to the expression of enhanced VEGF. A chemical signal from the tumor cells can displace the remaining endothelial cells into a rapid growth phase. Of the twelve known angiogenic proteins, the most commonly found in tumors appear to be the basic fibroblast growth factor (bFGF) and the vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF) (Folkman , J. New England J. Of Medicine, Vol 999 (26): 1757-1763 (1995) and references cited therein).

The understanding of tumor growth requires new blood vessels and the identification of the chemical factors that mediate neovascularization or angiogenesis, have expanded the understanding of pathological processes and new ways open to the treatment of these diseases. New inhibitors are currently being studied different angiogenesis in phase 1 or 2 clinical trials as a treatment for a broad spectrum of solid tumors, including breast, colon, lung and prostate cancer, as well as Kaposi's disease. (Folkman, J. Tumor angiogenesis In Mendelsohn J. Howley PM, Israel MA Liotta LA Eds The Molecular Basis of Cancer, Flladelfia: W.B. Saunders. 1995.206-232) One of these drugs, TNP-170, an analogue Synthetic Fumagillin (Denekamp J. Br J Radial 66.181-196, 1993) has been approved by the FDA for phase 1 testing in many patients with solid tumors. Other inhibitors of angiogenesis currently in clinical trials in patients with advanced cancer include platelet factor 4; carboxyamotriazole; BB-94 and BB-2516; metalloproteinase inhibitors; tecogalan sulfated polysaccharide (DS-152); talldomide; Etherlequin-12, and linomide. (Flier et al, The New England Journal of Medicine, vol 333 pp1757-1763, 1995 and references cited herein) PKC inhibitors have also been proposed for cancer therapy, see U.S. Patent 5,552,396. However, the effectiveness of inhibitors of PKC β isozyme against particular neoplastic diseases was not known. Given the role that VEGF plays in certain neoplastic and other diseases, there is a need in the art to identify additional drugs that are specifically targeted at the VEGF function.

BRIEF DESCRIPTION OF THE INVENTION It is an object of the invention to provide a method for treating neoplasia. Yet another object of the invention is to provide a method for treating rheumatoid arthritis. Still another object of the invention is to provide a method for treating keloid.

Still another object of the invention is to provide a method for treating conditions associated with pulmonary edema such as Adult Respiratory Distress Syndrome (ARDS). Still another object of the invention is to provide a method for treating carpal tunnel syndrome. These and other objectives of the invention are provided by one or more of the embodiments described below. In one embodiment of the invention there is provided a method of treating neoplasia which comprises administering to said mammal a therapeutically effective amount of an inhibitor of the ß isozyme of the kinase of protein C Still another embodiment of the invention provides a method of treating rheumatoid arthritis which comprises administering to said mammal a therapeutically effective amount of an inhibitor of the ß isozyme of protein kinase C Still another embodiment of the invention is provided a method to treat keloid the c The method comprises administering to said mammal a therapeutically effective amount of a β-isozyme inhibitor of protein kinase C. In yet another embodiment of the invention there is provided a method of treating pulmonary edema which comprises administering to said mammal a therapeutically effective amount of an inhibitor of the ß isozyme of protein kinase C The present invention provides the technique with the identity of compounds which are prophylactic and effective in treating neoplasia and other disorders associated with endothelial growth factor (VEGF).

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the inhibitory effect of the PKC inhibitor, (S) - 3,4- [N, N'-1, 1 '- ((2"-ethox?) - 3'" (0 ) -4 '"- (N, Nd? Met? Lamino) -butapo) -bis- (3,3'-indole?) - 1 (H) -pyrrole-2,5-dione in cell growth stimulated endothelial human recombinant VEGF Figure 2 further illustrates the inhibitory effect of the PKC inhibitor, (S) -3,4- [N, N'-1, 1 '- ((2"-ethoxy) -3'" ( 0) -4 '"- (N, N-dimethylamino) -butane) -bis- (3,3'-indole?) - 1 (H) -p? Rrol-2,5-dione in cell growth endothelial stimulated recombinant human VEGF. Figure 3 shows the im of the PKC inhibitor on the activity of endogenous VEGF expressed on the culture of retinal pepts under hypoxic conditions. Figure 4 further illustrates the inhibitory effect of the PKC inhibitor on stimulated endothelial cell growth. Recombinant human VEGF.

