WO2014087037A1 - Use of the growth factor derived from platelets or a selective agonist of the at2 receptor for the treatment of vascular calcifications - Google Patents

Abstract

The invention relates to the use of the growth factor derived from platelets or any of the like, biologically active fragments or variants, or from the agonists of the AT2 receptor, angiotensin, angiotensin II or any of the like, biologically active fragments or variants, or any of the combinations thereof, and the pharmaceutical compositions thereof, in the production of a medicament for the prevention or treatment of vascular calcification.

Description

 USE OF THE GROWTH FACTOR DERIVED FROM PLATELETS OR A SELECTIVE AGONIST OF THE AT2 RECEPTOR FOR THE TREATMENT OF

 VASCULAR CALCIFICATIONS.

FIRST INVENTION

USE OF THE GROWTH FACTOR DERIVED FROM PLATES FOR THE TREATMENT OF VASCULAR CALCIFICATIONS.

The present invention is within the field of medicine and pharmacy, and refers to the use of platelet-derived growth factor for the preparation of a medicament or pharmaceutical composition aimed at combating the calcification of blood vessels and especially the treatment of vascular calcifications associated with calcitriol treatment.

STATE OF THE PREVIOUS TECHNIQUE

Cardiovascular disease is the leading cause of death in patients with chronic renal failure (CRF). Hemodialysis patients present with atherosclerosis and arteriosclerosis. These vascular diseases begin to develop in the early stages of kidney disease, and as the kidney disease progresses, progressive worsening of cardiovascular disease occurs. Cardiovascular disease of patients with advanced renal failure is characterized by calcifications in the area of the intimate and middle layers of the arterial wall. Calcifications of the arteries cause decreased elasticity of the vessels and the degree of calcification correlates with mortality.

For many years, vascular calcifications of patients with renal insufficiency were considered to be due to passive deposition of calcium salts, the result of an oversaturation due to the increase in plasma concentrations of calcium and phosphorus (increase in Ca x P product) . Undoubtedly, hyperphosphatemia and the increase in the Ca x P product contribute significantly to vascular calcification. Hyperphosphatemia is a predictor of mortality, possibly due to its role in the pathogenesis of ratings. To prevent vascular calcifications in renal patients, it is essential to control plasma phosphate without providing an excess of calcium, that is, avoiding using intestinal phosphorus chelants that contain calcium. However, at present, it is known that vascular calcification is an active process regulated in the cellular field and by certain components of the plasma, such as certain proteins and other pathogenic factors.

Therefore, there are proteins that regulate vascular calcification, favoring or inhibiting it. It is evident that in patients with renal pathologies the inhibitory mechanisms are overcome by the other mechanisms that promote vascular calcification, which results in an imbalance.

Among the proteins that modulate extraosseous calcification are: Matrix gla-protein

It is an extracellular protein that has an affinity for hydroxyapatite. In bone, it promotes bone formation and, in the artery, inhibits calcification. The matrix gla-protein (MGP) is dependent on vitamin K and its function is affected by warfarin or acenocoumarol. MGP inhibits the activity of bone matrix protein 2a (BMP2a), which is a procalcifying protein. It has been proven that a mouse deficient in MGP shows diffuse arterial calcifications.

Fetish

It is a2-Heremans-Schmid (Ahsg) glycoprotein or fetuin A. This serum protein is an important inhibitor of extraosseous calcifications. The concentration of fetuin decreases in situations of inflammation (immune cell response). This protein decreases the precipitation of calcium and phosphorus in vitro. It has been seen that a mouse deficient in fetuin shows diffuse arterial calcifications. The clinical importance of fetuin was illustrated in a study that demonstrated an association between cardiovascular mortality and low fetuin values in hemodialysis patients. It has been described that the fetuin / MGP protein complex inhibits the precipitation of calcium salts and the formation of hydroxyapatite. It also seems that the inhibition of calcification by fetuin depends on the formation of stable protein complexes that incorporate the mineral nuclei. Osteoprotegerin

In bone, the interaction of osteoprotegerin (OPG-L) with its RANK receptor (the English receptor-activator of NF-κΒ) regulates the differentiation, activation and apoptosis of osteoclasts. It has been seen that in OPG-deficient mice, osteoporosis and arterial calcification occur.

Osteopontin

It is a potent inhibitor of hydroxyapatite deposition in vascular smooth muscle cell (VSMC) cultures. In addition, it protects endothelial cells from apoptosis that is caused by deprivation of growth factors. The most important proteins that favor calcification are alkaline phosphatase, osteonectin, osteocalcin and BMP2. These proteins are expressed in abundance in calcified vascular wall and in vascular smooth muscle cell cultures in which calcifications are induced.

