MXPA98009160A - Therapeutic treatment of ocular diseases related to the vascular endothelial growth factor (ve - Google Patents

Abstract

The present invention relates to a method for inhibiting stimulated endothelial cell growth (VEGF), such as that associated with macular degeneration and stimulated capillary permeability (VEGF), such as that associated with macular edema, particularly using a selective PKC inhibitor. of isozyme, hydrochloride salt of (S) -3-, 4-. { N, N'-1,1 '- ((2-ETOXI) -3' '' (O) -4 '' '- (N, N-dimethylamino) -butane) -bis (3,3'-indole) ) -1- (H) -pyrrole-2,5-dio

Description

THERAPEUTIC TREATMENT OF OCULAR DISEASES RELATED TO THE ENDOTHELIAL GROWTH FACTOR VASCULAR (VEGF) BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention is broadly directed to a method for inhibiting e! endothelial cell growth and capillary permeability associated with vascular endothelial growth factor (VEGF), eg, increased cell growth and permeability induced by (VEGF) using a protein isozyme inhibitor of Protein Kinase C (PKC, for its acronym in English). These conditions induced by VEGF are closely associated with a variety of ocular vascular disorders. The present invention is particularly directed to the use of an inhibitor of Protein Kinase C (PKC) isozyme (PKC) to treat ocular vascular disorders, including, macular degeneration, macular edema, vascular retinopathy, retinal vein occlusion, iris neovascularization, histoplasmosis and Ischemic retinal diseases. 2. Description of the Prior Art VPF / VEGF is a glycosylated multifunctional cytokine, the overexpression of VPF / VEGF is associated with a variety of ocular vascular disorders. VPF / VEGF induces the proliferation of endothelial cells, excessive permeability via the activation of transport mediated by the vesicular-vacuolar organelle, migration and reorganization of actin with changes in shape and pleating. It alters the expression of endothelial cell genes, inducing the increased production of tissue factor and several proteases, including interstitial collagenase and both activators if my cells are urokinase and tissue plasminogen. The majority of these same genes are induced by the activation stimulated by phorbol myristate acetate (PMA) of PKC. It is thought that vascular endothelial growth factor (VEG F) together with fibroblast growth factors (FGF) and transformation growth factor (TGFß) play a major role in mediating active intraocular neovascularization in patients with retinal diseases. ischemic (Aíello et al., New England Jour. Medicine, 331 (22): 1480-1487 (1994); Amin, et al., Invest Ophthalmol Vis Sci., 35: 3178-3188 (1994)). One of the ocular vascular disorders associated with increased VEGF expression is macular degeneration. Macular degeneration related to age is the main cause of blindness in old age. It is estimated that macular degeneration afflicts more than 16% of people aged 85 and older, and 6% of people between the ages of 65 and 74 years. More than 20% of patients older than 75 years have macular degeneration. The disease is more frequent in women (Liebowitz HM, Krueer DE, Maunder LE, and others, "The Framingham Eye Study: VI Macular Degeneration," Surv. Opthalmol 24 (supp 10: 428-457, 1980); Klein; others, "Prevalence of Age Related Maculopathy: The Beaver Dam Study," Ophthalmology, 99 (6): 933-943, 1992). Macular degeneration can be divided into dry type or wet type, the dry type being 10 times more common, but generally less severe in its clinical manifestations. More severe wettability, or exudative macular degeneration, is associated with abnormal growth of choroidal vessels (choroidal neovascularization) in the subretinal pigment epithelium or subretinal space and often leads to severe visual damage. Macular degeneration has the initial pathological lesion with a drusiform appearance that represents the deposit of abnormal tissue within the retinal pigment epithelial layer (RPE) and was thought to be secondary to vascular insufficiency. The new blood vessels then grow through the Bruch membrane that is between the RPE and the choriocapillaries to invade the retina. This retinal invasion causes the destruction of the photoreceptors and can lead to hemorrhage which reduces vision. Laser therapy is one of the most common forms of treatment for macular degeneration. Laser therapy is used to treat areas of neovascularization that do not extend into the central macular area (fovea). Nevertheless, the recurrence of the disease is common after laser therapy. (MPS Group, Aren Ophthalmol., Vol. 109, pp. 1232-1241 (1991)). In addition, laser therapy may result in residual scotomas and is therefore not an optimal treatment for neovascularization in the central macular region. Only a limited number of patients adequately meet the criteria for this form of treatment, mainly due to the defined disease, or occult nature of the commonly observed choroidal neovascularization. (Freund et al., Amer, Jour, Ophthalmol., 1 15: 786-791 (1993)). Interferon has also been treated as a therapeutic agent based on the known activity of growth factors such as fibroblast growth factor on the stimulation of pathological angiogenesis. However, no consistent effects have been observed. (Kirkpatrick et al., Br. J. Ophthalmol., 77: 766-770 (1993); Chan et al., Ophthalmology, 101: 289-300 (1994)). More recently, an analysis using the transforming growth factor (TGF) beta-2 to treat macular degeneration was unsuccessful. Biotechnology Newswatch, January 1, 1996. With the rapid aging of the population, macular degeneration has a substantial public health problem. Currently, there is no cure and the less than satisfactory results obtained with laser treatment is the only therapy accepted, although the FDA recently approved thalidomide for use in a clinical study with human patients. ("Researchers Focus on Macular Degeneration: Common Eye Problems, Causes and Treatment Get New Attention" by Steven Sternberg, Washington Post Health, October 31, 1995). There is still a strong need in the art for effective drug therapy for macular degeneration.