DETAILED DESCRIPTION OF THE INVENTION It is a discovery of the present invention that the therapeutic use of a particular class of protein C kinase inhibitors, ie, inhibitors of protein isozyme kinase C, and especially selective isozyme inhibitors of PKC counteracts the effects of VEGF In particular, it is a discovery of this invention that the use of this particular class of protein kinase C inhibitors counteracts endothelial cell growth and capillary permeability by growth factor VEGF. Accordingly, such compounds can be used therapeutically to treat disorders associated with VEGF, such as neoplasia, and other disease conditions that are associated with VEGF. The method of this invention preferably uses those C protein kinase inhibitors that effectively inhibit the β-isozyme. A suitable group of compounds is generally described in the prior art, such as bis-indolylmaleimides or macrocyclic bis-mdolylmaleimides. The bis-indolylmaleimides well recognized in the prior art, include those compounds described in US Pat. Nos. 5621098, 5552396, 5545636, 5481003, 5491242, and 5057614, all incorporated herein by reference. The macrocyclic bis-indolylmaleimides are represented in particular by the compounds of formula I. These compounds, and methods for their preparation, have been described in US Pat. No. 5,552,396, which is incorporated herein by reference. These compounds are administered in a therapeutically effective amount to a mammal to inhibit endothelial cell growth or capillary permeability associated with VEGF, to inhibit the effects of VEGF associated with neoplasia, and other disease conditions, eg, rheumatoid arthritis, keloid, syndrome of carpal tunnel and pulmonary edema. These compounds can also be administered to patients at risk of the disease conditions mentioned above as prophylactics A preferred class of compounds for use in the method of the invention has the formula wherein: W is -O-, -S-, -SO-, -SO2-, -CO-, C2-C6 alkylene, substituted alkylene, C2-C3 alkenylene, -aryl-, -ar? l (CH2) mO-, -heterocycle-, -heterocycle- (CH2) mO-, -bicyclic fused-, -cyclic-fused- (CH2) mO-, -NR3-, -ÑOR3-, -CONH-, or NHCO-; X and Y are independently C1-C4 alkylene, substituted alkylene, or together X, Y, and W combine to form - (CH -) "- AA-; R s are hydrogen or up to four optional substituents independently selected from halo, C 1 -C 4 alkyl, hydroxy, C 1 -C akoxy < , haloalkyl, nitro, NR4R5, or -NHCO (C-C4 alkyl); R2 is hydrogen, CH3CO-, NH2, or hydroxy; R3 is hydrogen, (CH2) maryl, C1-C4 alkyl, -COO (C-C alky), -CONR R5, - (C = NH) NH2, -SO (C, -C alkyl), - S02 (NR4Rs), or -S02 (C1-C4 alkyl); R 4 and R 5 are independently hydrogen, C 1 -C 4 alkyl, phenyl, benzyl or combine with the nitrogen to which they are bound to form a saturated or unsaturated 5 or 6 membered ring; AA is an amino acid residue; m is independently 0, 1, 2 or 3; and n is independently 2, 3, 4 or 5 or a pharmaceutically acceptable salt, prodrug or ester thereof. A more preferred class of compounds for use in this invention is represented by formula I, wherein the -X- W-Y- moieties contain 4 to 8 atoms, which may be substituted or unsubstituted. Most preferably, the -X-W-Y- portions contain 6 atoms. Other preferred compounds for use in the method of this invention are those compounds of formula I, wherein R 1 and R 2 are hydrogen; and W is a substituted alkylene, -O-, -S-, -CONH-, -N HCO- or -NR3-. Particularly preferred compounds for use in the invention are compounds of the formula la: ) where Z is - (CH2) P- or - (CH2) p-0- (CH2) p-; R 4 is hydroxy, -SH, C, -C 4 alkyl, (CH 2) maryl, -NH (aryl), -N (CH 3) (CF 3), -N H (CF 3), or -NR 5 R 6; Rs is hydrogen or C1-C4 alkyl; R6 is hydrogen, C1-C4 alkyl or benzyl; p is 0, 1, or 2; and m is independently 2 or 3, or a pharmaceutically acceptable salt, prodrug or ester thereof. The most preferred compounds of the formula la are those wherein Z is CH2; and R 4 is -NH 2, -NH (CF 3), or -N (CH 3) 2. Other preferred compounds for use in the method of the present invention are compounds wherein W in formula I is -O-, Y is a substituted alkylene, and X is an alkylene. These preferred compounds are represented by the formula Ib. wherein Z is - (CH2) P-; R4 is -NRdR6, -NH (CF3), or -N (CH3) (CF3), R5 and Rd are independently H or C1-C4 alkyl; p is 0, 1, or 2; and m is independently 2 or 3 or a pharmaceutically acceptable salt, promedicamento or ester of the same. The most preferred compounds of formula Ib are those wherein p is 1; and R5 and R6 are methyl. Because they contain a basic portion, the compounds of the formulas I, la and Ib may also exist as pharmaceutically acceptable acid addition salts. Acids commonly employed to form such salts include inorganic acids such as hydrochloric, hydrobromic, hydroiodic, sulfuric and phosphoric acids, as well as organic acids, such as para-toluenesulfonic, methanesulfonic, oxalic, para-bromophenesulfonic, carbonic, succinic, citric acids, benzoic, acetic, and related organic and inorganic acids. Such pharmaceutically acceptable salts include in this manner sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, capplate, acrylate, formate, sobutyrate. , heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, 2-butyn-1,4-dioate, 3-hex? no-2,5-dioate, benzoate, chlorobenzoate, hydroxybenzoate, methoxybenzoate, phthalate , xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, hippurate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and the like. Particularly the hydrochloric and mesylate salts are used.

In addition to pharmaceutically acceptable salts, other salts may also exist. It can serve as intermediates in the purification of the compounds, in the preparation of other salts, or in the identification and characterization of the compounds or intermediates.

The pharmaceutically acceptable salts of the compounds of the formulas I, la and Ib can also exist as solvated solvents, such as with water, methanol, ethanol, dimethylformamide, ethyl acetate and the like. Mixtures of such solvates can also be prepared. The source of such a solvate can be from the crystallization solvent, inherent in the preparation or crystallization solvent, or adventitious to such a solvent.

