ES2350998B1 - COMPOSITION FOR THE PREVENTION OR TREATMENT OF PATHOLOGICAL PROCESSES RELATED TO ANGIOGENESIS AND CELLULAR PROLIFERATION - Google Patents

Abstract

Composition for the prevention or treatment of pathological processes related to angiogenesis and cell proliferation. # Composition comprising a statin and a vascular endothelial activating agent. Said composition can be used for the preparation of drugs or pharmaceutical compositions, preferably, for the prevention or treatment of pathological processes associated with abnormal cell proliferation and pathological or unwanted angiogenesis such as cancer or arteriosclerosis.

Description

Composition for the prevention or treatment of pathological processes related to angiogenesis and cell proliferation.

The present invention is within the field of biomedicine. Specifically, it refers to a composition comprising a statin and an activating agent of the vascular endothelium and its use for the preparation of a medicament, preferably, for the prevention or treatment of pathological processes associated with abnormal or abnormal pathological or proliferative cell proliferation such as for example the arteriosclerosis cancer.

Prior art

Angiogenesis is the biological process, thanks to which blood vessels, ex novo, are generated from previous vascular structures.To do this, it is necessary to maintain a synergistic coupling of the mechanisms of morpho-functional proliferation and organization.The processes involved in the mechanisms ofangiogenesis and vasculargenesis represent a fundamental role both in healing phenomena, as in the development of neoplasms or in diabetic retinopathy (Scholz et al. 2001. Angiogenesis; 4: 246-257; Shuklaet al. 2007. Ann Thorac Surg; 84: 4349; Tang et al. 2006 EurJCardiothorac Surg; 30: 353-360).

There are many pathologies associated with alterations in vascularization or myo-endothelial proliferation, either by excess or by defect. One of the most common problems in this regard, within the histopathological context that represents atherosclerosis, silk after performing surgical interventions of vascularization (surgery on vascular structures). Surgery to eliminate cardiovascular obstructive problems can fail in the short, medium or long term due, in many cases, to cellular overproliferation (myo-endothelial proliferation at the anastomotic edges) and the appearance of atherosclerotic disease of the graft, which causes the vessel to restenosis. Similarly, restenosis phenomena can occur after endovascular revascularization procedures (also used in peripheral arteriopathies) due to myo-endothelial proliferation phenomena.

or thrombosis phenomena of the treated arterial segment. These pathological events have been tried to be solved by multiple strategies, such as the application of stents that partially reduce stenosis (reduce the scarring of elastic retraction), but do not avoid restenosis due to myo-endothelial proliferation. The latter is treated by avoiding other mechanisms such as the pharmacological treatments (WO / 2003/026492).

Another pathology that is characterized by an unwanted pathological angiogenesis is cancer. For the development of the tumor mass, a constant supply of nutrients is necessary. The neovascularization of the tumor allows nutrients to reach the proliferative cells of the tumor, allowing it to maintain its proliferative rate. It has been shown that inhibition of this neovascularization causes arrest in the development of the tumor. In addition, the neo-vessels formed have an altered structure and function, with obvious distortions in the endothelial permeability barrier, facilitating tumor resection and added symptomatology to tissue dematization.

In recent years the number of angiogenesis modulators and endothelial function studied for the treatment of these pathologies has increased, including statinasymodulators of the lysophosphatidic acid receptor.

Statins are widely prescribed drugs for cholesterol reduction in patients with hypercholesterolemia or cardiovascular disease (Liao et al. 2004. Ann Rev PharmacolToxicol; 45: 89-118). They act primarily by competitive inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a limiting enzyme in the process of cholesterol synthesis. These statins also have other non-dependent effects of the reduction of cholesterol levels (pleiotropic effects) such as the improvement of cardiovascular functionality (O'Driscoll et al. 1997. Circulation; 95: 1126-1131), the decrease in the expression of proinflationary cytokines (Masón et al. 2004. Circulation; 109: 1134-1141), or its effects on tumor growth and differentiation (Wong et al. 2002. Leukemia; 16; 508-519).

It has been shown that statins can reduce cancer death in patients with long treatments for cardiovascular disease (Pedersen et al. 2000. AmJCardiol; 86: 257-262). They also have the ability to inhibit tumor growth by activating apoptosis of cancer cells (Causal et al. 2003. Endothelium; 10: 4958) Cycle inhibition is produced by multiple strain-dependent mechanisms, such as through the inhibition of activity of cyclin-dependent kinase-2 (CDK-2) together with the activation of CDK inhibitors. Apoptosis occurs preferentially in proliferative cells and occurs through the control of various signaling pathways, such as the activation of cassettes, the inhibition of antiapoptotic protein bcl-2, or Raf / MEK / ERK signaling route (Agarwal et al. 1999. Clin Cancer Res; 5: 2223-2229; Wuet al. 2004. Cancer Res; 64: 6461-6468). In addition, it has been shown that the ability to inhibit protein geranylgeranylation is an indispensable element for statin death in acute myeloid leukemia cells (Xia et al. 2001. Leukemia; 15: 13981407). On the other hand it has also been indicated that statins are capable of inhibiting tumor growth by overexpression of the p21 protein (Ukomadu et al. 2003. JBiol Chem, 278: 43586-43594). Another anti-cancer effect that has been demonstrated by statins is the inhibition of epidermal growth factor receptor (EGFR) function, preventing the development of some lung tumors (Mantha et al. 2005.Clin Cancer Res; 11: 2398-2407 ).

The application of statins to the vascular endothelium, depending on the dose, may imply a marked decrease in the synthesis of VEGF-A, its specific FLT-1 receptor, as well as VEGF-C (Jones et al. 1999. AmJPhysiol Gastrointestinal Liver Physiol; 276: 1345-1355) Despite this, statins have been shown to have a proangiogenic effect when administered at concentrations within the nanomolar or picomolar range, while at high concentrations, within the micromolar range, they demonstrate a clearly antiangiogenic effect ( Dulak et al. 2005. Endothelium; 12: 233-241; Fricket al. 2003. Atherosclerosis; 170: 229-236).

Another modulator of endothelial function is lysophosphatidic acid (LPA). This is involved in cellular processes such as differentiation or inhibition of apoptosis in various cell types (Duriex et al. 1993. Trnds Pharmacol Sci; 14: 249-254). Furthermore, this compound has been demonstrated as a fundamental element for vascular development in mice (van Meeteren et al. 2006. Mol Cell Biol; 26: 5015-5022). This factor is also implicated in both proliferation and endothelial cell migration (Lee et al. 2000. AmJ Physiol Cell Physiol; 278: C612-C618), which demonstrates its proangiogenic character (Rivera-Lópezet al. 2008. Angiogenesis; 11: 301-310). This factor also induces expression of VEGF-C, which promotes neovascularization (Lin et al. 2008. Cell Signal; 20: 1804-1814). All this is endorsed by the fact that LPA receptor antagonists are being used as agents for the prevention of proliferative or associated diseases. carcinomas (US2008 / 0213274).

Modulation of unwanted angiogenesis and proliferation is therefore shown as a fundamental element when controlling various pathological conditions that, such as the development of solid tumors or arteriosclerosis, require both neovascularization and myo-endothelial proliferation for their development. These pathologies have developed numerous angiogenesis inhibitors and endothelial modulators that have shown excellent results in preclinical trials. Despite these promising results, subsequent clinical trials have not yet confirmed the expectations created around these treatments (Sivakumar et al. 2004. JAMA 292: 972-977). Therefore, there is a need for effective treatment to prevent and treat diseases related to the anomalous development of angiogenesis and myoendothelial proliferation.