Macular edema is associated with many types of ocular vascular diseases, such as retinitis pigmentosa, diabetic retinopathy, pars planitis, retinal vein obstruction, senile hyalitis and with intraocular surgical procedures (Henkind, Surv. Ophthalmol 28: 431-2 ( 1984), Bird, Surv Ophthalmol, 28: 433-6 (1984), Cunha-Vaz), Surv Ophthalmol. 28: 485-92 (1984)). Cystoid macular edema is the most common complication after cataract surgery (Yannuzzi, Surv Ophthalmol 2_8: 540-53 (1984)) and probably the most common cause of vision loss in patients suffering from lens extraction (Jampol and others, Surv Ophthalmol 2_8: 535-9 (1984)). Cystoid macular edema is usually self-limited and can even be improved spontaneously in chronic cases (Yannuzzi, Surv Ophthalmol, 28: 540-53 (1984)). However, a small proportion of patients (from 1 to 15%) can develop irreversible damage and permanent visual impairment (Yannuzzi, et al., Opthalmology, 88: 847-54 (1981)). Macular edema is also a cause of vision loss in patients with Vogt-Koyanagi-Harada syndrome (VKH) (Rutzen et al., J. Ret. And Vit. Dis., 1_5 (6): 475-479 ( nineteen ninety five)) . Macular edema is closely related to microaneurysms in diabetic retinopathy and non-diabetic persons with phalaroid cell anemia, branched-vein occlusion, carotid artery disease or severe hypertension (Klein, Med. Clin. N. Am., 72: 1415-1437 (1989)). Microaneurysms often release protein material, which results in the formation of heavy exudates. These exudates appear in a diffused, aggregated or ring-shaped configuration. When exudates and fluids are recovered in the posterior part of the retina, macular edema can result, which can cause significant vision blurring and lead to loss of visual acuity. A laser procedure, called focal photocoagulation, is used to treat areas of retinal swelling adjacent to microaneurysms. Focal photocoagulation has been shown to decrease the incidence of visual acuity deterioration by 60% in patients with clinically significant macular edema, but no benefit of photocoagulation has been shown in patients with mild to moderate macular edema (Raskin, et al. others, Ann. Int. Med, 117 .: (3) 226-233 (1992)). Vitreous surgery can improve visual diagnosis only in cases of diabetic macular edema associated with a pathological vitreous-macular interface (Vaneffenterre, et al., J. Francais D Ophtalmol., 16. (11): 602-610 (1993) ). Treatments with drugs such as oral and topical indomethacin (Miwa, Drug Intell. Clin. Pharm., 20: 548-550 (1986 ^), as well as erythropoietin (Friedman, et al., Amer. J. Kidney Dis., 26 ( 1): 202-208 (1995)) have been evaluated for macular edema but no significant effect has been observed.There is a need in the field for an effective drug therapy for macular edema.While VEG F was known to have some role In the pathology of certain ocular vascular disorders, it was still necessary to determine whether the inhibition of the function provided by VEGF could provide a therapeutic benefit for the treatment of said vascular ocular disorders .The present invention demonstrates that inhibiting the activity of VEGF can be diminished the pathology of a variety of these ocular vascular disorders COMPENDIUM OF THE INVENTION It is an object of the invention to provide methods for treating an ocular vascular disorder. invention to provide the method for inhibiting capillary permeability associated with macular edema in a mammal.

It is still another object of the invention to provide a method for inhibiting neovascularization induced by vascular endothelial growth factor (VEGF). These and other objects of the invention are provided by one or more of the modalities described below. In an embodiment of the invention, a method is provided for treating an ocular disorder comprising administering to a mammal in need of such treatment a therapeutically effective amount of a protein kinase C isozyme inhibitor. In another embodiment of the invention, a method for treating degeneration is provided. macular in a mammal comprising administering to said mammal a therapeutically effective amount of a β isozyme inhibitor of protein kinase C. In yet another embodiment of the invention, a method for inhibiting capillary permeability associated with macular adema in a mammal is provided. which comprises administering to said mammal a. capillary permeability inhibitory amount of an inhibitor of protein kinase β-isozyme C. In another embodiment of the invention, a method is provided for inhibiting vascular endothelial growth factor (VEGF) which comprises administering to said mammal an inhibitory amount of VEGF of an inhibitor of the protein kinase C isozyme. The present invention provides the art with the identity of compounds that are prophylactic and effective in treating a variety of ocular vascular disorders. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the inhibitory effect of the PKC inhibitor, (S) -3,4- [N, N'-1,1 '- ((2"-ethoxy) -3'" (O ) -4 ,,, - (N, Ndmethylamino) -butane) -bis- (3,3'-indolyl)] - 1 (H) -pyrrole-2,5-dione on endothelial cell growth stimulated by VEGF recombinant human. Figure 2 further illustrates the inhibitory effect of the PKC inhibitor, (S) -3,4- [N, N'-1,1 '- ((2"-ethoxy) -3"' (O) -4 ' "- (N, N-dimethylamino) -butane) -bis- (3,3'-indolyl)] - 1 (H) -pyrrole-2,5-dione on the endothelial growth cell stimulated by recombinant human VEGF Figure 3 shows the impact of the PKC inhibitor on the activity of endogenous VEGF expressed on the culture of retinal pericytes under hypoxic conditions, Figure 4 also illustrates the inhibitory effect of the PKC inhibitor on the growth of endothelial cells stimulated by Recombinant human VEGF.

