WO2011161295A2

WO2011161295A2 - Use of anticalcineurin compounds for the treatment of pathologies involving ocular neovascularisation

Info

Publication number: WO2011161295A2
Application number: PCT/ES2011/070453
Authority: WO
Inventors: Juan Miguel Redondo Moya; Arántzazu ALFRANCA GONZÁLEZ
Original assignee: Fundación Centro Nacional De Investigaciones Cardiovasculares Carlos Iii
Priority date: 2010-06-23
Filing date: 2011-06-22
Publication date: 2011-12-29
Also published as: WO2011161295A3

Abstract

The present invention relates to the use of a compound having anticalcineurin activity for the production of a medicament for the treatment of pathologies involving neovascularisation, in particular for those involving ocular neovascularisation.

Description

USE OF ANTICALCINEURINE COMPOUNDS FOR THE TREATMENT OF PATHOLOGIES THAT COURSE WITH OCULAR NEOVASCULARIZATION

FIELD OF THE INVENTION

The present invention relates to the use of an anticalcineurin compound for the manufacture of a medicament for the treatment of pathologies that occur with neovascularization, in particular for those that occur with ocular neovascularization.

BACKGROUND OF THE INVENTION

There are several eye diseases that present with retinal neovascularization processes, such as retinopathy of prematurity, diabetic retinopathy, the exudative form of macular degeneration and Eales disease among others.

Retinopathy of prematurity (ROP) is a disease caused by proliferative vascularization of the retina in premature babies (born before 37 weeks gestation), being one of the first causes of blindness in childhood. The incidence of this retinopathy is 83%, 60% and 50% for babies born between weeks 28-29, 30-31 and 32-33 respectively.

The vasculature of the human retina develops almost completely in the uterus and is completed approximately between 36 and 40 weeks of gestation. In week 12 the process of vasculogenesis takes place, in which a network of superficial vessels develops through the retina. From the 17-18 weeks, an angiogenesis process begins that involves the development of new vessels from the existing ones. The disease develops when physiological conditions are altered at the stage of angiogenesis, not only if there is hyperoxia but also if there is a loss of factors derived from the mother that contribute to the formation of vessels in the retina (Heidary G et al ., Semin Ophthalmol. 2009 Mar-Apr; 24 (2): 77-81). The disruption of the growth of retinal vessels in premature babies occurs in two sequential phases. The First, which takes place after premature birth, is triggered by higher partial oxygen pressures in the extrauterine environment and by the incubator's oxygen supplement. In response to the increase in oxygen levels, the levels of hypoxia-dependent angiogenic factors decrease, such as VEGF (Vascular-endothelial growth factor), stopping the growth of the vessels causing them to contract and retract. The second phase, vaso-proliferative, which occurs in week 32, occurs when the incompletely vascularized areas of the retina do not have enough oxygen supply and the growth of new vessels is induced in response to hypoxia in these areas, stopping this growth as soon as there is a good supply of oxygen. Neovascularization or fibrotic changes and scars on the retina, called "tufts", that occur after the retraction of the vessels can lead to detachment of the macula or retina and potentially trigger blindness. The current treatment of retinopathy of prematurity consists of ablation of the peripheral retina in the early stages by laser photocoagulation (or cryotherapy in very selected cases); the more advanced phases that present a partial or total detachment of the retina, require surgery with vitrectomy or the placement of an episcleral band (Sylvester CL. Seminars in Ophthalmology, Volume 23, Issue 5 & 6 September 2008 318-323). Children who have suffered ROP may suffer from other visual disorders such as myopia, loss of visual field, strabismus, retinal detachment, glaucous and cataracts.

Diabetic retinopathy is a specific microvascular complication of diabetes. The prevalence of diabetic retinopathy is directly related to the duration of diabetes so that almost all people with type I diabetes and 60% of those with type 2 diabetes suffer from retinopathy after 20 years of illness.

The disease progresses through different stages (Mohamed Q. et al, JAMA. 2007 Aug 22; 298 (8): 902-16). In the first there is a slight non-proliferative alteration in which microaneurysms may appear and is characterized by an increase in vascular permeability. It then progresses to a non-proliferative disorder moderate in which a vascular obstruction occurs. At a later proliferative stage, signals that trigger the growth of new vessels in the retina or on the posterior surface of the vitreous are induced in the retina areas deprived of blood supply. Finally, these changes can be accompanied by macular edema, which is characterized by a thinning of the retina producing an increase in vascular permeability, and as a consequence an edema appears in the retina adjacent to the altered vessel causing the filtration.

During the early stages of the disease no treatment is needed unless there is macular edema, although it is necessary to prevent the progression of the disease through blood glucose, cholesterol and blood pressure controls. In the proliferative stages, the usual treatment is performed with panretinal laser photocoagulation in the early stages and vitrectomy in case of vitreoretinal adhesions and retinal detachment.

Senile macular degeneration (DMS) is a disease that affects the central area of the retina (macula). It is suffered by approximately 10% of people between 65 and 70 years and 30% of those over 75, being the main cause of blindness of the elderly in developed countries.