It is recognized that there may be several stereoisomeric forms of the compounds of formulas I, la and Ib; for example, W can contain a chiral carbon atom in the substituted alkylene moiety. The compounds are normally prepared as racemates and can conveniently be used as such. Alternatively, both individual enantiomers can be isolated or synthesized by conventional techniques, if desired. Such racemates and individual enantiomers and mixtures thereof are part of the compounds used in the methods of the present invention. The compounds used in this invention also encompass pharmaceutically acceptable prodrugs of the compounds of formulas I, la and Ib. A prodrug is a drug, which has been chemically modified and may be biologically inactive at its site of action, but which can be degraded or modified by one or more enzymatic processes or other living processes to the parent bioactive form. This prodrug may probably have a different pharmacokinetic profile than the parent, allowing easier absorption through the mucosal epithelium, better salt formation or solubility, and / or improved systemic stability (an increase in the half-life of the plasma, for example), Normally, such chemical modifications include the following: 1) ester or amide derivatives, which can be cut by esterases or lipases; 2) peptides which can be recognized by specific or non-specific proteases; or 3) derivatives that accumulate at a site of action through membrane selection of a promedicamento form or a modified form of prodrug; or any combination of 1 to 3, supra. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in H.

Bundgaard, Pesian of Prodrugs. (1985) The synthesis of several bis-indole-N-maleimide derivatives is described in Davis et al. , U.S. Patent 5,057,614 and the synthesis of preferred compounds suitable for use in this invention are described in previously identified U.S. Patent 5,552,396 and Faul et al. EP 0 657 411 A1, all incorporated herein by reference. A particularly preferred protein C kinase inhibitor for use in the method of this invention is the compound described in Example 5g (Hydrochloride salt of (S-3,4- [N, N'-1, 1 '- ((2"-ethoxy) -3'" (0) -4 '"- (N, N- dimethylamine) -butane) -bis- (3,3'-indole?) - 1 (H) -pyrrole-2,5-dione) of the aforementioned U.S. Patent 5,552,396 This compound is an inhibitor of protein C kinase potent.It is selective for protein C kinase on other kinases and is highly selective for isozyme, it is say, it is selective for beta-1 and beta-2 isozymes. Other salts of this compound would also be favored, especially mesylate salts.

A preferred mesylate salt can be prepared by reacting a compound of the formula I I with methanesulfonic acid in a non-reactive organic solvent, preferably an organic / water mixture, and most preferably acetone acetone. Other solvents are operable, such as methanol, acetone, ethyl acetate and mixtures thereof. The ratio of solvent to water is not critical and is generally determined by the solubility of the reagents. The preferred ratios of solvent to water are generally from 0.1: 1 to 100: 1 solvent to water by volume. Preferably, the ratio is 1 1 to 20: 1 and most preferably 5: 1 to 10.1. The optimum ratio is dependent on the selected solvent and preferably it is acetone in a ratio of 9.1 solvent to water. The reaction usually involves approximately equimolar amounts of the two reactants, although other proportions, especially those where the methanesulfonic acid is in excess, they are operational. The addition rate of methanesulfonic acid is not critical to the reaction and can be added quickly (<5 minutes) or slowly over 6 hours or more. The reaction is carried out at temperatures ranging from 0 ° C to reflux. The reaction mixture is stirred until the formation of the salt is completed, as determined by X-ray powder diffraction and can take from 5 minutes to 12 hours. The salts of the present invention are preferably and easily prepared as a crystalline form. The trihydrate form of the salt can be easily converted to the monohydrate upon drying or exposure to 20-60% relative humidity. The sai is substantially crystalline demonstrating a defined melting point, double refraction, and an X-ray diffraction pattern. Generally, the crystals have less than 10% amorphous solids and preferably less than 5% and most preferably less than 1% of amorphous solids The mesylate salt is isolated by filtration or other separation techniques appreciated in the art directly from the reaction mixture in yields ranging from 50% to 100%. Recrystallization and other purification techniques known in the art can be used to purify the salt additionally, if desired.

Endothelial cells in the tissue culture stimulated by growth factors, such as VEGF, show a growth rate greater than the basal cell growth rate. Experiments performed in the present invention have shown that when administered in vitro, at a concentration of approximately 0. 1 to 100 nM, the rotein kinase inhibitor C, acid salt of (S) -3,4- [N, N'-1, 1, - ((2"-ethox?) - 3 '" (0 ) -4 '"- (N, N-dimet? Lam? No) -butane) -b? S- (3,3'-indolyl)] - 1 (H) -? Irol-2,5-d? Ona , significantly inhibits non-basal cell growth stimulated by growth factor (such as, VEGF)., other tests have shown that normal endothelial cell growth in the tissue culture is not inhibited by this compound, as is shown by the lack of inhibition of endothelial cell growth without VEGF stimulation in the middle of normoxic conditions. In a hypoxic conditioned medium, the rate of cell growth increases due to the increase in the content of the endogenous growth factor, VEGF, produced by the hypoxic cells. Again, the isozyme selective protein kinase inhibitor C, acid salt of (S) -3,4- [N, N'-1, 1, - ((2"-ethoxy) -3 '" (0) ) -4 '"- (N, N-dimethylamino) -butane) -bis- (3,3'-indole?) - 1 (H) -p? Rrol-2,5-dione, normalizes cell growth induced by such hypoxic conditions The experiments provided in the present invention demonstrate that capillary permeability is also affected by growth factors, such as VEGF The test has shown that in an animal model, VEGF significantly increases capillary permeability up to 3 times. VEGF-dependent capillary permeability is also dose-dependent.According to animal testing in vivo, administering protein C kinase inhibitor at a concentration of approximately 25 mg / kg / day before challenge with VEGF, greatly inhibited the VEGF-induced capillary permeability The use of concentrations from 1 nM to 5 mM, and preferably from 1 nM to 500 nM are specifically contemplated. The inhibition can be up to 80% and is generally specific for capillary permeability induced by growth factor. The capillary permeability can be measured by fluorescein angiography. The PKC-β inhibitors of the present invention can be used to treat conditions of diseases associated with endothelial cell growth and capillary permeability, especially neoplasia, and other VEGF related diseases. Pulmonary edema is also treatable by the compounds of the present invention Pulmonary edema is characterized by increases in the interstitial fluid content of the lungs due to increased capillary permeability Pulmonary edema can be associated with various disease conditions including Affliction Syndrome Respiratory in Adults (ARDS). It is probably associated mainly with rupture of the alveolar-capillary membranes, which could induce hypoxia and the subsequent increase in VEGF content. Such a breakdown could also activate PKC β. Therefore, the compounds identified in the present invention can interfere with the stimulation of capillary permeability by growth factors and / or PKC β and improve the conditions that lead to pulmonary edema The PKC inhibitors of the present invention can also be used to treat neoplasia and other VEGF-related diseases in a mammal. The VEGF signal transduction pathway has direct effects on tumor cells, as well as mediates angiogenic activities in a wide range of conditions.