Figures 5A, 5B show the time course of retinal permeability induced by VEGF. Figure 6 demonstrates the response of retinal permeability of fluorescein to VEG F. Figure 7 demonstrates the effect of inhibition of intravitreal PKC and stimulation on retinal permeability. Figures 8A, 8B show the inhibition of retinal permeability in response to VEG F by the protein kinase C β-inhibitor administered orally. DETAILED DESCRIPTION OF THE INVENTION It is a discovery of the present invention that the therapeutic use of a particular class of protein kinase C inhibitors, ie inhibitors of the protein isozyme protein kinase C, and especially inhibitors of protein isozyme kinase C and especially selective inhibitors of ß isozyme of PKC, counteract the effects of VEGF. In particular, it is a discovery of the present invention that the use of this particular class of protein kinase C inhibitors counteracts the growth of endothelial cells and capillary permeability especially the growth of endothelial cells and the capillary permeability stimulated by the growth factor of VEGF. Consequently, said compounds can be used therapeutically to treat disorders associated with VEGF, in particular, a variety of ocular vascular disorders.

The method of this invention preferably utilizes those inhibitors of protein kinase C that effectively inhibit the β-isozyme. A suitable group of compounds are generally described in the prior art as bis-indolylmaleimides or macrocyclic bis-indolylmaleimides. Bis-indolylmaleimides well recognized in the prior art include those compounds described in U.S. 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 the formula I. These compounds and methods for their preparation have been described in the patent of E.U.A. 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 and to inhibit the effects of VEGF associated with ocular disorders. These compounds can also be administered to patients who are at risk of 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-C6 alkenylene, -aryl-, aryl (CH2) mO-, -heterocycle, -heterocycle- (CH2) mO-, -cyclic fused, bicyclic fused- (CH2) mO, NR3-, -ÑOR3-, CONH-, or -NHCO-; X and Y are independently C? -C4 alkylene, substituted alkylene or together X, Y, and W combine to form - (CH2) p-AA-; R1 is hydrogen or up to four optional substituents independently selected from halo, C? -C4 alkyl, hydroxy, C? -C4 alkoxy, holoalkyl, nitro, NR4R5, or NHCO (C? -C4 alkyl); R2 is hydrogen, CH3CO-, NH2 or hydroxy; R3 is hydrogen, (CH2) maryl, C? -C alkyl, -COO (C? -C alkyl), CONR R, 5- (C = NH) NH2, -SO (C? -C4 alkyl), -SO2 (NR4R5), or -SO2 (d-C4 alkyl); R4 and Rs are independently hydrogen, d-C4 alkyl, phenyl, benzyl or combine with the nitrogen to which they are attached 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 most preferred class of compounds for use in this invention is represented by formula I wherein the -X- W-Y- moieties contain from 4 to 8 atoms, which may be substituted or unsubstituted. More 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-, -CON H-, N HCO- or -N R3-. Particularly preferred compounds for use in the invention are compounds of the formula la: (la) wherein Z is - (CH2) P- or - (CH2) p-O- (CH2) p-; R 4 is hydroxide, -SH, C 1 -C 4 alkyl, (CH 2) maryl, -NH (aryl), -N (CH 3) (CF 3) or -NR 5 R 6; R5 is hydrogen or C? -C4 alkyl, Rd is hydrogen, alkyl or benzyl of C? -C4; 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 formula IA are those wherein Z is CH2; and R 4 is -NH 2, -NH (CF 3), or -N (CH 3) 2 or a pharmaceutically acceptable salt, prodrug or ester thereof. Other preferred compounds for use in the method of the present invention with 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 -NRbR6, -NH (CF3), or -N (CH3) (CF3); R & and R6 are independently H or C? -C alkyl; 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 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 can also exist as pharmaceutically acceptable acid addition salts. Acids commonly employed to form said 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-bromophenylsufonic, carbonic, succinic, citric acids, benzoic, acetic and related inorganic and organic acids. Said pharmaceutically acceptable salts include, therefore, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, mono-hydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, 2-butyne-1,4-dioate, 3-hexin-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 the pharmaceutically acceptable salts, other salts may also exist other salts. They can serve as intermediates in the purification of the compounds, in the preparation of other salts or in the identification and characterization of the intermediates. The pharmaceutically acceptable salts of the compounds of the formulas I, la and I b, may also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, ethyl acetate and the like. Mres of said solvates can also be prepared. The source of said solvate may come from the crystallization solvent, inherent in the preparation or crystallization solvent or adventitious to said solvent. It is recognized that there may be several stereoisomeric forms of the compounds of formulas I, Ia and Ib, for example, W may contain a chiral carbon atom in the substituted alkylene portion. The compounds are normally prepared as racemates and conveniently can be used as such. Alternatively, both individual enantiomers can be isolated or synthesized by conventional techniques if so desired. Said individual racemates and enantiomers and mres thereof are part of the compounds used in the methods of the present invention. The compounds used in this invention also encompass the pharmaceutically acceptable prodrugs of the compounds of the formulas I, the and Ib. A prodrug is a drug that has been chemically modified and may be biologically inactive at its site of action, but which may be degraded or modified by one or more enzymatic processes or other processes in vivo to the mother's bioactive form. This prodrug can similarly have a different pharmacokinetic profile than the mother, allowing easier absorption through mucosal epithelium, better salt formation or improved systemic solubility and / or stability (an increase in plasma half-life, for example) . Typically, said chemical modifications include the following: 1) ester or amide derivatives which can be separated by esterases or lipases. 2) peptides that can be recognized by specific or non-specific proteases. 