Explanation of the invention.

The present invention refers to a composition comprising a statin and an activator of the vascular endothelium and its use for the preparation of a medicament, preferably, for the prevention or treatment of pathological processes associated with a desired myo-endothelial angiogenesis and proliferation such as, for example, cancer

or arteriosclerosis.

In the examples of the present invention it is demonstrated how the stimulation of the endothelium with an endothelial activator causes hyperproliferative changes in that endothelium, resulting in an altered endothelial situation. In this altered situation there is an increase in the expression of genes related to cell proliferation, as well as in the expression of proinflammatory cytokines.

On the other hand, lavastatin, used in concentrations of the micromolar range causes, in the endotheliovascular, a modification of the gene expression that makes the endothelium show a physiologically normal state.Unfortunately, the use of statin in the micromolar range also leads to a decrease in the cell viability of the endothelium with the consequent alteration of the tissue.

In the present invention it is demonstrated that the application of statins such as atorvastatin within the micromolar range to the endothelium, after activation with an endothelial activator such as lysophosphatidic acid, continues to produce reversion of the endothelium to a physiological state similar to the treatment with statin of independently. This reversal is observed at the gene level, and there is a decrease in the proliferative signals induced by lysophosphatidic acid, as well as in the indicative signs of endothelial dysfunction. Surprisingly, this pre-incubation of the endothelium with lysophosphatidic acid produces a protection of the apoptotic effect produced by the treatment independently with statins in micromolar concentrations. Therefore, this co-addition of compounds causes the reversion of an activated endothelium to one with normalized physiological functions without causing apoptosis but slowing its cell kinetics by significantly decreasing its proliferation.

Therefore this treatment reduces the toxicity of the use of high concentrations of statins, while maintaining their beneficial effects.

Thus, a first aspect of the invention refers to a composition (hereinafter, invention composition) comprising a statin, a saloon itself, and an endothelial activator.

Statins are a family of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase enzyme inhibitor compounds. They also stimulate the absorption of cholesterol by the liver, which reduces levels of the liver, as well as triglycerides in the circulating blood. In a preferred embodiment of this aspect of the invention, the statin is selected from the list comprising: atorvastatin, fl uvastatin, lovastatin, pitavastatin, pravastatin, simvastatin, or rosuvastatin.

In a still more preferred embodiment of this aspect of the invention, the statin is atorvastatin. Atorvastatin is a synthetic compound of formula (I). Atorvastatin salts and esters are also known because they can also be used.

The term "endothelial activator", as used in this description, refers to a compound capable of triggering specific and recognizable processes in the endothelium called endothelial activation. These effects on endothelial cells generally consist of overexpression of molecules such as VCAM, ICAM, E-selectin and von Willebrand factor, and a decrease in cAMP production. This leads to a loss of the barrier function of the endothelium and alterations in the endovascular motor. Within endothelial activators, there are, for example, but not limited to TNFα, hemodynamic forces, as well as sphyngosine-1-phosphate, and its analogues, or lysophosphatidic acid, an analogue or a salt of said acid.

In a preferred embodiment of this aspect of the invention, the endothelial activator is selected from the list comprising TNFα, fi ngosin-1-phosphate, an analogue thereof, or lysophosphatidic acid, an analogue or a salt of said acid.

In an even more preferred embodiment, the endothelial activator is lysophosphatidic acid, an analogue or a salt of said acid.

Lysophosphatidic acid, of formula (II), is a bioactive phospholipid with activity in various cell types. It induces proliferative effects and cellular morphological changes. It also acts as a mediator in membrane phospholipid synthesis processes.

The term "lysophosphatidic acid" herein includes a family of 1-acyl, 2-hydroxy-sn-glycerol-3-phosphate formulation compounds, which have a saturated (16: 0, 18: 0) or unsaturated (18: 1, 18: 2) fatty acid chain. , 20: 4).

The term "analog" as used herein refers to a chemical similar to another chemical in structure and / or function. For example, analogs of lysophosphatidic acid can be considered without limitation, saturated, unsaturated or polyunsaturated fatty esters or alkyl ethers. They can also be considered analogs of acidic acid.

sofosphatidic those compounds in which the phosphate group of lysophosphatidic acid is replaced by compounds on the list that includes, but are not limited to, phosphomomomics such as methylene, phosphonates, methylene phosphonates, phosphothioates, or phosphonothioates. Those similar to lysophosphatidic acid are also considered similar where the hydroxy groups in the sn-1 and sn-2 positions are replaced by fluoro methoxy groups. Those compounds that substitute the O-acyl group in the sn-1 position for an O-alkyl group are also included.

Another aspect of the invention refers to the use of the composition of the invention for the preparation of a medicine.

The composition of the present invention can be used, but not limited to, to normalize endothelial function in the processes of angiogenesis, abnormal or unwanted cell proliferation (such as myo-endothelial proliferation in atherosclerosis) or in situations of endothelial barrier dysfunction. Therefore, a preferred embodiment of this aspect of the The invention refers to the use of the composition of the invention for the preparation of a medicament for the prevention and / or treatment of a pathological process associated with abnormal or unwanted angiogenesis, cell proliferation, etc., or a dysfunction of The endothelial barrier.

It is understood by "abnormal myo-endothelial angiogenesis and proliferation" or "unwanted" that cardiovascular development that differs structurally, morphologically or functionally from the physiological development of the vessels and that is associated with a pathological process. This abnormal development leads to deterioration of normal tissue functions. There are many pathological processes related to the abnormal development of angiogenesis and myo-endothelial proliferation.

"Endothelial barrier dysfunction" is understood to be that situation in which the vascular endothelium partially or totally loses its functionality in maintaining the integrity of the vessels, causing an alteration in the permeability of the vessels. It is also characterized by an increase in vasoconstrictor signals such as the release of angiotensin II or the increase in the expression of endothelin-1, and by the reduction of the vasodilators such as the production of nitric oxide (NO) by the decrease in the expression and activity of the enzyme nitric oxide synthase endothelial (eNOS). It is also accompanied by the generation of a proinflammatory, proliferative and procoagulatory environment that favors atherogenesis.

A more preferred embodiment of this aspect of the invention refers to the use of the composition for the preparation of a medicament for the prevention or treatment of a pathological process associated with abnormal or unwanted angiogenesis, cell proliferation (such as myo-endothelial proliferation), or endothelial barrier dysfunction, selected from the list that includes arteriosclerosis, cancer, macular degeneration, neovascular glaucoma, endometriosis, rheumatoid arthritis, psoriasis, ocular herpes, trachoma, corneal graft neovascularization, viral interstitial keratitis, microbial keratoconjunctivitis, hypertrophic telangiectasia, vascular adhesions, acne fibrosis Rosacea, retrolental fibroplasia, primary or secondary pulmonary hypertension, shock lung, systemic inflammatory response syndrome, fibrosis, proliferative vitreoretinopathy, leukemia, ulcerative colitis, prematurity retinopathy, epidemic keratoconjunctivitis, atopic keratitis, keratitis of the upper limbs, dry keratitis of the pterygium, Sjögren's syndrome, phlectenulosis, syphilis, Mycobacterial infections, chemical burns, bacterial ulcers, fungal ulcers, Mooren ulcer, marginal degeneration of Terrien, keratolysis of marginal disease, polysrteritis, Stevens disease, polyarteritis Steve -Johnson, radial keratotomy, sickle cell anemia, elastic pseudoxanthoma, penfotoid, Paget's disease, chronic uveitis, chronic vitritis, Lyme disease, Eales disease, Behcet's disease, supposed ocular histoplasmosis, Best's disease, myopia, optic nerve wells, Stargardt's disease, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, post-laser complications, rubeosis, arthritis, synovitis, osteomyelitis, asthma, allergic dermatitis, DiGeorge syndrome, corneal graft neovascularization, warts, tumors, obstructive disease of carotid, age-related macular degeneration, or diabetic retinopathy.