Neoplastic and non-neoplastic diseases. The expression of VEGF has been demonstrated in a variety of human tumors, such as capillary hemangioblastoma, breast cancer, Kaposi's sarcoma, glioblastoma, angiomatous disorders, colorectal cancer, medulloblastoma, gastric carcinoma, adenocarcinomas of the gastrointerestinal tract, malignant melanoma, cancer of the ovarian cancer, non-small cell lung cancer, prostate cancer, bladder carcinoma, von Hippel Lindau syndrome, renal cell carcinoma, iel cancer, thyroid malignancies, cervical cancer, hepatocellular carcinoma, rhabdomyosarcoma and leiomisarcoma. The poor prognosis of a tumor is often associated with the degree of tumor vascularity coupled with the expression of VEGF. Without a vascular supply, tumor growth is limited Therefore, the use of an anti-angiogenic agent or anti-VEGF agent can prevent further growth and induce tumor regression by limiting vascular supply. Anti-VEGF agents may also have Effects on tumor cells, for example, VEGF directly affects malignant melanoma cells. VEGF expression is controlled by multiple mechanisms. VEGF production can be positively modulated by hypoxia, certain oncogenes and various cytokines including transforming growth factor-beta (TGF-β), platelet derived growth. In a preferred embodiment, PKC-β inhibitors can be used in anti-HIV therapy. -VEGF to treat a human with neoplasia. Any VEGF expressing neoplastic growth, for example, the tumors mentioned above, can be affected by the PKC-β inhibitors of the present invention. Anti-VEGF therapy is especially preferred for treating a human with unresectable primary tumors, primary tumors that are incompletely removed by surgical or radiotherapeutic techniques, primary tumors which have been adequately treated but who are at high risk to subsequently develop metastatic disease, and those with an established metastatic disease. Groups of tumors that have a worse prognosis conferred by a high degree of vascularity, for example, breast cancer, prostate cancer, colon cancer, melanoma cancer, non-small cell lung cancer and head / neck carcinoma, are especially good candidates for anti-VEGF therapy or PKC-β inhibitor treatment of the invention. The occurrence of childhood hemangioma is 10-12% of white infants. Generally, it is not a life-threatening disorder, but in some cases, either due to anatomical size or location it can cause significant morbidity and mortality. VEGF has been implicated in the growth of these tumors. Currently, interferon a-2a is used to induce the regression of this tumor. Given the angiogenic nature of this tumor, anti-VEGF therapy that uses inhibitors of PKC-β should be as effective as interferon a-2a or could be assessed as a wild-type therapy for use over interferon a-2a failure. PKC-β inhibitors or anti-VEGF therapy could also be used to treat tumor-induced ascites, malignant pleural effusions and peritumoral edema. Since VEGF is increased in the actitic fluid of females that have ovarian hyperstimulation syndrome after induction of ovulation, a PKC-β inhibitor could be used in this condition. VEGF is a vascular permeability factor with a high potency, for example, 50,000 times more than histamine. VEGF concentration is elevated in fluid removed from patients with pleural and peritoneal effusions due to malignancy. Intraperitoneal injection of tumor cells in shaved mice results in the accumulation of ascites that is temporally correlated with the increased secretion of VEGF in the peritoneum. . The peritumoral edema that occurs in neoplasms of the central nervous system, such as a glioblastoma, is associated with a high level of VEGF. Anti-VEGF therapy will reduce pleural effusions and ascites associated with malignancy and ovarian hyperstimulation syndrome. Such therapy will decrease the need for repeated paracentesis / thoracentesis and the associated morbidity associated with these procedures, eg, infection, protein suppression, collapsed lung, etc. Such therapy is especially preferred for the inhibition of peritumoral edema that occurs in closed anatomical areas, such as the central nervous system. The PKC-β inhibitors used in the present invention can also be used in anti-VEGF therapies to treat other diseases associated with VEGF expression. Rheumatoid arthritis is characterized by a hyperplastic synovial pannus with a high degree of vasculapity, which invades and destroys the normal architecture of the joint. In addition, the nature of Exudation of the synovial fluid suggests an enhanced degree of capillary permeability. VEGF can stimulate the expression of collagenase and further worsen the destructive process. VEGF levels are significantly elevated in synovial fluid derived from patients with rheumatoid arthritis, compared with patients with osteoarthritis. VEGF production has also been localized by infiltrating macrophages. Therefore, rheumatoid arthritis could be treated by administering inhibitors of PKC-β in anti-VEGF therapy. The keloid is characterized by the formation of exuberant granulation tissue during wound healing resulting in hypertrophic scarring. The disorder is normally seen in black patients and tends to be a recurrent disorder. Topical application of PKC inhibitors to hypertrophic granulation tissue could reduce angiogenesis and decrease subsequent scar formation. Carpal tunnel syndrome, also called entrapment neuropathy, is characterized by nerve compression, which can lead to sensory alternations, muscle weakness, and muscle loss. It is caused by pressure on the median nerve as it passes through the space formed by the wrist bones and the transverse carpal ligament. Carpal tunnel syndrome occurs either as a syndrome related to diabetes or in non-diabetic populations.