In the early stages of the disease, deposits of lipid material called drusen accumulate under the retinal pigment epithelium. As described in Co lemán HR. et al, Lancet. 2008 November 22; 372 (9652): 1835-1845, there are two types of senile macular degeneration: the atrophic or dry form is the most frequent, 90% of the DMS, has a slower evolution and allows to maintain an acceptable vision without treatment for longer and the exudative or wet form is less frequent, 10%> of the DMS, but is responsible for the majority of cases of severe vision loss. In the case of the exudative form of the disease there is a growth of new blood vessels under the retinal pigment epithelium and sometimes in the subretinal space. It is also frequent the detachment of the pigmentary epithelium and the appearance of hyperpigmentation. Finally, the lesion contracts and there is an elevated and well defined scar on the posterior pole of the eye. Traditionally the treatment of exudative macular degeneration consists of laser photocoagulation, photodynamic therapy with verteporfin, as well as intraocular administration of corticosteroids in cases of patients with persistent macular edema. Other alternative therapies such as the administration of intraocular anti-VEGF compounds (aptamers, antibodies and soluble forms of their receptors) are recently being used.

Eales disease, also known as juvenile retinal angiopathy, is a rare, usually bilateral, retinal vasculitis, relatively common in India, Pakistan and Afghanistan, being rare in Europe or America.

The pathophysiology of this disease is unknown. It seems to be a disorder of the walls of the peripheral vessels of the retina that give rise to vascular occlusions, peripheral neovascularization and produce a syndrome of repeated hemorrhages in the retina and in the vitreous (Das T. et al, Indian J Ophthalmol. 1994 Mar; 42 (l): 3-18).

There is no satisfactory medical treatment for this disease, the therapeutic objective being to eliminate the ischemic areas and destroy the neovases. Currently, the treatment of choice is laser panretinal photocoagulation to eliminate areas of retinal ischemia and induce regression of vasoproliferative lesions. Another option is the selective photocoagulation of the ischemic lesions and the bordering areas of the perfused retina. Photocoagulator treatment has also been combined with cryotherapy of the peripheral retina with good results. Vitrectomy via pars plana is used in those patients who present with persistent hemovitreous, retinal membranes or vitreous and traction detachment detachments.

So far there is no effective and uncomplicated therapy for the treatment of retinopathies. Photocoagulation does not allow you to recover lost vision, in fact it is possible to lose vision during this treatment, although it is possible to slow the progress of the disease. In addition, other complications such as corneal, iris and crystalline burns, hypema, retinal hemorrhages and choroidal rupture may occur. Vitrectomy allows vision recovery, but the main side effect It is the formation of neovascular glaucoma that can cause permanent blindness. After these treatments, 50% of patients do not recover a good central vision. If there has been macular edema, persistent hemovitreous, neovascular glaucoma or retinal detachment, patients have worse final visual acuities.

International patent application WO2004 / 027027 describes the ability of FK-506 to inhibit choroidal neovascularization in a rat model of choroidal neo-vascularization induced by Matrigel ™. The subretinal injection of said compound does not, however, completely block said neovascularization.

There is therefore a need to find an effective treatment for pathologies that occur with ocular neovascularization, thus avoiding complications and side effects of the therapies used to date. SUMMARY OF THE INVENTION

The invention relates to the use of an anticalcineurin compound for the manufacture of a medicament for the treatment of an ocular pathology that occurs with ocular neovascularization.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Systemic treatment with cyclosporine A (CsA) does not affect the development of neovascularization in the ROP model. N: normoxia; Hx: hyperoxia; ctrl: vehicle.

Figure 2. The intravitreal injection of CsA inhibits the development of neovascularization in the ROP model (1). N: normoxia; Hx: hyperoxia.

Figure 3. The intravitreal injection of CsA inhibits the development of neovascularization in the ROP model (2). N: normoxia; Hx: hyperoxia. Figure 4. Dose-response of the inhibition of the development of ROP by CsA. Hx: hyperoxia.

Figure 5. Administration of CsA under normoxia conditions does not alter the number of vessels or the structure of the retina. N: normoxia; Hx: hyperoxia.

Figure 6. The administration of CsA does not increase the number of apoptotic cells in the retina. N: normoxia; Hx: hyperoxia. DETAILED DESCRIPTION

The authors of the present invention have observed that the administration of a calcineurin inhibitor results in a significant decrease in the alterations that appear in an animal model of retinopathy of prematurity. Specifically, as observed in the examples of the present invention, sporin cycle treatment results in a blockage of the number of vessels in the internal plexiform layer (IPL or infernal plexiform layer) and a decrease in the complexity of the themselves, as well as in a blockage of the appearance of preretinal vascularization ("tufts"). Thus, in a first aspect, the invention relates to the use of at least one anticalcineurin compound for the manufacture of a medicament for the treatment of a pathology that occurs with neovascularization.

Alternatively, the invention relates to at least one anticalcineurin compound for use in the treatment of a pathology that occurs with neovascularization.

Alternatively, the invention relates to a method of treating a pathology that involves neovascularization in a subject comprising the administration to said subject of at least one anticalcineurin compound.

"Anticalcineurin compound" as used herein, refers to calcineurin inhibitors capable of causing a decrease in calcineurin activity, including those compounds that totally or partially inhibit the expression of the calcineurin gene, those that totally or partially inhibit the production of the protein by an inhibition in the translation of calcineurin mRNA as well as compounds that inhibit the activity of the enzyme.

The term "Calcineurin", as used in the present invention, refers to a protein of the Calcium-Calcium Serine / Threonine phosphatases family, discovered more than 30 years ago. Calcineurin, also called protein phosphatase 3 or 2B, is ubiquitously expressed and highly conserved among eukaryotes. It consists of 2 subunits, the enzymatic subunit A or alpha and the regulatory B or beta. In mammals, three iso-forms of calcineurin A (Aa (accession number Q08209), Αβ (accession number P 16298), Αγ (accession number P48454) and two iso-forms of calcineurin B (Bl (accession number P63098) have been described ) and B2 (accession number Q96LZ3). Subunit A contains a calmodulin binding site and a self-inhibition site. The binding of Ca ²⁺ and calmodulin allows a conformational change that unmasks the active site. Calcineurin can dephosphorylate a large amount of proteins among which: NFAT (nuclear factor of activated T cells), NF-κΒ, AP-1 and Elkl. (Rusnak F et al, Physiol Rev. 2000 Oct; 80 (4): 1483-521)

Compounds that produce reduced levels of calcineurin mRNA can be identified using standard assays to determine mRNA expression levels such as RT-PCR, RNA protection analysis, Northern procedure, in situ hybridization, microarray technology and the like.