3) Derivatives that accumulate in a site of action through the selection of membranes of a prodrug form or a modified prodrug form; 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 Produgs, (1985). The synthesis of various bis-indole-N-maleimide derivatives are described in Davis et al., Patent of E.U.A. 5, 057,614 and the synthesis of preferred compounds suitable for use in this invention are described in the Patents of E.U.A. previously identified 5, 552, 396 and in EP publication of Faul et al., 0 657 41 1 A 1, all of which are incorporated herein by reference. A particularly preferred protein kinase-β inhibitor for use in the method of this invention is the compound described in Example 5g (Sal de hydrochloride of (S) -3,4- [N, N'-1, 1 ' - ((2"-ethoxy) -3" '(O) -4' "- (N, N-dimethylamino) -butane) -bis- (3,3'-undolyl)] - 1 (H) -pyrrole -2,5-dione) of the above-mentioned U.S. Patent 5, 552, 366. This compound is a potent protein kinase C inhibitor.It is selective for protein kinase C on other kinases and is highly isozyme-selective. , ie, it is selective for beta-1 and beta-2 isozymes Other salts of this compound could also be favored, especially mesylate salts A preferred mesylate salt can be prepared by reacting a compound of formula II. with methanesulfonic acid in a non-reactive organic solvent, preferably an organic / water mixture and more preferably water-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 solvent to water ratios are generally from 0.1: 1 to 100: 1 solvent to water in volume. Preferably, the ratio is 1: 1 to 20: 1 and more preferably 5: 1 to 10: 1. The optimum ratio depends on the selected solvent and is preferably acetone at a solvent to water ratio of 9: 1. The reaction usually involves approximately equimolar amounts of the two reactants, although other ratios are operative, especially those in which methanesulfonic acid is in excess. The methanesulfonic acid addition rate is not critical to the reaction and can be added quickly (<5 minutes) or slowly for 6 or more hours. The reaction was carried out at temperatures ranging from 0 ° C to reflux temperature. The reaction mixture is stirred until the salt formation is complete, as determined by powder x-ray diffraction and may take from 5 minutes to 12 hours. The salts of the present invention are preferably and easily prepared as a crystal form ina. The trihydrate form of the salt can be easily converted to the monohydrate by drying or exposing to 20-60% relative humidity. The salt is substantially crystalline demonstrating a defined melting point, birefringence and a pattern of x-ray diffraction. Generally, the crystals have less than 10% amorphous solids and preferably less than 5% and more preferably less than 1% 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%. The recrystallization and other purification techniques known in the art can be used to purify the salt if desired. Endothelial cells in tissue culture stimulated by growth factors such as VEGF exhibit a growth rate greater than the basal cell growth rate. The experiments carried out in the present invention have shown that when administered in vitro, at a concentration of 0.1 to 100 nM, the protein kinase C inhibitor, acid salt of (S) -3,4- (N, N '-1,1, - ((2"-ethoxy) -3," (O) -4", - (N, N-dimethylamino) -butane) -bis (3,3'-indolyl)] - 1 ( H) -pyrro-2,5-dione, significantly inhibits the growth of new salts stimulated by growth factor (such as VEGF.) More importantly, other tests have shown that normal endothelial cell growth in culture of tissue is not inhibited by this compound, as shown by the lack of inhibition of endothelial cell growth without the stimulation of VEGF in normoxic conditional medium.In the hypoxic conditioned medium, the cell growth rate increases due to the increase in the Endogenous growth factor, VEGF, produced by hypoxic cells Again, the protein kinase C inhibitor acid salt of (S) -3,4- [N , N'-1, 1 '- ((2"-ethoxy) -3"' (O) -4 '"- (N, Nd.methylamino) -butane) -bis- (3,3'-indolyl)] -1 (H) -pyrrole-2,5-dione normalizes the growth of cells induced by said hypoxic conditions.

The experiments provided in the present invention demonstrate that capillary permeability is also affected by growth factors such as VEGF. Tests have shown that in an animal model, VEGF significantly increases capillary permeability up to 3 times. This increase in capillary permeability that depends on VEGF also depends on the dose. According to animal tests in vivo, administration of the protein kinase C inhibitor at a concentration of approximately 25 mg / kg / day prior to the confrontation with VEGF largely inhibited VEGF-induced capillary permeability. The use of concentration from 1nM to 5mM, and preferably from 1nM to 500nM is specifically contemplated. The inhibition can be up to 80% and is generally specific for capillary permeability induced by growth factor. Capillary permeability can be measured by fluorescein angiotherapy. Particularly in macular edema fluorescein angiography is a retinal photographic procedure that involves injection of a fluorescent dye into the bloodstream to detect areas of leakage in the retina. Although not wishing to be limited to any technical explanation, applicants think that alterations in retinal perfusion arising from decreased blood flow, retinal capillary loss, agenesis or obliteration of peripheral vasculature or separation of the choroidal blood supply from the retina may give as a result the relative retinal ischemia. This ischemia stimulates the synthesis and secretion of growth factors such as VEG F in retinal pericytes, endothelial cells, the retinal pigment epithelium, gual cells and possibly other cell types.; and subsequently leads to retinal neovascularization and increased capillary permeability. These conditions are associated with a variety of ocular vascular disorders. The PKC β isozyme inhibitors described in the present invention can be used to treat disease conditions associated with endothelial cell growth and capillary permeability, especially a variety of ocular vascular disorders. Ocular vascular disorders that can be treated by the compounds of the present invention include, but are not limited to, macular degeneration, macular adeno, vascular retinopathy, retinal vein occlusion, iris neovascularization, histoplasmosis, and ischemic retinal diseases. Macular degeneration can be related to age. Macular edema may be associated with diabetes or central retinal occlusion. As used herein, the phrase "vascular retinopathy" does not include diabetic retinopathy but includes vascular retinopathy associated with calciform cell anemia, premature infants and neovascularization of the angle or trabecular meshwork. The neovascuiarization of iri s may or may not be associated with diabetes.