The increased nervous hydration in carpal tunnel syndrome can be caused by the high level of VEGF. Increased VEGF levels in the tissues surrounding the nerve can cause nerve entrapment by inducing vascular permeability and emanation of fluid towards the pepneural tissues. The alteration of the synthesis and / or degradation of collagen in carpal tunnel syndrome can be caused by the high level of TGF-β production. The expression of increased TGF-β could intensify the synthesis of extracellular protein including collagen and reduce its degradation, which leads to an increased extracellular matrix deposition in the tissues surrounding the nerves. It has been shown that the activation of PKC induces the transcription of TGF-β by stimulating the activity of the activator of protein 1. Therefore,, the PKC-β inhibitors of the present invention can be used to counteract the activity of VEGF and / or TGF-β in carpal tunnel syndrome. One skilled in the art will recognize that a therapeutically effective amount of the C-β protein kinase inhibitors used in accordance with the present invention is sufficient to inhibit the growth of endothelial cells or to develop capillary permeability by inhibiting VEGF and that this amount varies inter alia, depending on the size of tissue affected, the concentration of the compound in the therapeutic formulation, and the patient's body weight. Generally, an amount of protein C kinase inhibitor to be administered as a therapeutic agent to treat neoplasia and other VEGF-related diseases discussed above will be determined on a case-by-case basis by the attending physician. As a guideline, the degree of neovascularization, the body weight and age of the patient should be considered when an appropriate dose is established.

Generally, a suitable dose is one that results in a concentration of protein C kinase inhibitor at the treatment site in the range of 0.5 nM to 200 μM, and more usually 0.5 nM to 200 nM. 0.5 nM to 100 nM should be sufficient in most circumstances. To obtain these treatment concentrations, a patient in need of treatment will probably be administered between approximately 0,001 mg per day per kg of body weight and 50 0 mg per day per kg. Usually, no more than about 1.0 to 10 mg per day per kg body weight of C-β protein kinase inhibitor should be needed. As noted above, the above amounts may vary on a case-by-case basis. The compounds of formula I and the preferred compounds of the formula la and Ib are preferably formulated before administration. Suitable pharmaceutical formulations are prepared by known procedures using well-known and readily available ingredients. To make compositions suitable for use in the method of the present invention, the active ingredient will usually be mixed with a carrier, or diluted by a carrier, or enclosed within a carrier, which may be in the form of a capsule, sachet , paper or other container. When the carrier serves as a diluent, it can be a solid, semi-solid or liquid material, which acts as a vehicle, excipient or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, capsules, elixirs, suspensions, emulsions, solutions, syrups, aerosol (as a solid or in a liquid medium), soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile powders packaged either for oral or topical application Some examples of suitable carriers, excipients and diluents include lactose, dextrose, saccharose sorbitol Mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcpstalin cellulose, polyvinylpyrrolidone, cellulose, water syrup, methylcellulose methyl and propylhydroxybenzoates, talc, magnesium stearate and mineral oil Formulations may include additionally lubricating agents, wetting agents, emulsifying and suspending agents, preservatives, sweetening agents or sabotagents. The compositions of the invention can be formulated in order to provide rapid, sustained or delayed release of the active ingredient after administration to the patient. compositions are formulated preferably in a unit dosage form containing each dosage from about 0.05 mg to about 3 g, more usually about 750 mg of the active ingredient. However, it will be understood that the therapeutic dosage administered will be determined by the physician in light of the relevant circumstances including the severity of the condition to be treated, the choice of the compound to be administered and the route of administration chosen. Therefore, the above dosage ranges are not intended to limit the scope of the invention in any way. The term " unit dosage form "refers to physically discrete units suitable as unit dosages for human subjects and other mammals, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier. In addition to the above formulations, most of which can be administered orally, the compounds used in the method of the present invention can also be administered topically. Topical formulations include ointments, creams and gels. Ointments are usually prepared using either (1) an oil base, i.e., one consisting of hydrocarbons or fixed oils, such as white petrolatum or mineral oil, or (2) an absorbent base, i.e., one consisting of an anhydrous substance or substances, which can absorb water, for example, lanolin anhydrous Normally, following the formation of the base, either oil or absorbent, the active ingredient (compound) is added to an amount to provide the desired concentration. The creams are oil / water emulsions. These consist of an oil phase (internal phase), usually comprising fixed oils, hydrocarbons, and the like, such as waxes, petrolatum, mineral oil, and the like, and an aqueous phase (continuous phase), comprising water and any substance soluble in water. water, such as added salts. The two phases are stabilized by the use of an emulsifying agent, for example, an active surface agent, such as sodium lauryl sulfate.; hydrophilic colloids, such as colloidal acacia clays, "veegum", and the like. About the formation of the emulsion, the active ingredient (compound) is usually added in an amount to achieve the desired concentration. The gels comprise a base selected from an oleaginous base, water or an emulsion-suspension base. To the base is added a gelling agent, which forms a matrix in the base, increasing its viscosity. Examples of gelling agents are hydroxypropylcellulose, acrylic acid polymers, and the like. Normally, the active ingredient (compound) is added to the formulation at the desired concentration at a point preceding the addition of the gelling agent. The amount of the compound incorporated in a topical formulation is not critical, the concentration must be within a sufficient range to allow easy application of the formulation to the area of affected tissue in an amount which will deliver the desired amount of compound to the treatment site wanted. The normal amount of a topical formulation to be applied to an affected tissue will depend on the size of tissue affected and concentration of the compound in the formulation. Generally, the formulation will be applied to the affected tissue in an amount that provides from about 1 to about 500 μg of compound. per cm2 of an affected tissue. Preferably, the applied amount of compound will vary from about 30 to about 300 μg / cm2, more preferably, from about 50 to about 200 μg / cm2, and, most preferably, from about 60 to about 100 μg / cm2 The following formulation examples are illustrative only and are not intended to limit the scope of the invention in any way.