Compounds that produce reduced levels of calcineurin protein can be identified using standard assays to determine protein expression levels such as immunoblot or Western blot, ELISA (adsorption enzyme immunoassay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS -ELISA (ELISA sandwich with double antibody), immunocytochemical and immunohistochemical techniques, techniques based on the use of biochip or Protein micromatrices that include specific antibodies or tests based on colloidal precipitation in formats such as test strips.

The determination of inhibitory capacity on the biological activity of calcineurin is detected using known standard assays to measure calcineurin activity such as methods based on the detection of phosphatase activity. For example, it is possible to determine if a compound is a calcineurin inhibitor by analyzing the effect of the compound on calcineurin phosphatase activity. The phosphatase activity assay can be easily carried out as described in Fruman DA., Et al, (Proc.Natl.Acad.Sci.USA, 1992, 89: 3686-3690). Specifically by using as a substrate the peptide with the sequence "Asp-Leu-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Val-Ser-Val-Ala-Ala-Glu" (SEQ ID NO : 1) corresponding to a sequence of the RII subunit of the cyclic AMP-dependent and phosphorylated kinase in the serine moiety with [γ- ³² ] ATP by a reaction catalyzed by the catalytic subunit of the cyclic AMP-dependent kinase.

For the test, it is necessary to make a mixture containing 100 nM of purified bovine brain calcineurin (Sigma), 100 nM calmodulin (Sigma) and 5 μΜ of the labeled phosphopeptide [γ- ³² ] in 60 μΐ of the reaction buffer containing TRIS pH 20 mM, 100 mM NaCl, 6 mM MgCl ₂ , 0.5 mM dithiothreitol, 0.1 mg / ml bovine serum albumin and 0.1 mM CaCl ₂ . After 15 min at 30 ° C, the reaction is stopped by adding 0.5 ml of a potassium phosphate buffer (pH 7.0) containing 5% trichloroacetic acid. Free inorganic phosphate is isolated by cation exchange chromatography with a Dowex AG 50 W-X8 column, type H ⁺ , washed sequentially with 10 ml of water, 1 ml of 1M NaOH, 2 ml of 1M HCI and 4 ml of water . The reaction mixture is loaded on the column and the elutions are collected in scintillation vials. After washing with 0.5 ml of water to recover all the phosphorus, 12 ml of Aquasol is added to each vial and the radioactivity of each vial is quantified by means of a scintillation counter. The number of phosphorus picomoles released is calculated with the specific activity of the substrate measured on the day of the test, for which the cpm is measured at 20 μΐ of 15 μΜ (300 pmol) of the labeled phosphopeptide [γ- ³² ]. In a particular embodiment the anticalcineurin compound is selected from Table 1.

TABLE 1: CALCINEURINE INHIBITORS SUITABLE FOR USE ACCORDING TO THE INVENTION

I Ciclosporin A (CsA) according to the formula

or cyclosporine variants that substantially maintain the ability to bind and inhibit calcineurin such as

[(R) a-Methylsarcosine ³ ] CsA and

[Dimethylaminoethylthiosarcosine ³ ] CsA

[MeBm ₂ t] ¹ -CsA

TABLE 1: CALCINEURINE INHIBITORS SUITABLE FOR USE ACCORDING TO THE INVENTION

II Voclosporin according to the formula

III FK-506, tacrolimus or fujimycin according to the formula

As well as tacrolimus analogs such as 31-O-Desmethyl-FK506; L- 683,590, L-685,818; 9-deoxo-31-O-desmethyl-FK506; L-688,617; A-119435; AP1903; dexamethasone-FK506 heterodimer; 13-O-desmethyl tacrolimus and conjugate FK 506-dextran.

IV Ascomycin according to the formula

TABLE 1: CALCINEURINE INHIBITORS SUITABLE FOR USE ACCORDING TO THE INVENTION

VII PD 144795, benzothiophene derivative compound as described in

Gualberto A., et al, (J Biol Chem 273: 7088-7093, 1998) with the general formula:

VIII

AKAP79 (Coghlan VM, et al, 1995, Science 267: 108-111, 1995) and sequences derived therefrom such as those described in W09616172 corresponding to the sequence peptides

RRKRSQSSKEEKP (SEQ ID NO: 3)

RRKRSQSSKEEKPLQ (SEQ ID NO: 4)

RRKRSQSSKEEKPFK (SEQ ID NO: 5)

Peptides from the autoinhibitorio IX calcineurin domain such as those peptides described CaN ₄₅₇ -482-AID (SEQ ID NO: 6) and CaN _424-521-AID (SEQ ID NO: 7) (Arch Biochem Biophys 1999, 372: 159- 165 and Biochem J 1996, 320 (Pt 3): 879-884).