Within the angiogenesis and abnormal myo-endothelial proliferation, one of the most important pathologies is arteriosclerosis characterized by thickening and dysfunction of the walls of the vessels. Within this, the most common and the most serious is the formation of the atheromatic placade characterized by the accumulation of lipids at the subendothelial level. This event has been associated in many cases with myocardial, cerebral, musculoskeletal or splanchnic ischemia, so the decrease in plaque would be a desirable event. In addition, the instability of the plaque formed can lead to the release of highly thrombogenic lipids that produce acute cardiovascular events (myocardial infarction, acute lower limb ischemia, acute splanchnic ischemia, cerebral infarctions, etc.). Statin treatment has shown a reducing effect on atheroma plaque formation, as well as plaque stabilizer. Due to the potent anti-arteriosclerotic effects of statins at high concentrations (within the micromolar range) and the elimination of the cytotoxic effect by preactivation with LPA, the present combination could be used for example, but not limited, for the prevention of treatment of arteriosclerotic plaques, ischemic heart disease, ischemic stroke, ischemic stroke vascular restenosis, hypertrophic scarring or scleroderma, peripheral arteriopathies, etc.

In addition, tumors are also found.For tumor progression, vital importance is the development of vascular structures. Due to the rapid tumor growth and the need for the vasculature to develop this rhythm, the developed vessels show dysfunctions and no correct development of the endothelial barrier. In this sense, treatment with statinase is at high concentrations together with the addition of the endothelial activator would cause a tumor growth arrest and only the tumor growth arrest. In addition, the functionalization of

Vascular tissue allows the recovery of the endothelial barrier preventing the spread of tumor cells, and therefore metastasis. Therefore, the present composition would be useful for the prevention and treatment of both solid tumors, such as blood-borne tumors, such as, but not limited to, childhood bronchial adenomas, childhood cerebellar astrocytoma, childhood malignant cerebralyglioma astrocytoma, pleuropulmonary blastoma, Burkitt lymphoma, cancer infantile colorectal cancer, anus cancer, head and neck cancer, nasal and paranasal sinus cancer, oral and lip cavity cancer, renal cell cancer, cervical cancer, colon cancer, cervical cancer, endometrial cancer, esophageal cancer, stomach (gastric) cancer, Salivary gland cancer, adult liver cancer (primary), hypopharyngeal cancer, small bowel cancer, larynx cancer, extrahepatic bile duct cancer, breast (breast) cancer, nasopharyngeal cancer, intraocular melanoma, cancer eye, retinoblastoma, oropharyngeal cancer, multiple cancer, cancer pancreas, parathyroid cancer, skin cancer (melanoma), skin cancer (not melanoma), prostate cancer, non-small cell lung cancer, small cell lung cancer, rectal cancer, testicular cancer, thyroid cancer, urethra, vaginal cancer, bladder cancer, gallbladder cancer, vulvar cancer, epithelial ovarian cancer, squamous neck cancer with metastatic occult primary tumor, bone cancer, osteosarcoma, basal cell carcinoma, Merkel cell carcinoma, cortex carcinoma adrenal, carcinomatymic and thymoma, leukemia, spinal cord and brain cancer, swing tumor, pheochromocytoma, glioma, cerebral astrocytoma, hypophysial tumors, bony malignant histiocytoma, Kaposi sarcoma, T-cell lymphoma, semoral syndrome medulloblastoma, mesothelioma, pineoblastoma, primitive neuroectodermal tumor su pratentorial, rhabdomyosarcoma, atypical rhabdoid / teratoid cancer of the central nervous system, soft tissue sarcoma, uterine uterine sarcoma, myelodysplastic syndromes, ependymoma, Wilms tumor, extracranial germ cell tumor, germ cell tumors, or macroglobulinemia.

An even more preferred embodiment of this aspect of the invention refers to the use of the composition for the preparation of a medicament for the prevention or treatment of a pathological process associated with angiogenesis, abnormal or unwanted myo-endothelial proliferation, or processes pathological associated with a dysfunction of the endothelial barrier, selected from the list that includes arteriosclerosis, restenosis of cardiovascular grafts, endovascular procedures, tumors of the Central Nervous System (CNS), solid tumors, hematopoietic tumors, pulmonary hypertension, shock lung or systemic inflammatory response syndrome.

Due to the high concentrations (within the micromolar range) in which it is required to administer the statin, it would be interesting, although not limited, that the administration of the composition be performed locally preferably for its action on the affected tissue. Therefore, a preferred embodiment of this aspect of the invention refers to the use of the composition in a form suitable for local administration.

Treatment of the endothelium with activators causes a characteristic response. This response is what allows the statin to exert its physiological normalization effects avoiding the proapoptotic effect and producing an inhibition of myo-endothelial proliferation. Therefore the use of statin in isolation on an already activated endothelium would exert the same effects as on an endothelium treated with an endothelial activator. Therefore, another aspect of the invention describes the use of a statin, a salt or an ester thereof for the prevention and / or treatment of a pathological process associated with abnormal myo-endothelial angiogenesis and / or proliferation or unwanted when endothelial cells are activated (abnormal tumor neovases).

A preferred embodiment of this aspect of the invention refers to the use of a statin, salt or ester thereof for the preparation of a medicament for the prevention or treatment of a pathological process associated with angiogenesis, desired abnormal or endothelial proliferation, or an endothelial barrier function, selected from the list that includes although without limitation, arteriosclerosis, cancer, macular degeneration, neovascular glaucoma, endometriosis, rheumatoid arthritis, psoriasis, ocular herpes, trachoma, corneal graft neovascularization, viral interstitial keratitis, microbial keratoconjunctivitis, hypertrophic telangiectasia, vascular adhesions, acne fibrosis retrolental, primary or secondary pulmonary hypertension, shock lung, systemic inflammatory response syndrome, fibrosis, proliferative vitreoretinopathy, leukemia, ulcerative colitis, retinopathy of prematurity, epidemic keratoconjunctivitis, qu atopic eratitis, keratitis of the upper limbs, dry keratitis of the pterygium, Sjögren's syndrome, phlectenulosis, syphilis, Mycobacterial infections, chemical burns, bacterial ulcers, fungal ulcers, Mooren ulcer, marginal Terrien degeneration, keratolysis, marginal polyarteritis of Stevens-Johnson, radial keratotomy, sickle cell anemia, elastic pseudoxanthoma, penfotoid, Paget's disease, obstructive carotid disease, chronic uveitis, chronic vitritis, Lyme disease, Eales disease, Behcet disease, supposed ocular histoplasmosis, Best's disease, myopia, optic nerve wells, Stargardt's disease, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, post-laser complications, rubeosis, arthritis, synovitis, osteomyelitis, asthma, allergic dermatitis, DiGeorge syndrome, corneal graft neovascularization, warts tumors, obstructive carotid disease, age-related macular degeneration, or diabetic retinopathy.