Formulation 1 Hard gelatin capsules are prepared using the following ingredients: Amount (mg / capsule) Active agent 250 Starch, dry 200 Magnesium stearate 10 Total 460 mg The above ingredients are mixed and filled into hard gelatin capsules in amounts of 460 mg Formulation 2 A tablet is prepared using the ingredients below- Quantity (mg / capsule) Active agent 250 Microcpstal cellulose a Silicon dioxide, smoked 400 Stearic acid 10 Total 665 mg The components are mixed and compressed to form tablets, each weighing 665 mg Formulation 3 Tablets containing 60 mg of active ingredient were made as follows Amount (mg / tablet) Active agent 60 mg Starch 45 mg Cellulose mtcrocpstalin 35 mg Polyvinyl pyrrolidone (as a 10% solution in water) 4 mg Sodium carboxymethyl starch 4 5 mg Magnesium stearate 0 5 mg Talc 1 mg Total 150 mg The active ingredient, starch and cellulose are passed through a US sieve of No. 45 mesh and mixed vigorously. The polyvinylpyrrolidone solution is mixed with the resulting powders, which are then passed through a US No. 14 mesh sieve. Granules produced in this way are dried at 50 ° C and passed through a US mesh No. 18 mesh.

Sodium carboxymethyl, magnesium stearate and talcum, previously passed through a US sieve No. 60 mesh, are then added to the granules, which, after mixing, are compressed into a tablet machine to produce tablets weighing each 150 mg.

EXAMPLES These examples all demonstrate that the use of hydrochloride salt of (S) -3,4- [N, N'-1, 1 '- ((2"-ethoxy) -3'" (0) -4 '" - (N, N-dimethylamino) -butane) -bis- (3,3'-indolyl)] - 1 (H) -pyrrole-2,5-dione inhibits endothelial cell growth in vitro and increased capillary permeability in vivo stimulated by VEGF.

Example 1 In this example, the inhibitory effect of the compound noted on endothelial cell growth stimulated by VEGF was examined using recombinant human VEGF. Bovine retina endothelial cells were isolated from fresh calf eyes by homogenization and a series of filtration steps. The primary endothelial cell cultures were grown on fibronectin-coated plates (NYBen Reagents, New York Blood Center) (Costar) containing Dulbecco's modified Eagle's medium (DMEM) with 5.5 mM glucose, 10% horse serum derived from plasma (Wheaton, Scientific). 50 mg of heparin per liter and 50 units of endothelial cell growth factor per liter (Boehringer Mannheim).

After the cells reached confluency, the medium was changed to include 5% fetal bovine serum (HyClonc). The medium was changed every 3 days. Endothelial cell homogeneity was confirmed with anti-HIV factor antibodies. The effect of the PKC inhibitor noted in the action of VEGF in vitro was evaluated by using sparingly platinum cultures of the bovine retina microvascular endothelial cells, which undergo growth stimulation on the VEGF additive. The bovine retina endothelial cells were patinated sparingly (~ 2500 cells per well) in 24-well plates (Costar), incubated overnight in DMEM containing 10% calf serum (GIBCO). The medium was changed the next day. To examine the impact of the PKC inhibitor noted on endothelial cell growth, a set of experiments was conducted in which cell growth in the absence of any active agent served as a control, and then the impact of the addition of the inhibitor was examined. of PKC noted both in the presence of VEGF (25 ng / ml; Genetech) and in the absence of VEGF. After incubation at 37 ° C for 4 days, the cells were lysed in 0.1% sodium dodecylsulfate (SDS) and the DNA content was measured using Hoechst 33258 dye and a fluorometer (model TKO-100; Hoefer) . All determinations were made at least in triplicate and the experiments were repeated a minimum of three times. The results are expressed as means ± SD for all experiments. The analysis of the in vitro results was carried out using the Student's test paired A P value of < 0 050 was considered statistically significant. Figure 1 illustrates the results obtained using recombinant VEGF. As shown by the three columns to the left of the figure, the addition of the PKC inhibitor noted to the endothelial cell culture essentially had no impact on the rate of basal growth (column one). The growth rate increased substantially over the addition of VEGF (fourth column). This growth rate was significantly shortened over the addition of > 0.5nM of the PKC inhibitor noted (four columns to the right) Example 2 This example is similar to the work reported in Figure 1 and further illustrates the inhibitory effect of the PKC inhibitor noted on endothelial cell growth stimulated by VEGF using recombinant human VEGF. Using the procedures of Example 1, cells were isolated and grown. bovine retina endothelial; then the sparsely platinum cultures were prepared. Again, using the procedure of Example 1, experiments were conducted in which the effect of the PKC inhibitor noted on endotellal cell growth was examined both in the presence of VEGF (25 ng / ml, Genetech) and in the absence of VEGF . After incubation at 37 ° C for 4 days, the cells were lysed in 0.1% sodium dodecylsulfate (SDS) and the DNA content using Hoechst 33258 dye and a fluorometer (model TKO-100, Hoefer) Figure 2 illustrates the results of this work As shown by the columns above the legend -VEGF, the addition of the PKC inhibitor noted to the culture of endothelial cells from 0.1 nM to 100 nM, essentially had no impact on the rate of basal cell growth. Stimulation of endothelial cells with recombinant human VEGF (25 ng / ml) produced a significant increase in cellular DNA content after 4 days, indicative of an increase in growth rate, compared with unstimulated cells (compare -VEGF) to 0 with + VEGF to 0). This rate of growth was significantly shortened by the addition of the PKC inhibitor noted (four columns to the right above the legend + VEGF). In particular, the stimulating capacity of VEGF was slightly reduced in the presence of 0.1 nM of the PKC inhibitor and essentially was completely eliminated by the simultaneous addition of 1 nM and more of the PKC inhibitor.