X Orthologs and variants of the protein cain / cabin 1, as described in Lai et al (J. BioLChem., 273: 18325-18331, 1998), including cain / Cabin 1 of Rattus norvegicus) (Accession number in NCBLO88480. 1) (SEQ ID NO: 8) and Cain / Cabin 1 human (NCBI access number: Q9Y6J0.1) (SEQ ID NO: 9) TABLE 1: CALCINEURINE INHIBITORS SUITABLE FOR USE ACCORDING TO THE INVENTION

XI Orthologs and functionally equivalent variants of CHP (calcineurin homologous protein), described in Lin X, et al, (J Biol Chem 274: 36125-36131, 1999) such as CHP1 proteins of human origin (accession number in NCBI: Q99653 ) (SEQ ID NO: 10), CHP1 of bovine origin (access number in NCBI: Q3SYS6.1) (SEQ ID NO: ll), CHP1 of Rattus norvegicus (access number in NCBI: P61023) (SEQ ID NO: 12), CHP2 of human origin (access number in NCBI: 043745) (SEQ ID NO: 13), CHP2 of Mus musculus (access number in NCBI: Q9D869) (SEQ ID NO: 14).

XII Orthologs and functionally equivalent variants of the African pig fever virus A238 protein described in Miskin JE, et al (Science 281: 562-565, 1998), including the protein whose accession number in NCBI is NP 042733 (SEQ ID NO: 15).

TABLE 1: CALCINEURINE INHIBITORS SUITABLE FOR USE

ACCORDING TO THE INVENTION

XIII Tirfostina A8, A23 and A48 as described in Martin BL. et al, (Biochem

Pharmacol 56: 483-488, 1998) with the general formulas

Thyrostin A8

Thyrostin A23

Thyrostin A48

XIV Quercetin, as described in H. Wang et al, (J. Biochem., February 1, TABLE 1: CALCINEURINE INHIBITORS SUITABLE FOR USE ACCORDING TO THE INVENTION

2010; 147 (2): 185-190) with the general formula

XV A peptide comprising the sequence

Where Ri is Tyr or Phe, R ₂ is Ala or Ser and R ₃ is any amino acid except proline. and, preferably, the sequence peptides

DQFLSVPSPFTWSKP (SEQ I D NO: 1 6)

DQYLAVPQHPYQWAK (SEQ I D NO: 17)

MDYLAVPSPLAWSKA (SEQ I D NO: 1 8) as described in EP 1975174

XVI An ApoE analog as described in WO09018477A selected from the group of

- Ac-As-Aib-LRKL-Aib-KRLL-NH ₂ (SEQ ID NO: 19)

- Ac-LRVRLAS-Aib-LRKLRK (nitro-Arg) LL-NH ₂ (SEQ ID NO: 20)

- Ac-LRVRLAS-Aib-LRKLRK (acetyl-Arg) LL-NH ₂ (SEQ ID NO: 21)

- AC-RQIKIWFQNRRMKWKKCLRVRLASHLRKLRKRLL-NH ₂ (SEQ ID NO: 22)

- Ac-Aib-LRKL-Aib- (n acetyl K) RLL-NH ₂ and (SEQ ID NO: 23)

- AC-RRLSYSRRRFLRVRLASHLRKLRKRLL-NH2 (SEQ ID NO: 24)

wherein Aib is iso-butyric amino acid, (nitro-Arg) is nitro arginine, (acetyl-Arg) is acetyl arginine, (n acetyl K) is N-acetyl lysine and Ac indicates a TABLE 1: CALCINEURINE INHIBITORS SUITABLE FOR USE ACCORDING TO THE INVENTION

N-terminal acetylated end.

XVII ISA247 and derivatives as described in US 7141648 with general formula:

XVIII Cyclosporine analogues without immunosuppressive activity

(NMeIle) - -Cyclosporin (NIM81 1) of formula:

and (R) -Methyl-3- (NEtVal) -4-cyclosporin (Debio-025) of formula:

TABLE 1: CALCINEURINE INHIBITORS SUITABLE FOR USE ACCORDING TO THE INVENTION

XXIV Inhibitors of calcineurin-mediated signaling such as:

1,5-dibenzoyloxymethyl-norcantharidin (Baba Y. et al, 2003, J. Am. Chem. Soc, 125: 9740-9749)

AM404 (Caballero F.J. et al, 2007, Biochem. Pharmacol., 73: 1013-1023)

BTP1 (J Biol Chem., 2001, 276: 48118-48126)

BTP2 (J. Immunol, 2003, 170: 4441-4449)

Dibefurin (J. Antibiot., 1996, 49: 124-128)

Dipyridamole (J. Biol. Chem., 2009, 284: 9394-9401)

Gossypol (J Biol. Chem. 2001, 276: 47914-47921)

INCAl (Proc. Nati. Acad. Sci. USA 2004, 101: 7554-7559.)

Kaempferol (IUBMB Life 2008, 60: 549-554)

Liel20 (Brain Res Mol Brain Res 2001, 97: 21-31)

NCI3 (Eur J Immunol 2007, 37: 2617-2626)

The term "antibody", as used in the present invention, refers to a monomeric or multimeric protein that is capable of specifically binding to an antigen and that comprises part or all of the heavy or light chain variable region. Antibodies inhibiting calcineurin activity suitable for use in the present invention include, without limitation, polyclonal antibodies, monoclonal antibodies as well as fragments of antibodies that substantially maintain the ability to bind and inhibit calcineurin activity such as, without limitation, Fab. , F (ab ') 2, Fab', scFv, Fv, diabodies. Likewise, the term antibody includes Fab and single chain Fv fragments (scFV) thereof, bispecific antibodies, heteroconjugates, humanized antibodies, and primatized antibodies and human antibodies. The term "inhibitory RNA" or "RNAi", as used in the present invention, refers to RNA molecules that are capable of silencing calcineurin expression or any gene necessary for calcineurin function. To do this, RNAi typically they are double chain oligonucleotides that are at least 30 base pairs in length and, more preferably, comprise about 25, 24, 23, 22, 21, 20, 19, 18 or 17 base pairs of ribonucleic acid. Several different types of molecules have been used effectively in RNAi technology, including small interference RNAs (siRNAs or siRNAs), sometimes known as short interfering RNAs or RNA silencers, microRNAs (miRNAs), which typically differ of siRNAs because they are processed from single-stranded RNA precursors and are shown only partially complementary to target mRNAs and short bracketed RNA (hRNA or shRNA). Suitable RNAi for calcineurin inhibition include, without limitation, RNAi directed against the catalytic subunit (Calcineurin A) or against the regulatory subunit (Calcineurin B) and, in particular, the sequence siRNA:

5 '-CAGAGUAUUUCACGUUUAAdTdT-3' and (SEQ ID NO: 25)

3'- -dTdTGUCUCAUAAAGUGCAAAUU-5 '(SEQ ID NO: 40) corresponding to positions 677-695 of the alpha chain of subunit A of rat calcineurin and sequence siRNA:

5 '-GGGUUGAUGUUCUGAAGAAdTdT-3' (SEQ ID NO: 27)

3 '-dTdTCCCAACUACAAGACUUCUU-5 (SEQ ID NO: 26)

corresponding to positions 448-466 of the beta chain of the subunit A of rat calcineurin as described in WO2007082909.

Other interfering RNAi suitable for use in the present invention can be obtained using standard algorithms for the design of RNAi molecules from the sequence of a target gene (eg, the criteria described by Elbashir, et al. EMBO J 2001 , 20: 6877-6888, by Taxman et al. (BMC Biotechnol. 2006; 6: 7) or the algorithms available online at:

http://www.ambion.com/techlib/misc/psilencer_converter.html,

http://www.protocol-online.org/cgi-bin/prot/jump. CGI? ID = 3411

or in

http://www.protocol-online.org/cgi-bin/prot/jump. cgi? ID = 3412). Once the region of the target mRNA that will serve as a basis for the synthesis of RNAi and the synthesis of said RNAi has been selected, these can be selected based on their ability to cause a decrease in the RNA levels of the subunits catalytic or regulatory calcineurin or cause a decrease in calcineurin phosphatase activity, determined as mentioned above.

The sequences of the catalytic and regulatory subunits of calcineurin are available in the databases. Thus, the sequences of the mRNAs of the alpha isoforms of the catalytic subunits of human, rat, mouse and bovine calcineurin correspond, respectively, to the sequences with access numbers L14778 (SEQ ID NO: 28), D90035 (SEQ ID NO: 29), J05479 (SEQ ID NO: 30) and U33868 (SEQ ID NO: 31) in NCBI. The sequences of the mRNAs of the beta isoforms of the catalytic subunits of human and mouse calcineurin correspond respectively to the sequences with access numbers M29551 (SEQ ID NO: 32) and BC066000 (SEQ ID NO: 33) in NCBI

Antisense oligonucleotides useful for calcineurin inhibition include, without limitation, the oligonucleotides described by Ikegami, S. et al. (Brain Res. Mol. Brain Res. 1996, 41: 183-191).

The term "antisense oligonucleotide," as used in the present invention, refers to a polynucleotide that is capable of specifically hybridizing with a particular DNA or RNA and includes both oligonucleotides of DNA, RNA or chimeric mixtures, as well as single chain or double chain oligonucleotides. Likewise, the term oligonucleotide includes variants of modified oligonucleotides in the base (5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5- (carboxyhydroxythiethyl) uracil, 5-carboxymethylaminomethyl -thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylkeosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, 5 , N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylkeosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, acid uracil-5-oxyacetic (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2- thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl ester of uracil-5-oxyacetic acid, acid uracil-5-oxyacetic (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2- carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine.), in the sugar group (arabinose, 2-fluoroarabinous, xylulose, hexose and morpholinos), or the phosphate skeleton (peptide nucleic acid), for example to improve the stability of the molecule, hybridization etc.

Antisense oligonucleotides useful for calcineurin inhibition include, without limitation, the oligonucleotides described by Ikegami, S. et al. (Brain Res. Mol. Brain Res. 1996, 41: 183-191) or the oligonucleotides described by Garver et al. (MOLECULAR PHARMACOLOGY, 1999, 55: 632-641) comprising the sequences:

5'-TGA CTG GAG ATG TCC GAG-3 '(SEQ ID NO: 34)

3'-ACT GAC CTC TAC AGG CTC-5 '(SEQ ID NO: 35)

5'-AGC ATG GCC GCC CCG GAG-3 '(SEQ ID NO: 36)

3'-TCG TAC CGG CGG GGC CTC-5 '(SEQ ID NO: 37)

5'-G AGC AAA ATG GGA AAT GA-3 '(SEQ ID NO: 38)

3'-C TCG TTT TAC CCT TTA CT-5 '(SEQ ID NO: 39)

and they can be designed from the aforementioned sequences according to protocols known to an expert and selected for their ability to inhibit calcineurin activity or to totally or partially block the production of calcineurin mRNA. The term "ribozyme," as used in the present invention, refers to a catalytic RNA molecule that is capable of processing a target RNA sequence. Specific ribozymes for calcineurin mRNA include, without limitation, hammerhead ribozymes, and "endoscopic Cech-like ribozymes" RNA such as that which occurs naturally in Tetrahymena thermophila (known as the IVS, or RNA L -19 IVS) and which has been extensively described by Thomas Cech et al. (Zaug et al, Science 224: 574-578, 1984; Zaug et al, Science 231: 470-475, 1986; Zaug et al, Nature 324: 429 -433, 1986; International Patent Application Published No. WO88 / 04300 from University Patents Inc .; Been, et al, Cell 47: 207-216, 1986). The gene-directed ribozymes necessarily contain a hybridization region complementary to two regions, each of at least 5 and preferably each of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19 or 20 contiguous nucleotides in length of a target AR, such as an mRNA of a sequence represented in the calcineurin genes.