A more preferred embodiment of this aspect of the invention refers to the real use of the statin, except the ester itself for the preparation of a medicament for the prevention or treatment of an associated pathological process, angiogenesis, myo-endothelial proliferation desired, or pathological processes associated with an endothelial barrier dysfunction, selected from the list that includes arteriosclerosis, endovascular stenosis, cardiovascular restenosis, endovascular stenosis, restenosis Central Nervous System (CNS), solid tumors, hematopoietic tumors, pulmonary hypertension, shock lung or systemic inflammatory response syndrome.

A still more preferred embodiment of this aspect of the invention is the real use of statin, except the ester itself in a form suitable for local administration.

It is interesting that the endothelial activator and the statin are also undergoing the form of the endothelial activator, act on the tissue before the statin and thus obtain the best results, so that the combined preparation of an endothelial vascular activator and a statin separately, sequentially, would make the preparation of a drug sequential.

Therefore, another aspect of the invention refers to a combined preparation (hereafter, combined preparation of the invention) for sequential use comprising:

a) first, an endothelial activator, and

b) second, a statin, or a salt or an ester thereof.

In a preferred embodiment of this aspect of the invention, the statin is selected from the list comprising: atorvastatin, fl uvastatin, lovastatin, pitavastatin, pravastatin, simvastatin, and yrosuvastatin. In an even more preferred embodiment, the statin selected is atorvastatin.

In another preferred embodiment of this aspect of the invention, among the endothelial activators, TNFα is fi ngosin-1-phosphate, an analogue thereof, or lysophosphatidic acid, an analogue or a salt of said acid.

In an even more preferred embodiment, the endothelial activator is lysophosphatidic acid, an analogue or a salt thereof.

It should be emphasized that the term "combined preparation" or also called "juxtaposition", herein, means that the components of the combined preparation need not be present as a joint, for example, in a composition, in order to be available for separate or sequential application. In this way, "juxtaposed" expression implies that it is not necessarily a true combination, in view of the physical separation of the components.

Another aspect of the invention relates to the use of the combined preparation of the invention for the preparation of a medicament.

The combined preparation of the present invention can be used, but not limited to, to normalize endothelial function in processes of angiogenesis, unwanted or abnormal myo-endothelial proliferation, or endothelial barrier dysfunction. Therefore, a preferred embodiment of this aspect of the invention refers to the use of the combined preparation of the invention for the preparation of a medicament for the prevention and / or treatment of a pathological process associated with angiogenesis and / or my proliferation. - abnormal or unwanted endothelial.

A more preferred embodiment of this aspect of the invention refers to the use of the combined preparation of the invention for the preparation of a medicament for the prevention or treatment of a pathological process associated with angiogenesis and / or unwanted myo-endothelial proliferation, selected from the list comprising, for example, but not limited to, arteriosclerosis, cancer, macular degeneration, neovascular glaucoma, endometriosis, rheumatoid arthritis, psoriasis, ocular herpes, trachoma, corneal graft neovascularization, viral interstitial keratitis, microbial keratoconjunctivitis, hypertrophic telangiectasia, hypertrophic vascular adhesions, angio fi broma, acne rosacea, retrolental fi broplasia, primary or secondary pulmonary hypertension, shock lung, systemic inflammatory response syndrome, fibrosis, proliferative vitreoretinopathy, leukemia, ulcerative colitis, retinopathy of prematurity, keratoconjunctivitis, keratic conjunctivitis atopic, upper limb keratitis, dry keratitis of the pterygium, Sjögren's syndrome, phlectenulosis, syphilis, Mycobacterial infections, chemical burns, bacterial ulcers, fungal ulcers, Mooren's ulcer, marginal Terrien degeneration, marginal keratolysis, polyarteritis, scleritis, sciarteritis Stevens-Johnson, radial keratotomy, sickle cell anemia, elastic pseudoxanthoma, penfigent, Paget's disease, obstructive carotid disease, chronic uveitis, chronic vitritis, Lyme disease, Eales disease, Behcet's disease, purported ocular histoplasmosis, disease Best, myopia, optic nerve wells, Stargardt disease, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, post-laser complications, rubeosis, arthritis, synovitis, osteomyelitis, asthma, allergic dermatitis, DiGeorge syndrome, graft neovascularization cornea, warts, tumo res, obstructive carotid disease, age-related macular degeneration, or diabetic retinopathy.

An even more preferred embodiment of this aspect of the invention refers to the use of the combined preparation for the preparation of a medicament for the prevention or the treatment of a pathological process associated with angiogenesis, a desired abnormal myo-endothelial proliferation or an endothelial barrier dysfunction, selected from the list that includes arteriosclerosis, cardiovascular graft restenosis and / or endovascular procedures, tumors of the Central Nervous System (CNS), solid tumors, hematopoietic tumors, pulmonary hypertension, shock lung or systemic inflammatory response syndrome.

A preferred embodiment of this aspect of the invention refers to the use of the combined preparation in a form suitable for local administration.

Another aspect of the invention relates to a pharmaceutical composition comprising the composition of the invention. Another aspect of the invention relates to a pharmaceutical composition comprising the combined preparation of the invention.In a preferred embodiment of this aspect of the invention the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

A more preferred embodiment of this aspect of the invention is a pharmaceutical composition that comprises the composition of the invention and, in addition, together with a pharmaceutically acceptable molecule, another active principle.

The compositions of the present invention may be formulated for unanimous administration, and more preferably a mammal, including man, in a variety of ways known in the state of the art, thus, they may be, without limitation, in aqueous solutions that are not aqueous, in emulsions or in suspensions. Examples of non-aqueous solutions are, for example, but not limited to, propylene glycol, polyethylene glycol, vegetable oils, such as mineral oil, or injectable organic esters, such as ethyl oleate. Examples aqueous solutions, are examples, but not limited to, water, alcoholic solutions in water, or media saline Aqueous solutions may be buffered or not, and may have additional active or inactive components. Additional components include salts to modulate ionic strength, preservatives including, but not limited to, antimicrobial agents, antioxidants, chelators, or the like, or nutrients, including glucose, dextrose, vitamin or minerals. Alternatively, the compositions can be prepared for administration in solid form. The compositions may be combined with various inert carriers or excipients, including but not limited to: binders, such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients, such as starch or lactose; dispersing agents, such as alginic acid or corn starch; lubricants, such as magnesium stearate, glidants such as colloidal silicon dioxide; sweetening agents, such as sucrose or saccharin; or flavoring agents, such as peppermint or methyl salicylate.

Such compositions and / or their formulations may be administered to an animal, including a mammal and, therefore, to man, in a variety of ways, including, but not limited to, parenteral, intraperitoneal, intravenous, intradermal, epidural, intraspinal, intrastromal. , intraarticular, intrasynovial, intrathecal, intralesional, intraarterial, intracardiac, intramuscular, intranasal, intracranial, subcutaneous, intraorbital, intracapsular, topical, through transdermal patches, rectal, vaginal or urethral, by administering a suppository, percutaneous, nasal spray , surgical implant, internal surgical paint, infusion pump or catheter.

The compositions of the present invention are suitable for application by medical devices that allow their release in concentrations suitable for treatment. These devices should be suitable for the administration of the drug in a local way that allows the treatment to be dispersed and to affect the affected area. These medical devices can allow drug release both jointly and sequentially, first releasing the endothelial activator and subsequently the statin. The devices can, for example, but not limited to, carry the drugs inside or be coated therewith.