Example 3 This example examines the impact of the PKC inhibitor noted on the activity of endogenous VEGF expressed on the culture of retinal pigments under hypoxic conditions. Bovine retinal cells and endothelial cells were isolated from fresh calf eyes by homogenization and a series of filtration steps. The endothelial cells were grown and they were poorly plated using the procedures of Example 1. Using similar techniques, bovine retinal pericytes were cultured in DMEM / 5.5 M glucose with 20% fetal bovine serum. Hypoxic conditioned medium was prepared for expression of endogenous VEGF and normoxic conditioned control medium, respectively according to the following procedures. Monolayers of confluent retinal pericytes were exposed for 24 h at 2% 02/5% C02 / 93% N2 using an advanced computer controlled infrared-water-cooled C02 incubator from Lab-LIne Instruments with reduced oxygen control ( 480 model). All cells were maintained at 37 ° C and showed no morphological changes in the light of the microscope, excluding the blue dye "trypan" (> 98%) and subsequently could be passed normally. Cells incubated under normoxic conditions (95% air / 5% C02) were used from the same batch and passage as controls. Subsequently, the medium was collected and filtered (Nalgene, 0.22 μm) before use. In this example, experiments were conducted in which the effect of the PKC inhibitor noted on epdotelial cell growth in the presence of either normoxic conditioned medium or hypoxic conditioned medium was examined. As was done in the previous examples, after incubation at 37 ° C for 4 days, the cells were lysed in 0.1% sodium dodecylsulfate (SDS) and the DNA content was measured using Hoechst 33258 dye and a fluorometer (model TKO-100; Hoefer).

In the tests reported in Figure 3, the PKC inhibitor noted at a concentration of 10 nM was used. As shown in Figure 3, retinal endothelial cell growth was stimulated by the conditioned medium of retinal pericytes grown under hypoxic conditions known to induce VEGF expression (compare column 1 with column 3 in Figure 3). The stimulation of growth was suppressed (normalized) in the presence of the hydrochloric acid salt of (S) -3,4- [N.N'-1. r - ^^ etox -S ^ OJ ^^ - ÍN. N-dimethylamino-J-butane-J-bis-SS.sup.-? Ndolyl)] - 1 (H) -p? Rrol-2,5-dione of the PKC inhibitor (compare column 3 with column 4) Example 4 This example is similar to the work reported in Figures 1 and 2 and further illustrates the inhibitory effect of the PKC inhibitor noted in endotehal cell growth stimulated by VEGF using recombinant human VEGF. Using the procedures of Example 1, they were isolated and made grow bovine retina endothelial cells; then the sparsely platinum cultures were prepared. Again, using the procedure of Example 1, experiments were conducted in which the effect of the noted inhibitor of PKC on endothelial cell growth was examined both in the presence of VEG (+ VEGF) (25 ng / ml, Geneptech) and the absence of VEGF (-VEGF) As before, after incubation at 370C for 4 days, the cells were lysed in 0.1% sodium dodecylsulfate (SDS) and the DNA content was measured using Hoechst 33258 dye and a fluorometer (model TKO-100, Hoefer) Figure 4 illustrates the results of this work As shown by the columns above the legend -VEGF, the addition of the inhibitor of PKC noted to the culture of endotehal cells at a concentration of 10 nM had essentially no impact on the rate of basal cell growth. The stimulation of endothelial cells with recombinant human VEGF (25 ng / ml) produced a significant increase in the DNA content, indicative of an increase in the rate of growth, compared with unstimulated cells (compare Control of -VEGF with Control of + VEGF) This growth rate was significantly shortened on the addition of the inhibitor of PKC noted at a concentration of 10 nM These results show that the described class of PKC inhibitors and particularly, (S) -3,4- [N, N'-1, r - ((2"-etox?) - 3 '" (0) -4' " - (N, Nd? Met? Lam? No) -butane) -b? S- (3,3 '-? Ndol? L)] - 1 (H) -p? Rrol-2,5-d? Ona, avoids mVT stimulation of retinal endothelial cell growth by both exogenous VEGF and VEGF induced by hypoxia. Since VEGF expression has been closely linked to neovascularization associated with macular degeneration, these results support the use of these PKC inhibitors as a Therapy for the treatment of macular degeneration The principles, preferred embodiments and modes of operation of the present invention have been described in the specification However, the invention which is intended to be protected in the present, does not It should be interpreted as limited to the forms described, as they should be seen as illustrative rather than restrictive. Variations and changes can be made by those skilled in the art without departing from the spirit of the invention.