By "disease associated with ocular neovascularization" is understood, according to the present invention, any disease in which an abnormal proliferation of a micro vascular network occurs in any compartment of the eye. Said ocular neovascularization may be choroidal, corneal or retinal.

"Choroidal neovascularization" refers to the formation of new vessels that originates in the choroid, and can access the subretinal space through the rupture of the Bruch membrane in the retinal pigmentary epithelium.

"Corneal neovascularization" refers to the process of formation of new vessels originating from the corneal vessels and extending from the limbus to the adjacent corneal stroma.

"Retinal neovascularization" means the formation of new vessels originating from the retinal vessels and extending into the vitreous from the surface of the retina.

In a particular embodiment of the invention the anticalcineurin compound for the manufacture of an anticalcineurin medicament for the treatment of a pathology that occurs with ocular neovascularization is cyclosporine A.

The term "cyclosporin A", as used in the present invention, refers to the molecule described in Table 1 (I) (CAS 55126-45-9) and also known as Sandimmune, Ramihyphin A, Sigmasporin, Consupren, Cyclokat , Mitogard, Neoplanta, Papilock, Pulminiq, Restasis, Sandimmun, Zyclorin, Equoral, Gengraf, Vekacia, Neoral, Cyclosporin H, Consupren S, Sandimmun Neoral and SangCyA. Compounds for use according to the present invention include not only the compounds as such but also pharmaceutically acceptable salts, solvates, prodrugs thereof. The term "pharmaceutically acceptable salts, solvates, prodrugs" refers to any salt, ester, solvate or any other pharmaceutical compound that when administered to a recipient subject is capable of providing (directly or indirectly) a compound with the activity described in the present document However, it will be noted that pharmaceutically unacceptable salts are also within the scope of the invention because the latter may be useful in the preparation of pharmaceutically acceptable salts. The preparation of salts, prodrugs and derivatives can be carried out by means of methods known in the art.

For example, it is possible to synthesize pharmaceutically acceptable salts of the compounds provided herein by conventional chemical methods from an original compound containing a basic or acidic residue. Such salts are generally prepared, for example, by reacting the free acid or base forms of the compounds with a suitable stoichiometric amount of the base or acid in water or in an organic solvent or a mixture of both. Non-aqueous media, such as DMSO (dimethylsulfoxide), ether, ethyl acetate, ethanol, isopropanol or acetonitrile are generally preferred. Examples of acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, iodhydrate, sulfate, nitrate, phosphate and organic acid addition salts such as, for example, acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, matanesulfonate, and p-toluenesulfonate. Examples of base addition salts include organic salts such as for example sodium, potassium, bromide, calcium, ammonium, magnesium, aluminum, lithium salts and salts of organic bases such as, for example, ethylenediamine, ethane, lamina, Ν salts. -dialquilenetane lamina, trietano lamina, glucamina and basic amino acids. Particularly preferred derivatives or prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a patient (for example, by having a compound administered from oral form is absorbed more easily in the blood), or by increasing the release of the original compound in a biological compartment (for example, the brain or lymphatic system) in relation to the original species. The invention also provides therapeutic uses of compositions wherein at least one of the compounds is found as prodrug. The term "prodrug" is used in its broadest sense and includes those derivatives that are converted in vivo into the compounds of the invention. Such derivatives are evident to those skilled in the art and depending on the functional groups present in the molecule and without limitation, include the following derivatives of the compounds present: esters, amino acid esters, phosphate esters, sulphonate esters of metal salts, carbamates and amides. Examples of methods for producing a prodrug of a given active ingredient are known to those skilled in the art and can be found for example in Krogsgaard-Larsen et al. "Textbook of Drug design and Discovery" Taylor & Francis (April 2002).

The compounds for use according to the present invention may be in crystalline form as free compounds or as solvates. Solvation methods are generally known in the art. Suitable solvates are pharmaceutically acceptable solvates. In a particular embodiment, the solvate is a hydrate.

Compounds for use in the present invention may include enantiomers, depending on the presence of chiral centers in a C, or isomers, depending on the presence of multiple bonds (eg, Z, E). The individual isomers, enantiomers or diastereomers and mixtures thereof are included within the scope of the present invention.

The different substituents selected for the different compounds for use in the invention provide a number of factors that significantly affect log P values. In this way, hydroxyl groups act as hydrogen bond donors and intra or intra linkages can be established. intermolecular even in the case of phenols The presence of carbonyl or carboxyl groups generates proton acceptor groups in the molecule. The presence of halogens generates very poor carbons and considerably modifies the biological properties. The amino groups generate good nucleophiles in the molecule and in most cases significantly modify their polarity and polarizability, and the presence of additional alkyl and / or aryl groups increases the lipophilicity of the molecule.

In another aspect, the invention provides use of anticalcineurin compounds (as well as their pharmaceutically acceptable salts, derivatives, prodrugs, solvates or steroisomers thereof) together with a pharmaceutically acceptable carrier, adjuvant or vehicle for administration to a patient. The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a filler, diluent, excipient, solvent or liquid or solid encapsulation material, involved in carrying or transporting the agents object of an organ. , or part of the body, to another organ, or part of the body. Each support must be "acceptable" in the sense of being compatible with the other ingredients of the formulation. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as potato starch and corn starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, solutol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline solution; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other compatible non-toxic substances used in pharmaceutical formulations such as DMSO (dimethylsulfoxide) and its derivatives. The compounds for use according to the present invention may be administered by any suitable route of administration, for example an oral, topical, rectal or parenteral route (including subcutaneous, intraperitoneal, intradermal, intramuscular and intravenous route). In a preferred embodiment, the anticalcineurin compound is administered ocularly.