Within these devices, for example, circulatory assistance devices, endovascular procedures and cardiovascular surgery can be found, and within these, for example, but not limited to, stents, valves, rings, sutures, vascular graft patches.

Throughout the description and the claims the word "comprises" and its variants is not intended to exclude other technical characteristics, additives, components or steps. For experts in the field, other objects, advantages and features of the invention will be derived in part from the description in part of the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.

Description of the fi gures

Figure 1. Show the ratio Y = Pstd / Bstd.Z2 / A as a function of Cstd for the patterns of S, Na, P, K, MgyCl at 10 kV. Each value represents the mean ± SD of 15-20 analysis. Calibration standards were prepared identically to cells grown on gold grids covered with pyoloform. The 20% dextran solutions with the different concentrations of the inorganic salts used were deposited on polycarbonate fi lters (pore size of 0.4 µm) isolated from their polystyrene supports, cryo-fixed in liquid nitrogen, cryo-dried and analyzed in a Philips XL30 microscope using identical analytical and analytical conditions as indicated above.

Figure 2. It shows the changes in ion concentrations in HUVEC cells treated "in vitro" analyzed by microanalysis. CE: HUVEC grown in standard medium. LPA: HUVEC grown 90 minutes with 10 µM lysophosphatidic acid and subsequently 24 hours in standard medium. ATS: HUVEC grown with Atorvastatin (ATS) 1 µ for 24 hours. LPA + ATS: HUVEC cultured with 10µ lysophosphatidic acid for 90 minutes and post

subsequently with Atorvastatin1 µ for 24 hours.ATS + LPAHUVEC grown with Atorvastatin1µ for 24 hours and then with LP10 µ for 90 minutes. The data is the mean ± SEM. Experiments performed in triplicate by condition. * p <0.05 vs. EC.

Figure 3. It shows the relationship potassium concentration / sodium concentration (millimoles / kg dry weight) (cell viability indicator) in HUVEC cells analyzed by microanalysis. CE: HUVEC grown in standard medium. LPA: HUVEC grown 90 minutes with 10 µM lysophosphatidic acid and subsequently 24 hours in standard medium. ATS: HUVEC grown with 1 µ Atorvastatin (ATS) for 24 hours. LPA + ATS: HUVEC cultured with 10 µ lysophosphatidic acid for 90 minutes and subsequently with Atorvastatin 1 µm for 24 hours. ATS + LPA HUVEC grown with Atorvastatin 1 µm for 24 hours and subsequently with LPA10 µ for 90 minutes.

Figure 4. It shows the changes in gene expression of endothelin-1 (A) and eNOS (B), genes involved in endothelial function in HUVEC cells, analyzed by microarray. The results are shown in fluorescent units. CE: HUVEC grown in standard medium. LPA: HUVEC grown 90 minutes with 10 µM lysophosphatidic acid and subsequently 24 hours in standard medium. ATS: HUVEC grown with 1 µM Atorvastatin (ATS) for 24 hours. LPA + ATS: HUVEC cultured with 10 µ lysophosphatidic acid for 90 minutes and subsequently with Atorvastatin 1 µM for 24 hours.ATS + LPAHUVEC grown with Atorvastatin1µ for 24 hours and subsequently with LPA10 µ for 90 minutes. * p <0.05 vs. EC.

Figure 5. Shows the changes in gene expression of genes involved in the permeability of the endothelial barrier in HUVEC cells analyzed by microarrays. The results are shown in fluorescent units. CE: HUVEC grown in standard medium. LPA: HUVEC grown 90 minutes with 10 µM lysophosphatidic acid and subsequently 24 hours in standard medium. ATS: HUVEC grown with 1 µ Atorvastatin (ATS) for 24 hours. LPA + ATS: HUVEC cultured with 10 µ lysophosphatidic acid for 90 minutes and subsequently with 1 µAvavastatin for 24 hours. ATS + LPAHUVEC grown with Atorvastatin 1 µ for 24 hours and subsequently with 10 µL LPA for 90 minutes. * p <0.05 vs. EC.

Figure 6. It shows the changes in gene expression of growth factors in HUVEC cells analyzed by microarrays. The results are shown in fluorescent units. CE: HUVEC grown in standard medium. LPA: HUVEC grown 90 minutes with 10 µM lysophosphatidic acid and subsequently 24 hours in standard medium. ATS: HUVEC grown with 1 µ Atorvastatin (ATS) for 24 hours. LPA + ATS: HUVEC cultured with 10 µ lysophosphatidic acid for 90 minutes and subsequently with Atorvastatin 1 µm for 24 hours. ATS + LPA HUVEC grown with Atorvastatin 1 µm for 24 hours and subsequently with LPA10 µ for 90 minutes. * p <0.05 vs. EC.

Figure 7. It shows the changes in gene expression of genes involved in the development of the cell cycle in HUVEC cells analyzed by microarrays. The results are shown in fluorescent units. CE: HUVEC grown in standard medium. LPA: HUVEC grown 90 minutes with 10 µM lysophosphatidic acid and subsequently 24 hours in standard medium. ATS: HUVEC grown with 1 µM Atorvastatin (ATS) for 24 hours. LPA + ATS: HUVEC cultured with 10 µM lysophosphatidic acid for 90 minutes and subsequently with Atorvastatin1 µM for 24 hours.ATS + LPAHUVEC grown with Atorvastatin1 µ for 24 hours and subsequently with LPA

10 µFor 90 minutes. * p <0.05 vs. EC;

p <0.05ATS vs. E1.

Figure 8. It shows the changes in gene expression of genes involved in apoptosis in HUVEC cells analyzed by microarrays. The results are shown in fluorescent units CE: HUVEC grown in standard medium. LPA: HUVEC grown 90 minutes with 10 µM lysophosphatidic acid and subsequently 24 hours in standard medium. ATS: HUVEC grown with 1 µ Atorvastatin (ATS) for 24 hours. LPA + ATS: HUVEC cultured with 10 µ lysophosphatidic acid for 90 minutes and subsequently with Atorvastatin 1 µm for 24 hours. ATS + LPA HUVEC grown with Atorvastatin 1 µm for 24 hours and subsequently with LPA10 µ for 90 minutes. * p <0.05 vs. EC.

Examples

The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations to the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.

Example 1

HUVEC cell culture and lysophosphatidic acid administration protocol 10 µMyvatorvastatin 1 µM

For the study of the effects of endothelial activation, prior to the administration of statins, on cell death, HUVEC cells marketed by Sigma Aldrich were used. These cells were cultured in F12K medium (modified by Kaighn) with 2 mM glutamine + 1.5 g / l sodium bicarbonate + 0.10 mg / ml heparin + 0.03 mg / ml endothelial growth supplement (ECGS) + 20 % of inactivated fetal serum bovine (by thermal shock at 56 ° C for 30 minutes). The culture medium is changed every 72 hours. The incubation is carried

It is carried out at a temperature of 37 ° C and in an atmosphere of 5% CO2. 3 cells are carried out from the cells prior to their experimental use. This cell detachment is carried out by means of three washes with PBS (sterile buffered solution), in order to eliminate fetal bovine serum remains. Subsequently, Tripsin-EDTA is added 0.03% (incubating at 37 ° C, 90% humidity and 5% CO 2 for 5-10 minutes, examining the degree of cellular individualization). Once the cell suspension is obtained, neutralization of the trypsin-EDTA is carried out by means of culture medium. Subsequently, it has been centrifuged at 1000 rpm. for 10 minutes, the supernatant is removed, and the cell precipitate is diluted by adding new culture medium to reseed in a 1: 2 ratio.