One skilled in the art will recognize that a therapeutically effective amount of an inhibitor of the ß isozyme of protein kinase C according to the present invention is sufficient to inhibit the growth of endothelial cells or development of capillary permeability by inhibiting VEG F and this amount varies, inter alia, depending on the size of tissue affected, the concentration of the compound in the therapeutic formulation and the body weight of the patient. Generally, an amount of a protein kinase C isozyme inhibitor that will be administered as a therapeutic agent to treat ocular vascular disorders will be determined on a case-by-case basis by the physician. As a baseline, the degree of neovascularization, the body weight and age of the patient will be considered when an appropriate dose is established. Generally, a suitable dose is one which results in a concentration of the inhibitor of the isozyme of protein kinase C at the treatment site in the range of 0.5 nM to 200 μM, and more usually 0.5 nM to 200 nM. It is expected that serum concentrations of 0.5 nM to 100 nM will be sufficient in most circumstances. To obtain these treatment concentrations, a patient in need of treatment will probably be administered between about 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 10.0 mg per day per kg of body weight of the protein kinase inhibitor Cβ could be needed. As noted above, the above amounts will vary on a case-by-case basis. The compounds of the formula I and the preferred compounds of the formula la and Ib are preferably formulated before their administration. Suitable pharmaceutical formulations are prepared by known procedures using well-known and readily available ingredients. To form the compositions suitable for use in the method of the present invention, the active ingredient will usually be mixed with a vehicle or diluted by a vehicle, or enclosed within a vehicle which may have the shape of a capsule, sack, paper or another container. When the vehicle serves as a diluent, it can be a solid, semi-solid or liquid material that acts as a vehicle, excipient or medium for the active ingredient. Thus, the compositions can take the form of tablets, pills, powders, troches, sacks, wafers, elixirs, suspensions, emulsions, solutions, syrups, aerosol (as a solid or liquid form), soft gelatin capsules and hard, suppositories, sterile injectable solutions and sterile packaged powders for oral or topical application. Some examples of suitable vehicles, excipient, and suitable diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia gum, calcium phosphates, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose ina, polyvinylpyrrolidone. , cellulose, water syrup, methyl cellulose, methyl and propylhydroxybenzoates, talc, magnesium stearate and mineral oil. The formulations may additionally include lubricating agents, wetting agents, emulsifying and suspending agents, sweetening agents, or flavoring agents. The compositions of the invention may be formulated so as to provide rapid, sustained or delayed duration of the active ingredient after administration to the patient. The compositions are preferably formulated in a unit dose form, each dose containing 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 dose administered will be determined by the physician in view of the relevant circumstances which include the severity of the condition to be treated, the choice of compound to be administered and the route of administration chosen. Thus, the above dose scales are not intended to limit the scope of the invention in any way. The term "unit dose form" refers to physically discrete units suitable as unit doses for human subjects and other mammals, each unit containing a predetermined quantity 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 generally prepared using (1) an oily base, i.e. one consisting of fixed oils or hydrocarbons, such as white petrolatum or mineral oil, or (2) an absorbent base, i.e., one consisting of an anhydrous substance or substances that can absorb water, for example lanolin anhydride. Commonly, after the formation of the base, whether it is oily or absorbent, the active ingredient (compound) is added in an amount that gives the desired concentration. The creams are oil / water emulsions. They consist of an oil phase (internal phase), which normally comprises fixed oils, hydrocarbons and the like, such as waxes, petrolatum, mineral oil and the like and an aqueous phase (continuous phase), which comprises water and any water-soluble substance, such as added salts. The two phases are stabilized by an emulsifying agent, for example, an active surface agent such as sodium lauryl sulfate.; hydrophilic colloids, such as colloidal acacia clays, gum V and the like. Under the formation of the emulsion, the active ingredient (compound) is commonly added an amount to achieve the desired concentration. The gels comprise a base selected from an oily base, water or an emulsion-suspension base. To the base is added a gelling agent that forms a matrix in the base, increasing its viscosity. Examples of gelling agents are hydroxypropyl cellulose, acrylic acid polymers and the like. Commonly, the active ingredient (compounds) is added to the formulation at the desired concentration at a point prior to the addition of the gelling agent. The amount of compound incorporated in a topical formulation is not critical; the concentration must be within a sufficient scale to allow easy application of the formulation to the area of affected tissue in an amount that will supply the desired amount of compound to the desired treatment site. The common amount of a topical formulation that will be applied to an affected tissue will depend on an affected tissue size and the concentration of the compound in the formulation. Generally, the formulation will be applied to the affected tissue in an amount that gives 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 more preferably from about 60 to about 100 μg / cm2. The following examples of formulations are illustrative only and are not intended to limit the scope of the invention in any way. Formulation 1 Hard gelatine capsules are prepared using the following ingredients: Amount (mg / capsule) Active agent 250 dry starch 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 following ingredients: Amount (mg / capsule) Active agent 250 microcrystalline cellulose 400 silicon dioxide, smoked 10 stearic acid 5 Total 450 mg The compounds are mixed and compressed to form tablets each weighing 665 mg. Formulation 3 The tablets each containing 60 mg of active ingredient are made as follows Amount (mg / capsule) Active agent 60 mg starch 45 mg microcrystalline cellulose 35 mg polyvinylpyrrolidone (as a 10% solution in water) 4 mg starch sodium carboxymethyl 4.5 mg magnesium stearate 0.5 mg talc 1 mg Total 150 mg The active ingredient, starch and cellulose are passed through a sieve of E.U.A. No. 45 mesh are mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resulting powders and then passed through a screen of E.U.A. No. 14. The granules thus produced are dried at 50 ° C and passed through a sieve of E.U.A. of mesh No.18. Sodium carboxymethyl starch, magnesium stearate and talc, previously passed through an E.U.A. No. 60 mesh, are then added to the granules which, after mixing, are compressed in a tabletting machine to give tablets each weighing 150 mg.