Claims (14)

1. EIVINDICATIONS
2. A method for treating a neoplasm, which comprises administering to a mammal in need of such treatment, a therapeutically effective amount of an inhibitor of the C 2 protein kinase isozyme.
3. The method of claim 1, wherein the inhibitor of the isozyme of protein kinase C is a bis-indolylmaleimide or a macrocyclic bis-indolylmaleimide The method of claim 1, wherein the inhibitor is isozyme-selective and wherein the selectivity of isozyme is selected from the group consisting of beta -1 and beta-2 isozymes 4 The method of claim 3, wherein the cmase inhibitor of protein C has the following formula
4. wherein W is -O-, -S-, -SO-, -S02-, -CO-, C2-C6 alkylene, substituted alkylene, C2-C6 alkenylene, -aplo-, -ar? l (CH2) rrlO -, -heterocycle-, -
5. heterocycle- (CH2) mO-, -bicylic-fused-, -cyclic-fused- (CH2) mO-, -NR3-, -ÑOR3-, -CONH-, or NHCO-, X and Y are independently alkylene C1-C4, substituted alkylene, or together X, Y, and W combine to form - (CH2) n-AA-, R1s are hydrogen or up to four optional substituents independently selected from halo, C1-C4 alkyl, hydroxy, alkoxy of C1-C4, haloalkyl, nitro, NR R5, or -NHCO (alkyl of C-C4),
6. R2 is hydrogen, CH3CO-, NH2, or hydroxy, R3 is hydrogen, (CH2) maplo, C1-C4 alkyl, -COO (C1-C4 alkyl), -CONR4Rs, - (C = NH) NH2, -SO (C1-C4 alkylene), -S02 (NR4R5), or -S02 (C1-C4 alkyl), R4 and Rs are independently hydrogen, C, -C, phenyl, benzyl alkyl or combine with the nitrogen to which they are bound to form a saturated or unsaturated 5 or 6 membered ring, AA is an amino acid residue, m is independently 0, 1, 2 or 3, and n is independently 2, 3, 4 or 5 or a pharmaceutically acceptable salt, prodrug or ester thereof The method of claim 4, wherein the protein C kinase inhibitor has the following formula
7. wherein Z is - (CH2) P- or - (CH2) p-0- (CH2) p-, R4 is hydroxy, -SH, alkyl of C, -C4, (CH2) mar-lo, -NH (ar ?), -N (CH3) (CF3), -NH (CF3), or -NR5R6, R5 is hydrogen or C1-C4 alkyl, R? is hydrogen, C1-C4 alkyl or benzyl, p is 0.1 , or 2, and m is independently 2 or 3, or a pharmaceutically acceptable salt, prodrug or ester thereof The method of claim 4, wherein the protein kinase C inhibitor has the following formula
8. (Ib)
9. wherein Z is - (CH2) P-, R4 is -N RSR6, -NH (CF3), or -N (CH3) (CF3), R5 and R6 are independently H or C1-C4 alkyl, p is 0, 1, or 2, and m is independently 2 or 3, or a pharmaceutically acceptable salt, prodrug or ester thereof The method of claim 5, wherein the protein C kinase inhibitor comprises (S) -3,4- [ N, N'-1, 1 '- ((2"-ethoxy?) - 3'" (0) -4 '"- (N, Nd? Met? Lam? No) -butane) -b? S- ( 3,3 '-? Ndol? L)] - 1 (H) -p? Rrol-2,5-d? Ona or its pharmaceutically acceptable acid salt The method of claim 1, wherein the neoplasm is selected from the group consisting of capillary hemangioblastomy, breast cancer, Kaposi's sarcoma, glioblastoma, angiomatous disorders, childhood hemangioma, colorectal cancer, medulloblastoma, gastric carcinoma, adenocarcinomas of the gastrointestinal tract, malignant melanoma, ovarian cancer, non-cell lung cancer small, prostate cancer, malignant effusions, pre-cumulative edema, bladder carcinoma, Yes von Hippel Lindau syndrome, renal cell carcinoma, skin cancer, thyroid malignancies, cervical cancer, hepatocellular carcinoma, rhabdomyosarcoma, and leiomisarcoma The method of claim 10, wherein the neoplasm is selected from the group consisting of capillary hemangioblastomy , breast cancer, Kaposi's sarcoma, glioblastoma, angiomatous disorders, childhood hemangioma, colorectal cancer, malignant melanoma, ovapco cancer, non-small cell lung cancer, prostate cancer, malignant effusions, pre-tumoral edema, bladder carcinoma
10. 10. A method of treating rheumatoid arthritis, which comprises administering to a mammal in need of such treatment, a therapeutically effective amount of an ß isozyme inhibitor of protein C kinase.
11. 11. A method of treating pulmonary edema, which comprises administering to a mammal in need of such treatment, a therapeutically effective amount of a protein C kinase β isozyme inhibitor.
12. 12. A method for inhibiting VEGF-stimulated capillary permeability associated with pulmonary edema, which comprises administering to a mammal in need of such treatment, a therapeutically effective amount of an inhibitor of protein C kinase β isozyme.
13. 13. A method of treating keloid, which comprises administering to a mammal in need of such treatment, a therapeutically effective amount of an inhibitor of protein C kinase ß isozyme
14. 14. A method to treat carpal tunnel syndrome, the which comprises administering to a mammal in need of such treatment, a therapeutically effective amount of an inhibitor of protein C kinase β isozyme.