For the conduct of the microanalysis, after the third pass, the cells are divided into 5 groups and incubated in wells of experimentation, on microanalysis grids 37 ° C and 5% CO 2 under the following conditions:

-

CONTROL GROUP (EC): umbilical vein endothelial cells, cultured in standard medium

-

LPA GROUP: umbilical vein endothelial cells, from the third subculture, treated with 10 µL LPA for 90 minutes and subsequent incubation for 24 hours in standard medium.

-

GRUPOATS: umbilical endothelial cells, from the third subculture, treated pharmacologically with Atorvastatin (ATS) 1 µ for 24 hours.

-

GROUP E1: umbilical endothelial cells, from the third subculture, treated pharmacologically with 10 µL LPA for 90 minutes and subsequently with SS1 µ for 24 hours.

-

GROUP E2: umbilical endothelial cells, from the third subculture, treated pharmacologically with AS1 1 µM for 24 hours and then with 10 µLPA for 90 minutes.

Example 2

Effects of treatment with lysophosphatic acid from HUV cells prior to the administration of atorvastatin 1 µM on cell death

For the conduct of the microanalytical study, the sample of endothelial cells must be physically on the microanalysis gold grids. To this end, it stretches a thin layer of pioloform resin in ultrapure water and precisely placing the microanalysis grids and an 11 mm diameter coverslip on the grid. -pioloform, are sterilized by irradiation with ultraviolet rays for 24 hours. Then, 2 gratings are deposited in each well of experimentation and cell seeding is proceeded.

Cell seeding is carried out according to the protocol followed in example 1. The cells come from a third trypsinization of the culture.

The experimental test (treatment with LPA, atorvastatin, both or without treatment) was carried out after 24 hours of culture, thus ensuring correct adhesion and viability of the endothelial cells used.

Subsequently, the microanalysis lattices are washed in double-distilled water autoclaved at 4 ° C for 4 seconds, generating a gentle movement by means of a magnetic stirrer. Excess water is removed with rapid drying on fi lter paper.

Subsequently, the microanalysis gratings are cryo fi xed, after removing the corresponding grating covers. The cellular material is fixed by freezing in liquid nitrogen, lowering its temperature from 37 ° C to -196 ° C, in less than 10 seconds.

Cryophobic endothelial cells are transferred to a high-vacuum cryo-drying chamber, at 10-5 mbar pressure (EmitechK775 system) for complete cell extraction by sublimation, following a 6-stage protocol (Warley et al. 2000. JMicrosc; 198: 116-123).

The sample is then covered with a carbon film, using an Emitech evaporator (Watfor, United Kingdom) provided with a graphite wire under high vacuum conditions, which facilitates scanning of the electron beam.

The visualization of the samples in the scanning electron microscope by secondary electrons was carried out using the following analytical parameters:

- Microscope voltage

10 kV

-Angulation of surface

0º

-Work distance

10 mm

The instrumental conditions set for microanalytical detection were as follows:

- Microscope voltage - Increases

10 kV 10000

-Tilt of surface (tilt) 0º -Accounts per second (CPS) registered by the detector -Acquisition time 200 s -Electron beam size (spot size) -Working distance 10 mm -Static and static analysis area

500 6

Prior to the microanalytical study, a thorough evaluation of the endothelial cells was performed by scanning electron microscopy, discarding those that, due to their special proximity or anchorage to the metal structure, could generate an obvious distortion of the microanalysis patterns.

In order to quantitatively determine the elementary content of the cells, the bottom rape method was used - P / B method - with reference to previously known organic matrix calibration standards (Boekestein et al. 1980. Scan Electron Microsc; pt-2: 321-334 and Boekesteinet al. 1984. JMicrosc; 134: 327-334; Roomanset al. 1988. JElectron MicroscTech; 9: 3-17; Statham 1998. Microanc Microanal; 4: 605-615). The concentration of each element was determined according to the following equation:

where:

-

Cspc is the concentration of the element to quantify.

-

It corresponds to the net counts of the characteristic signal of the element.

-

Bes the continuous radiation measured below the peak, that is, taken between the same energy levels of the characteristic signal.

-

Z2 / A is the mean value of the squared atomic number divided by the atomic weight, corresponding to a specific value for 20% index (standard standard) and cells (Warley, 1997. Practical Methods in Electron Microscopy).

The calibration standards were prepared according to the pre-set guidelines in the laboratory of analytical electron microscopy of the Department of Histology of the Faculty of Medicine of the University of Granada. (Crespo et al. 1993. Acta Otolaryngol; 113: 176-180; López-Escámez et al. 1994. Scanning Microsc Suppl; 8: 171-185) using various salts (NaHPO4, MgCl2, CaCl2 · H2O and K2SO4) dissolved in Organic matrix as a test analysis.To do this, solutions from each room were prepared with different concentrations in 20% transtraalal (300 kD). The patterns were mounted on microporous membranes of the Millicell® units, and the same procedure was set forth for the preparation of endothelial cells. The standards were analyzed in the electron microscope immediately after preparation to avoid contamination or chemical modification, obtaining between 15 and 20 spectra for each standard concentration, using the same conditions of analysis previously considered, which were used for endothelial cells.

The analysis of the 20% dextran standards containing known concentrations of Na, Cl, Mg, P, S, and KyCa salts, allowed the calibration of the concentrations of the aforementioned elements against the P / B signal.The concentration of each element in the standards directly provides the P / B ratio. according to the following formula:

where:

-

Cts is the known concentration of the element in the pattern.

-

(Pstd / Bstd) is the P / B ratio, where the background has been measured between the same energy range of the characteristic signal obtained from the standards analysis.

-

K is the characteristic calibration constant for each element and instrumental configuration used.

The relationship between Pstd / Bstd versus the concentration of each element was studied using simple linear regression. Taking into account that the so-called calibration constant K, depends on the difference in Z2 / A (factor G) between the cell, it is necessary to use the following equation to assess the influence that this factor exerts on the curve adjustment:

where: -Z2 / A is the average of the Z2 / A value for all the elements present in the standard analyzed volume:

where:

-

Zi is the atomic number of element i.

-

Ai is the atomic weight of that element.

-

fi is the mass fraction of the elementi expressed as elementary mass divided by the total mass (Hall

and Gupta, 1986).

Table 1 shows the regression equations obtained for all the elements when Y = Pstd / Bstd · Z2 / A, calibrated against Cstd, for the elements Na, Mg, P, S, Cl, KyCa analyzed at 10kV.

TABLE 1

Equations of regression obtained when Y = Pstd / Bstd · Z2 / A were calibrated against Cstd analyzed at 10 kV

Table 2 shows the values of the standard pattern expressed in molds / kg of mass, for the different elements were the following:

TABLE 2

Standard standard values expressed in moles / kg dry mass

• To carry out the statistical analysis of the data obtained, we propose the following hypothesis test:

◦

Ho → there are no statistically significant differences between the values of each element in the different experimental groups.

◦

Hi → there are statistically significant differences between the values of each element in the different experimental groups.

The problem posed is solved by using the non-parametric Kruskal-Wallis test for multiple samples, which allows us to perform the global statistical analysis.

Then we perform the statistical analysis of the different experimental groups through two-to-two comparisons, using the non-parametric Mann-Whitney test (W. Wilcoxon).