EXAMPLES These examples demonstrate the use of hydrochloride salt of (S) -3,4- [N, N'-1,1 '- ((2"-ethoxy) -3'" (O) -4 '"- ( N, N-dimethylamino) -butane) -bis- (3,3'-indolyl)] - 1 (H) -pyrrole-2,5-dione to inhibit the growth of endothelial cells in vitro and to inhibit capillary permeability increased in vivo by VEGF Example 1 In this example, the effect of the inhibitor of the compound observed on the growth of endothelial cells stimulated by VEGF was examined using recombinant human VEGF.Retinal endothelial cells of bovine were isolated from fresh calf eyes by homogenization and a series of filtration steps.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% plasma-derived horse serum (Wheaton, Scientific), 50 mg heparin per l and 50 units of endothelial cell growth factor per book (Boehringer Mannheim). After the cells reached the frequency, the medium was changed to include 5% fetal bovine serum (HyClonc). The medium was changed every 3 days. The homogeneity of endothelial cells was confirmed with anti-HIV factor antibodies. The effect of the PKC inhibitor has been observed on the action of VEGF in vitro was evaluated using plaque-plated cultures dispersed from the retinal microvascular endothelial cells of bovine which are subjected to growth stimulation by the addition of VEGF. Bovine retinal endothelial cells were sparsely seeded (approximately 2500 cells per well) in 24-well plates (Costar), incubated overnight in DMEM containing 10% calf serum (GIBCO). The medium was changed the following day. To examine the impact of the PKC inhibitor observed on the growth of endothelial cells, a set of experiments was carried out in which the growth of the cells in the absence of any active agent served as a control, and the impact of the addition of the PKC inhibitor observed both in the presence of VEGF (25 ng / ml, Genentech) and in the absence of VEGF. After incubation at 37 ° C for 4 days, the cells were used in 0.1% sodium dodecyl sulfate (SDS) and the DNA content was measured using Hoechst 33258 dye and a fluorometer (model TKO-100; Hoefer) . All determinations were carried out at least in triplicate and the experiments were repeated a minimum of three times. The results were expressed as mean +. SD for all experiments. Analyzes of in vitro results were carried out by Odd Student's t test. 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 on the left, the addition of the PKC inhibitor observed for endothelial cell culture essentially had no impact on the basal growth rate (one column). The growth rate was substantially increased by the addition of VEGF (fourth column). This growth regime was made significantly by the addition of > 0.5nM of the observed PKC inhibitor (the four columns on 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 observed on the growth of VEGF stimulated endothelial cells using recombinant human VEGF. Using the procedures of Example 1, the bovine reticular endothelial cells were isolated and developed; then cultures specifically planted on the plates were prepared. Again, using the procedure of Example 1, experiments were carried out in which the effect of the PKC inhibitor observed on the growth of endothelial cells was examined both in the presence of VEGF (25 ng / ml, Genentech) and in the absence of of VEGF. After incubation at 37 ° C for 4 days, the cells were used in 0.1% sodium dodecyl sulfate (SDS) and the DNA content was measured 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 observed for the culture of endothelial cells from 0.1 nM to 100 nM did not have an impact essentially on the basal growth of the cells. Stimulation of endothelial cells with recombinant human VEGF (25 ng / ml) produced a significant increase in cellular DNA content after 4 days, indicating an increase in the growth rate, compared with unstimulated cells (compare - VEGF at 0 with + VEGF at 0). This regimen of growth was made significantly by the addition of the observed PKC inhibitor (four column on the right above the legend + VEGF). In particular, the stimulatory capacity of VEGF was slightly reduced in the presence of 0.1 nM of the PKC inhibitor and was essentially eliminated completely by the simultaneous addition of 1 nM of the PKC inhibitor. EXAMPLE 3 This example examines the impact of the PKC inhibitor observed on the endogenous VEGF activity expressed by culturing the retinal pericytes under hypoxic conditions. The retinal endothelial cells of bovine and retinal pericytes were isolated from fresh calf eyes by homogenization and a series of filtration steps. Endothelial cells were grown and sparsely plated using the procedures of Example 1. Using similar techniques, retinal bovine pericytes were cultured in DMEM / 5.5 mM glucose with 20% fetal bovine serum.