By "ocular administration" as used herein refers to the administration of a compound in or on the eye and includes, without limitation, topical, subretinal, periocular, retrobulbar, sub-scleral, intrachoroidal, subconjunctival, intracameral, juxtaescleral, subtenon administration. , or intravitreal.

Compositions for ocular administration comprise conventionally prepared injectables, liquid solutions, suspensions, solid forms suitable for solution or suspension in a liquid prior to injection as well as an emulsion.

In another additional particular embodiment the anticalcineurin compound is administered intravitreally. The term "intravitreal administration", as used in the present invention, refers to direct administration in vitreous humor, this being understood as the gel-like substance that fills the space between the lens and the cornea.

For application in therapy, the compositions of the invention will preferably be in a pharmaceutically acceptable or substantially pure form, that is, the compositions of the invention have a pharmaceutically acceptable level of purity excluding pharmaceutically acceptable excipients and not including material considered toxic at levels. of normal dosage. The purity levels for calcineurin inhibitors preferably exceed 50%, more preferably exceed 70%, more preferably exceed 90%. In a preferred embodiment, they exceed 95%. Thus, the invention contemplates the administration of anticalcineurin compounds in the form of a solution of a hyaluronic acid polymer at a concentration between 1 mg / ml and 60 mg / ml. The therapeutically effective amounts of calcineurin inhibitors in the compositions of the invention will generally depend, among other factors, on the individual to be treated, on the severity of the disease that said individual suffers, on the chosen form of administration, etc. For this reason, the doses mentioned in this invention should be considered as guidelines for the person skilled in the art and the latter must adjust the doses according to the variables mentioned previously.

In a particular embodiment the anticalcineurin compound of the invention is administered intravitreally so that a concentration in the vitreous humor of at least 1 to 2 µg / ml or more preferably of at least 100-200 ng / ml is obtained.

The composition according to the present invention can be formulated as a single preparation or, alternatively, it can be provided as a product for simultaneous, concurrent, separate or sequential administration.

The anticalcineurin compounds of this invention, their pharmaceutically acceptable salts, prodrugs and / or solvates, as well as the pharmaceutical compositions containing them can be used together with additional drugs to provide a combination therapy. Such additional drugs may be part of the same pharmaceutical composition or alternatively they may be provided in the form of a separate composition for simultaneous or non-simultaneous administration with the pharmaceutical composition comprising a calcineurin inhibitor or a pharmaceutically acceptable prodrug, solvate or salt of the same. The other drugs may be part of the same composition or be provided as a separate composition for administration at the same time or at different times.

Thus, administration of anticalcineurin compounds with compounds capable of inhibiting signaling by vascular epithelial growth factor (VEGF) is possible, either by blocking VEGF interaction with its receptor using monoclonal antibodies such as ranibizumab (LUCENTIS (R); rhuFab V2) and bevacizumab (AVASTIN (R); rhuMab-VEGF) or by inhibiting receptor expression using, for example, nucleic acids (aptamers such as MACUGEN (R), (pegaptanib) a PEGylated RNA aptamer and specific siRNAs for VEGF RNA). Alternatively, it is possible to inhibit VEGF-mediated signaling by blocking receptor signaling once it has been stimulated by VEGF using, for example, inhibitors of receptor tyrosine kinase activity such as PTK787, sunitinib (Sutent), sorafenib (Nexavar), axitinib and pazopanib. It is also possible to inhibit VEGF-mediated signaling by PI3K inhibitors such as wortmanin and LY294002, PLC inhibitors such as U73122 or neomycin and PKC inhibitors such as staurosporine, bisindolylmaleimide I and LY333531 among others.

Other agents with activity against neovascularization include anti-inflammatory compounds, rapamycin, anti-TNF agents, anti-complement agents, non-steroidal anti-inflammatory agents.

In a particular embodiment, the invention relates to the use of an anticalcineurin compound for the manufacture of a medicament for the treatment of a pathology that involves ocular neovascularization, wherein ocular neovascularization is a retinal neovascularization.

In another particular embodiment, the pathology with retinal neovascularization is selected from retinopathy of prematurity, diabetic retinopathy, exudative form of senile macular degeneration and Eales disease.

The term "retinopathy of prematurity", as used in the present invention, refers to an alteration of the development of retinal capillaries that occurs in premature babies. In contrast to what happens in normal development, where the capillaries grow from the back central part of the eye to the ends, in premature infants the process does not complete resulting in an abnormal proliferation of the capillaries that give rise to the formation of scars, retinal detachment and possible blindness. The term "diabetic retinopathy", as used in the present invention, refers to a progressive disease characterized by the appearance of alterations in the capillaries of the retina caused by diabetes and which include the weakening of the capillary walls, the Increased permeability of capillaries and bleeding and scar formation around new capillaries.

The term "exudative form of macular degeneration" (or wet form), as used in the present invention, refers to an alteration associated with age and characterized by the formation of abnormal permeable capillaries in the macula of the eye, which results in bleeding and edema in the macular region that can lead to blindness.