The data indicate that treatment with Atorvastatin1 µM causes a decrease in the indicators of cell viability (K / Na ratio, concentration of chlorointracellular) (Figures 2 and 3 respectively) .Culture treatment with lysophosphatidic acid 10 µM for 90 minutes did not induce just variations in terms of cell viability with respect to control culture. The addition to the culture of lysophosphatic acid 10 µM for 90 minutes after incubation with atorvastatin1 µFor 24 hours, there were no variations related to survival with respect to treatment alone with atorvastatin1 µM. On the contrary, the preactivation of the endothelium by preincubation of the cells with LPA10 µM for 90 minutes prior to the administration of Atorvastatin causes a protection of the tissue against the damage generated by the atorvastatin concentration1 µM.

Example 3

Effect of treatment with lysophosphatic acid of HUV cells prior to administration of Atorvastatin 1 µM on gene expression

The culture and treatment protocol of the cells is the same as in the example 1. The experimental groups used are:

-

CONTROL GROUP (EC): umbilical vein endothelial cells, cultured in standard medium

-

GROUP A: umbilical vein endothelial cells, from the third subculture, treated with 10 µL LPA for 90 minutes and subsequent incubation for 24 hours in standard medium.

-

GRUPOATS1: umbilical endothelial cells, from the third subculture, treated pharmacologically with Atorvastatin (ATS) 1 µ for 24 hours.

-

GROUP E1: umbilical endothelial cells, from the third subculture, treated pharmacologically with 10 µL LPA for 90 minutes and subsequently with SS1 µ for 24 hours.

-

GROUP E2: umbilical endothelial cells, from the third subculture, treated pharmacologically with AS1 1 µM for 24 hours and then with 10 µLPA for 90 minutes.

For gene expression analysis, total RNA was extracted from each of the samples to be analyzed using the Trizol system (LifeTechnologies, Inc., Gaithersburg, MD, USA) and purification through the commercial system Qiagen RNeasy System (Qiagen, Mississauga, Ontario, Canada). , the integrity and quality of the latter was checked by direct visualization of the 28 and 18S ribosomal RNA by 1.2% agaroseal gel electrophoresis and ethidium bromide infection.

For the analysis, all RNAs were transformed into DNC by a reverse transcriptase (Superscript II, LifeTechnologies, Inc., Carlsbad, CA, USA) with an oligonucleotide rich in thymine tails (T7-polyT first), which allowed amplification of any messenger RNA present in the cell. Then, all ARs were synthesized cDNAs by in vitro transcription, using biotin-labeled UTP and CTP (Enzo Diagnostics, Farmingdale, NY). Once synthesized, and to favor hybridization, these RNAs were chemically fragmented by adding a concentration of high temperatures. Finally, the labeled and fragmented RNAs hybridized in front of the chips that constitute the Affymetrix Human Genome U133 plus 2.0® microarray system of the Affymetrix commercial house, for 16 hours at 45 ° C. After a standardized and automated process, the chips were washed and scanned to obtain absolute values of expressed expression. All expression values were normalized using the Affymetrix Microarray Suite 5.0 software supplied by the Affymetrix trading house.

The results represented in Figure 4A show that treatment with LPA causes an increase in the expression rate of endothelin-1, which determines a clear pattern of endothelial dysfunction. This result is endorsed with the decrease in the expression of endothelial nitric oxide synthase (eNOS) (Figure 4B). On the other hand, treatment with atorvastatin induces a clear increase in the expression of eNOS that also occurs in the case of pretreatment with LPA. This indicates that Atorvastatin induces a recovery of endothelial function, even with a preactivation of endothelial tissue.

Figure 5 shows that treatment with atorvastatin potentiates overexpression molecules involved in intercellular junctions. This indicates that atorvastatin induces potentiation of the endothelial barrier. This potentiation occurs both in the case of treating tissue treated only with atorvastatin and in that activated by preincubation with LPA. This indicates that the beneficial effects of improving the endothelial barrier are not eliminated by tissue preactivation.

In the fi gure, it is observed that the activation of the endothelium with the LPA causes an increase in the expression of the cardiovascular-vascular growth factor A. This induction is reversed by the treatment with atorvastatin. Atorvastatin itself also produces a decrease in both the expression of the growth factor of the endotheliovascular Cyen and the cell receptor with the pre-treatment of the FLT-1PA. That shows

that atorvastatin is able to exert a negative regulation on autocrine and paracrine cell proliferation mechanisms that is not altered by pretreatment with LPA.

Figure 7 shows that in cell groups treated with atorvastatin, either independently or with pre or post-incubation with LPA, there is a reduction in the expression of the genes involved in cell cycle progression such as cyclin C1, A1, A2, B1, B2, FyH. Also reduce the expression of cyclin-dependent kinases1,2 and 4. This indicates that treatment with atorvastatin causes a blockage of the cell cycle that prevents the proliferation of cells treated with the compound. This effect is independent of the preactivation of the endothelium with lysophosphatidic acid.

Figure 8 shows that cells treated with atorvastatin1 µMonically or with subsequent application of LPA, have an increased expression of indicators of lost viability such as caspase3, caspase6caspase7, Rac-1 and Cdc-42, with respect to untreated cells. This severely limited or reversed increase in the physiological situation in which the cells have been pre-incubated with the prior APA at the use of atorvastatin. These data endorse the cell viability data obtained by microanalysis. Another gene increased in the ATSyATS + LPA groups is a programmed cell death 4 (PDCD-4), which has an important role in apoptosis. Expression is also reduced by treatment with LPA prior to the administration of atorvastatin.

Therefore, combining the results it is observed that the treatment of endothelial cells with high concentrations of atorvastatin promotes the physiological recovery of altered endothelial tissues. The problem is that this treatment also promotes the apoptosis of a large part of the treated cells causing tissue disorganization.Activation of the endothelium with APL prior to treatment with high concentrations of atorvastatin, causes modifications in the endothelium that lead to a reduction in the death produced For this compound, on the contrary, this protection does not prevent the recovery of the physiological expression pattern that makes it an adequate treatment for the recovery of altered tissues.

Claims (26)

1. Composition comprising: a) an endothelial activator, and b) a statin, or a salolester itself.

2.

Composition according to claim 1 wherein the statin is selected from the list comprising: pravastatin, simvastatin, fl uvastatin, atorvastatin, pitavastatin, rosuvastatin or lovastatin.

3. Composition according to claims 1-2 where the statin is atorvastatin.

Four.

Composition according to any one of claims 1 to 3 wherein the endothelial activator is selected from the list comprising TNFα, phygosine-1-phosphate, an analogue thereof, or lysophosphatidic acid, an analogue or a salt of said acid.

5. Composition according to claim 4 wherein the endothelial activator is lysophosphatidic acid, an analogue or a salt thereof. 6. Use of the composition according to any of claims 1 to 5 for the preparation of a medicament. 7. Use of the composition according to claim 6 for the preparation of a medicament for the prevention and / or treatment of a pathological process associated with angiogenesis, abnormal or unwanted myo-endothelial proliferation, or to a dysfunction of the endothelial barrier. 8. Use of the composition according to claim 7 where the pathological process associated with angiogenesis, abnormal or unwanted myo-endothelial proliferation, or endothelial barrier dysfunction, is from the list comprising: arteriosclerosis, cardiovascular graft restenosis, endovascular procedures, tumors of the Central Nervous System (CNS), solid tumors, hematopoietic tumors, pulmonary hypertension, lung shock, systemic inflammatory response syndrome. 9. Use the composition according to any of claims 6 to 8 in a form suitable for local administration.