The hypoxic conditioned medium for endogenous VEGF expression and normoxic conditioned control medium were prepared respectively according to the following procedures. The monolayers of confluent retinal pericytes were exposed for 24 hours at 2% O2 / 5% CO2 / 93% N2 using a CO2 incubator with infrared water jacket controlled by an advanced Lab-Line I nstruments computer with reduced oxygen control (model 480). All cells were maintained at 37 ° C and showed no morphological changes by luminous microscopy, trypan blue dye excluded (> 98%) and subsequently could be passed normally. Cells incubated under normoxic conditions (95% air / 5% CO2) of the same batch and passage were used as controls. The medium was subsequently recovered and filtered before use (Nalgene, 0.22μm). In this example, experiments were carried out in which the effect of the PKC inhibitor observed on the growth of endothelial cells in the presence of 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 used in 0.1% sodium dodecyl sulfate (SDS) and the DNA content was measured using a Hoechst 33258 dye and a fluorometer (model TKO- 100; Hoefer). In the tests reported in Figure 3, the observed PKC inhibitor was used as a concentration of 10 nM. As shown in Figure 3, the growth of retinal endothelial cells was stimulated by conditioned medium from retinal pericytes grown under epoxy conditions known to induce VEGF expression (compare column 1 with column 3 in Figure 3). This stimulation of growth was suppressed (normalized) in the presence of hydrochloric acid salt of (S) -3,4- [N, N'-1, 1 '- ((2"-ethoxy) -3," (O ) -4", - (N, N-dimethylamino) -butane) -bis- (3,3'-indolyl)] - 1 (H) -pyrrole-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 observed on the growth of endothelial cells stimulated by VEGF using recombinant human VEGF. Using the procedures of Example 1, bovine reticular endothelial cells were isolated and developed; scarcely planted crops were plated. Again, using the procedure of Example 1, experiments were carried out in which the effect of the PKC inhibitor observed on the growth of endothelial cells in the presence of (+ VEGF) (25 ng / ml, Genentech) was examined. in the absence of VEGF (-VEGF). As before, after incubation at 37 ° C for 4 days, the cells were used in 0.1% sodium dodecyl sulfate (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 on the legend -VEGF, the addition of PKC observed for the culture of endothelial cells at a concentration of 10 nM essentially had no impact on the basal growth rate of the cells. Stimulation of the endothelial cells with recombinant human VEGF (25 ng / ml) produced a significant increase in the cellular DNA content indicating an increase in the growth rate, compared with the unstimulated cells (compare control of -VEGF with + VEGF control). This growth regime was made significantly by the addition of the PKC inhibitor observed at a concentration of 10 nM. These results demonstrate that the described class of PKC inhibitors and particularly, (S) -3,4- [N, N'-1, 1 '- ((2"-ethoxy) -3"' (O) -4 ' "- (N, N-dimethylamino) -butane) -bis- (3,3'indolyl)] - 1 (H) -pyrrole-2,5-dione, prevents in vitro stimulation of retinal endothelial cell growth by VEGF exogenous hypoxia-induced expression Since VEGF expression has been closely linked to neovascularization associated with macular degeneration, these results support the use of PKC inhibitors as a therapy for the treatment of macular degeneration Example 6 This example demonstrates the course of the retinal permeability time induced by VEGF.

One eye of each rat received an intervitrea injection of 2.0 ng of VEG F (calculated final concentration of 25 ng / ml). The contralateral eye received a similar volume of control solution. After 10 minutes, 30 microliters of fluorescein was injected through a catheter into the right jugular vein. The fluorometry of the vitreous was carried out at the indicated times after the injection of fluorescein. As shown in Figure 5A, there is a clear increase in fluorescein permeability in the vitreous of eyes treated with VEGF. This becomes statistically significant within 10 minutes of angiogram or fluorescein injection and is maintained for at least 30 minutes. Figure 5B shows that this stimulation expressed as a percentage of control demonstrating that there is leakage of additional fluorescein over eyes treated with VEGF over time. Example 6 This example shows the dose response of retinal permeability to fluorescein in response to VEGF. One eye of each animal was injected with control while the contralateral was injected with several doses of VEG F. 10 minutes later intravenous fluorescein was injected and the amount of the vitreous leak was analyzed after 30 minutes. As shown in Figure 6, there is an increase that depends on the VEGF dose of retinal permeability. The stimulation was maximum by 14 to 20 ng / ml that is known to be obtained in human beings.