The term "Eales disease", as used in the present invention, refers to an idiopathic obliterative vasculopathy that primarily affects the peripheral retina of young adults and which is characterized by the appearance of avascular areas on the periphery of the retina followed of microaneurysms, dilation of the capillaries, tortuosity of the neighboring vessels and spontaneous chorioretinal scars.

The invention is now described in detail by means of the following examples that should be considered as merely illustrative and not limiting the scope of the invention.

EXAMPLE 1 Retinopathy model of prematurity

The model used in the present invention has been widely used to study the molecular mechanisms involved in neovascularization in general, and more specifically in the pathogenesis of diseases such as ROP and proliferative diabetic retinopathy. For the realization of the present invention, seven-day-old mouse pups (C57BL / 6 NRj; Janvier, France) were introduced into a sealed chamber, in which an oxygen concentration of 75% was maintained for five days. These changes in the concentration of 0 ₂ induce uncontrolled secretion of proangiogenic factors, which results in abnormal retinal vascularization. This process translates into an increase in the number and complexity of the retinal vessels, and in the appearance of preretinal vascular structures called "tufts" similar to those that occur in the first phase of the ROP. Subsequently, the mice were maintained at an ambient oxygen concentration (21%) for five days. During this stage the avascular retina becomes hypoxic and directs the neovascular retina mimicking the second phase of ROP. Mice were sacrificed by inhalation of C0 ₂ to analyze the angiogenic response in the retina.

To assess the angiogenic response, the eyes were removed and placed in Fekete fixative (70% ethanol, 5% A. Acetic, 4% formaldehyde) for 24 hours. After including the eyes in paraffin, cuts of 5μιη thick were made and subsequently stained with hematoxylin-eosin.

EXAMPLE 2

Intravitreal injection of Cyclosporin A inhibits the development of neovascularization in the retinopathy model of prematurity.

Initially, CsA was administered systemically, by intraperitoneal injection of the drug at a dose of 20mg / Kg every 2 days. This dose has been previously used successfully in various studies (Hernández et al. 2001 J. Exp. Med 193: 607-20). On the other hand, controls were made of the functioning of the administered CsA, by western blotting of NFATcl in thymic tissue of treated mice (not shown). As shown in Figure 1, the CsA has no effect on the number of vessels in the IPL (A.) or in the number of tufts (B.). N: normoxia; Hx: hyperoxia; ctrl: vehicle.

Then it was decided to inject CsA intravitreally when the mice were removed from the chamber, at which point the neovascularization process is triggered. A single injection of Ιμΐ / eye of CsA was administered at

Taking into account the volume of the vitreous in these mice, it is estimated that the actual concentration in the tissue is l-2μg / ml. As shown in Figure 2, the CsA totally blocks the increase in the number of vessels in the IPL (and its complexity, represented by the number of nuclei), as well as the appearance of tufts. N: normoxia; Hx: hyperoxia.

The inhibitory effect of CsA on retinal neovascularization was also evaluated by staining whole-mount retinas with isolectin B4 (which specifically recognizes murine endothelium). Figure 3 shows the vascular pattern of retinas from mice under conditions of normoxia (N), hyperoxia (Hx) and hyperoxia plus CsA 10μg / ml Ιμΐ / eye (CsA Hx). You can see the formation of tufts (arrows) in the case of Hx retinas, which disappear when treating mice with CsA.

EXAMPLE 3

Dose-response of the inhibition of the development of ROP by cyclosporine A.

Next, it was decided to check if the inhibition of the development of ROP also took place at lower doses of CsA, closer to those used in vitro, and that lack toxicity in cell cultures. For this, Ι μΙ / eye of CsA ^ g / ml (corresponding to a concentration of 100-200ng / ml; CsA 1) was administered, in parallel with CsA 10μg / ml (CsA 10). As shown in Figure 4, CsA inhibits the development of ROP at both concentrations, although the reduction in the number of tufts is somewhat smaller than in the case of CsA 10. EXAMPLE 4

CsA does not produce morphological changes in the retina indicative of toxicity nor does it cause an increase in the number of apoptotic cells

To check if the administration of CsA produces morphological changes in the retina that could be indicative of toxicity at the dose used, Ιμΐ / eye of CsA l (^ g / ml was injected under both normoxia (N) and hyperoxia (Hx conditions) As shown in Figure 5, the injection of CsA into normoxia did not cause an increase in the number of retinal vessels or the appearance of tufts (A.) Likewise, no morphological changes suggestive of retinal toxicity were observed after treatment with CsA (B.).

To confirm the absence of CsA toxicity in the retina, specific stains were performed for apoptotic cell nuclei (Apoptag®) in tissues of mice treated with CsA. As seen in Figure 6, no significant differences were detected in the number of apoptotic cells in the retinas of mice subjected to the different treatments (A.); B., representative images of retinas stained for apoptotic nuclei (in red, arrow).

Claims

1. Use of an anticalcineurin compound or combination of anticalcineurin compounds for the manufacture of a medicament for the treatment or prevention of a pathology that involves ocular neovascularization.

2. Use according to claim 1 wherein the compound is selected from Table 1.

3. Use according to claim 2 wherein the compound is sporin A cycle.

4. Use according to any of claims 1 to 3 wherein the compound is administered ocularly.

Use according to claim 4 wherein the ocular administration intravitreal administration.

Use according to claim 4 wherein the anticalcineurin compound is administered so as to obtain a concentration in the vitreous humor of 1 to 2 μg / ml or 100-200 ng / ml.

Use according to claim 1 wherein the pathology with ocular neovascularization is a retinal pathology.

Use according to claim 7 wherein the retinal pathology is selected from the

group consisting of retinopathy of prematurity, diabetic retinopathy, exudative form of senile macular degeneration and Eales disease.