10.

Use of a composition comprising a statin, or a salt or an ester thereof for the preparation of a medicament for the prevention and / or treatment of a pathological process associated with angiogenesis, abnormal or unwanted myo-endothelial proliferation, or endothelial barrier dysfunction, when Endothelial cells are activated.

eleven.

Use of the statin according to claim 10 wherein the pathological process associated with angiogenesis, abnormal or unwanted myo-endothelial proliferation, or endothelial barrier dysfunction is from the list comprising: arteriosclerosis, cardiovascular graft restenosis, endovascular procedures, tumors del Central Nervous System (CNS), solid tumors, hematopoietic tumors, pulmonary hypertension, lung shock systemic inflammatory response syndrome.

12.

Use of the statin according to any of claims 10 or 11 in a form suitable for local administration.

13. Combined preparation for sequential use comprising: c) first, an endothelial activator, and d) second, a statin, or a salt or an ester thereof.

14.

Combined preparation according to claim 13 wherein the statin is selected from the list comprising: pravastatin, simvastatin, fl uvastatin, atorvastatin, pitavastatin, rosuvastatin or lovastatin.

15. Combined preparation according to claims 13 or 14 wherein the statin is atorvastatin.

16.

Combined preparation according to any one of claims 14 to 15 wherein the endothelial activator is selected from the list comprising TNFα, fi ngosin-1-phosphate, an analogue thereof, or lysophosphatidic acid, an analogue or a said acidic acid.

17.

Combined preparation according to claim 16 wherein the endothelial activator is lysophosphatidic acid, an analogue or a salt thereof.

18.

Use of the combined preparation according to any of claims 13 to 17 for the preparation of a medicament.

19.

Use of the combined preparation according to claim 18 for the preparation of a medicament for the prevention and / or prevention treatment of a pathological process associated with angiogenesis, abnormal or unwanted myoendothelial proliferation, or a dysfunction of the endothelial barrier.

twenty.

Use of the combined preparation according to claim 19 wherein the pathological process associated with angiogenesis, abnormal or unwanted myo-endothelial proliferation, or an endothelial barrier dysfunction is from the list comprising: arteriosclerosis, cardiovascular graft restenosis, endovascular procedures, Central Nervous System (CNS) tumors, solid tumors, hematopoietic tumors, pulmonary hypertension, shock lung or systemic inflammatory response syndrome.

21. Use the combined preparation according to any of claims 18 to 20 in a form suitable for local administration. 22. Pharmaceutical composition comprising a composition according to any of claims 1 to 9.

2. 3.

Pharmaceutical composition comprising a combined preparation according to any of claims 13 to 21.

24.

Pharmaceutical composition according to any of the claims 22 or 23 which further comprises a pharmaceutically acceptable vehicle.

25.

Pharmaceutical composition according to any of claims 22-24 further comprising another active ingredient.

17 18 19 20 21 22 SPANISH PATENTS AND BRANDS OFFICE Application no .: 200930321 SPAIN Date of submission of the application: 16.06.2009

REPORT ON THE STATE OF THE TECHNIQUE

Priority Date: 00-00-0000 00-00-0000 00-00-0000

51 Int. Cl.:

A61K 31/40 (2006.01)

A61K 31/661 (2006.01)

 RELEVANT DOCUMENTS  

Category

56 Documents cited Claims Affected

Y

FRICK, M. et al .: "Statins differentially regulate vascular endothelial growth factor synthesis in endothelial and vascular smooth muscle cells". Atherosclerosis, 2003, vol. 170, pages 229-236, the entire document. 1-12

Y

RIVERA-LOPEZ, C.M. et al .: "Lysophosphatidic acid (LPA) and angiogenesis". Angiogenesis, 2008, vol. 11, pages 301-310, the entire document. 1-12

Y

DULAK, J. et al .: "Atorvastatin affects several angiogenic mediators in human endothelial cells". Endothelium, 2005, vol. 12, pages 233-241, the entire document. 1-12

Y

CHI-LOU LIN et al .: &quot; Lysophosphatidic acid upregulates vascular endothelial growth factor-C and tube formation in human endothelial cells through LPA 1/3, COX-2, and NF-KB activation and EGFR transactivation dependent mechanisms &quot;. Cellullar Signaling, 2008, vol. 20, pages 1804-1814, the whole document. 1-12

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims □ for claims no:

Date of completion of the report 15.10.2010

Examiner H. Aylagas Cancio Page 1/4

Application number: 200930321 REPORT ON THE STATE OF THE TECHNIQUE Minimum documentation sought (classification system followed by classification symbols) A61K Electronic databases consulted during the search (name of the database and, if possible, search terms used) INVENES, EPODOC, WPI, NPL, BIOSIS, EMBASE, MEDLINE  WRITTEN OPINION Application number: 200930321 Date of Completion of Written Opinion: 15.10.2010

Statement

Novelty (Art. 6.1 LP 11/1986)

Claims 1-25 YES

Claims _____________________________________

NO

Inventive activity

Claims 13-25 YES

(Art. 8.1 LP11 / 1986)

Claims 1-12 NO

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published.  WRITTEN OPINION Application number: 200930321 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

Atherosclerosis, vol. 170, pages 229-236. 2003

D02

Endothelium, vol.12, pages 233-241 2005

D03

Angiogenesis, vol.11, pages 301-310 2008

D04

Cellular Signaling vol. 20, pages 1804-1814. 2008

2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The present application refers to a composition comprising an endothelial activator and a statin, the latter being selected from pravastatin, atorvastatin, pitavastatin, rosuvastatin or lovastatin and the endothelial activator between sphingosine 1 phosphate, TNF-alpha or lysophosphatidic acid. It also refers to a combined preparation for sequential use that first comprises an endothelial activator and second a statin and its application in the prevention or treatment of a pathological process associated with angiogenesis, abnormal or unwanted myo-endothelial proliferation or dysfunction of the endothelial barrier Documents D1 and D2 refer to the application of statins in the vascular endothelium whose effect depends on the dose, thus they have a proangiogenic effect when administered at concentrations within the picomolar or nanomolar range while at high concentrations within the micromolar range they demonstrate a clearly antiangiogenic effect. Documents D3 and D4 refer to lysophosphatidic acid as a modulator of endothelial function. It is indicated as an angiogenesis promoter and neovascularization promoter. Claims 1-12 that refer to a composition formed by an endothelial activator and a statin and to the use in pathological processes associated with angiogenesis lack inventive activity for the following reasons: The joint administration of an endothelial activator and a statin for the prevention or treatment of a pathological process associated with angiogenesis implies a mere juxtaposition of elements known in the state of the art. In general, the fact of combining 2 or more active ingredients to treat a particular disease cannot be considered inventive in the event that said active agents are individually known as therapeutically effective in the treatment of that disease. With regard to claims 13-25 which refer to the combined preparation for sequential use, which first comprises an endothelial activator and secondly a statin and its application in pathological processes associated with angiogenesis, said preparation has not been found in any of the cited documents. There is an advantage with the previous administration of lysophosphatidic since in view of the examples it follows that the previous administration of it to atorvastatin causes modifications in the endothelium that lead to a reduction in cell death caused by atorvastine. Accordingly, claims 13-25 present novelty, inventive activity according to articles 6.1 and 8.1 of the L.P. Report on the State of the Art (Written Opinion) Page 4/4