Example 7 This example demonstrates that the effect of the intravitreous PKC inhibitor, (S) -3,4- [N, N'-1, 1 '- ((2"-ethoxy) -3'" hydrochloride salt (O ) -4"'- (N, N-dimethylamino) -butane) -bis- (3,3'-indolyl)] - 1 (H) -pyrrole-2,5-dione and its stimulation on retinal permeability. The eyes of rats were further injected with 2.0 ng of VEGF per eye, 10 nM of the PKC β inhibitor, or one microgram of PDBU (a PKC agonist) as indicated in Figure 7. The PKCβ inhibitor was injected 15 minutes before the addition of VEGF, 10 minutes after the addition of VEGF, intravenous fluorescein was given and fluorescein in the vitreous was evaluated after 30 minutes.As shown in Figure 7, the intravitreal injection of VEGF showed the expected stimulation of retinal patency Intravitreal injection of PKCß inhibitor 15 minutes before VEGF injection eliminated most of the permeability response Direct stimulation of protein kinase C by injection of PDBU demonstrated an increase in permeability very similar to VEGF. EXAMPLE 8 This example demonstrates the inhibition of retinal permeability in response to VEGF by an inhibitor of orally administered protein kinase C β, hydrochloride salt of (S) -3,4- [N, N'-1, 1 '- (( 2"-ethoxy) -3 '" (O) -4", - (N, N-dimethylamino) -butane) -bis- (3,3'-indolyl)] - 1 (H) -pyrrole-2, 5-dione.

The rats were fed croquettes mixed with inhibitor of protein kinase C β at the doses indicated in Figure 8A and 8B. After one week with these croquettes, the retinal permeability in response to 2.0 ng of intravitreally injected VEGF was determined as previously treated. Oral administration of the PKCß inhibitor of this invention for one week reduced retinal permeability in response to VEG F. This was more noticeable at higher doses. The principles, preferred embodiments and modes of operation of the present invention have been described in the above specification. The invention which is intended to be protected in the present, however, should not be construed as limited to the particular forms described, since they should be considered 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 (12)

1. CLAIMS 1. A method for treating an ocular vascular disorder which comprises administering to a mammal in need of such treatment, a therapeutically effective amount of an inhibitor of protein isozyme β-kinase C.
2. 2. The method of claim 1, wherein the inhibitor of the ß isozyme of protein kinase C is a bis-indolylmaleimide or a macrocyclic bis-indolylmaleimide.
3. The method of claim 1, wherein the inhibitor is selective isozyme and wherein the selective isozyme is selectively selected from the group consisting of beta-1 and beta-2 isozymes
4. 4. The method of claim 3 wherein the inhibitor of protein kinase C has the following formula: wherein W is -O-, -S-, -SO-, -SO2-, -CO-, C2-C6 alkylene, substituted alkylene, C2-C6 alkenylene, -aryl-, aryl (CH2) mO-, -heterocycle, -heterocycle- (CH2) mO-, -cyclic fused, bicyclic fused- (CH2) mO, NR3-, -ÑOR3-, CONH-, or -NHCO-; X and Y are independently C 1 -C 4 alkylene, substituted alkylene or together X, Y, and W combine to form - (CH 2) n-AA-; R1 is hydrogen or up to four optional substituents independently selected from halo, C? -C4 alkyl, hydroxy, d-C4 alkoxy, holoalkyl, nitro, NR4R5, or NHCO (C? -C4 alkyl); R2 is hydrogen, CH3CO-, NH2 or hydroxy; R3 is hydrogen, (CH2) maryl, C? -C4 alkyl, -COO (C1-C4 alkyl), CONR R, 5- (C = NH) NH2, -SO (C1-C4 alkyl), -SO2 (NR4R5), or -SO2 (d-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 attached 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 kinase C inhibitor has the following formula: wherein Z is - (CH2) P- or - (CH2) p-O- (CH2) p-; R 4 is hydroxide, -SH, C 1 -C 4 alkyl, (CH 2) maryl, -NH (aryl), -N (CH 3) (CF 3) or -NR
5. 5 R 6; R 5 is hydrogen or C 1 -C 4 alkyl, R 6 is hydrogen, alkyl or C 4 -C 4 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 formula IA are those wherein Z is CH2; and R 4 is -NH 2, -NH (CF 3), or -N (CH 3) 2 or a pharmaceutically acceptable salt, prodrug or ester thereof.
6. 6. The method of claim 4, wherein the protein kinase C inhibitor has the following formula: wherein Z is (-CH2) P-; R4 is -NR5R6, -NH (CF3), or -N (CH3) (CF3); R5 and R6 are independently H or C? -C4 alkyl; p is 0, 1 or 2; and m is independently 2 or 3, or a pharmaceutically acceptable salt, prodrug or ester thereof.
7. The method of claim 4, wherein the inhibitor of protein kinase C comprises (S) -3,4- [N, N'-1, 1 '- ((2"-ethoxy) -3"' - (N, N-dimethylamino) -butane) -b] - (3,3, -indolyl)] - 1 (H) -pyrrole-2,5-dione or its pharmaceutically acceptable acid salt.
8. The method of claim 1, wherein the ocular vascular disorder is selected from the group consisting of macular degeneration, macular adeno, vascular retinopathy, iris neovascularization, retinal vein occlusion, histoplasmosis, and ischemic retinal disease.
9. The method of claim 1, wherein the ocular vascular disorder is selected from the group consisting of macular degeneration, macular edema, retinal vein occlusion.
10. 10. The method of claim 8, wherein the vascular retinopathy is retinopathy in premature infants. eleven .
11. A method for inhibiting the growth of endothelial cells stimulated by VEGF which comprises administering to a mammal in need of such treatment, a therapeutically effective amount of a protein kinase C β isozyme inhibitor.
12. 12. A method for inhibiting capillary permeability stimulated by VEGF associated with edema, which comprises administering to a mammal in need of such treatment, a therapeutically effective amount of an inhibitor of the protein isoenzyme kinase C.