Among the pathogenic factors are: Hyperiosphatemia

Increasing the concentration of phosphorus in the medium in VSMC cultures induces the calcification of these cells. In order for the phosphorus to induce the calcification of the VSMC, it needs to have an active transport from outside to the inside of the cell (Pit-1), and that subsequently Cbfa-1, an osteoblast-specific transcription factor that regulates the expression, is induced of procalcifying proteins. It has been observed that using phosphonorphic acid (PFA) can prevent phosphorus cell transport. The calcifying effect of phosphorus requires a prior alteration of the vascular wall that facilitates or promotes the phenotypic transformation of VSMC into osteoblast.

Uremia

In vitro studies with VSMC show that uremic serum is capable of inducing its calcification, independently of phosphorus concentration. In the calcified arteries of uremic patients, there is an increase in Cbfa-1 expression in the middle and intimate layers. Hypercalcemia

It has also been shown that an increase in calcium concentration exerts a direct stimulating effect on calcification. In VSMC cultures, the high concentration induces calcification even when the phosphorus is not increased. Like phosphorus, elevated calcium also stimulates the expression of the Na-P transporter (Pit-1), and inhibition of this transporter inhibits calcium-induced calcification. Also high calcium induces Cbfa-1 expression. Hyperphosphoremia and hypercalcemia induce vascular calcifications independently, but together they exert a synergistic effect that favors vascular calcifications. It is important to note that, in the presence of a high phosphorus, a small increase in calcium means an exaggerated calcification.

Microvesicles

The first step in the calcification process is the accumulation of calcium and phosphorus in vesicles that come from the apoptosis of VSMC (apoptotic bodies), or that are released by cells of the osteoblast line vascular wall that express Cbfa-1 (matrix vesicles)

In addition to these factors, there are drugs that induce vascular calcifications. Thus, calcitriol (DCI) or 1-alpha, 25-dihydroxycholecalciferol (abbreviated 1, 25- (OH) 2D3) is the active form of vitamin D found in the body (vitamin D3). It is used to treat secondary hyperparathyroidism in uremic patients. But calcitriol increases the intestinal absorption of calcium and phosphorus, and produces hypercalcemia and hyperphosphatemia, which can favor vascular calcification. The danger of vitamin D calcification is greater if it is administered to patients with hyperphosphatemia who are being treated simultaneously with phosphorus chelators containing calcium. In addition, some authors have found that calcitriol stimulates vascular calcification by direct action on the vascular wall.

Currently, other vitamin D analogues, such as paricalcitol or 22-oxacalcitriol (OCT), can be used in the treatment of secondary hyperparathyroidism, which, while continuing to inhibit PTH, appear to have less effect on calcium intestinal absorption and phosphorus In a recent study, lower mortality has been shown in hemodialysis patients who are being treated with paricalcitol, compared to those treated with calcitriol One of the possible reasons that explain the beneficial effect of paricalcitol on mortality may be a lower hypercalcemic and hyperphosphatemic effect.

An alternative to calcitriol treatment is the use of calcimimetics, allosteric calcium receptor activators (RCa), which has been used for a few years to control secondary hyperparathyroidism in uremic patients. The effect of calcitriol and calcimimetic on vascular calcifications in uremic rats with secondary hyperparathyroidism has been analyzed (López et al., 2006. J Am Soc Nephrol 17 (3): 795-804). The administration of calcitriol in doses necessary to control hyperparathyroidism, produced severe calcifications of the vessel wall; Calcimimetic administration controlled hyperparathyroidism and did not cause calcifications. In addition, the most important thing is that in rats that received the combination of calcitriol and calcimimetics calcifications were not observed, that is, that calcimimetic is able to avoid calcifications induced by calcitriol. A possible explanation would be that in animals treated with calcimimetics there is a moderate decrease in calcium and phosphorus compared to animals treated with calcitriol, however rats treated with calcitriol and calcimimetics had high values of calcium and phosphorus. The fact that rats treated with calcimimetics have an increase in MGP (anticalcifying protein) suggests a direct effect of calcimimetic on the vascular wall. In bovine VSMC (BVSMC) cultures the presence of RCa has been demonstrated and calcification is associated with a decrease in RCa expression levels, which could be inherent to osteoblastic transformation (Block et al., 2004. N Engl J Med; 350: 1516-25).

In recent years the implication of the Wnt ^ -catenin pathway has been characterized in the calcification processes. Activation of Wnt ^ -catenin leads to increased proliferation and osteogenesis (Dahlóf 2010. Am J Cardiol. 4; 105 (1 Suppl): SAGA). Its activation has also been related to methylation and inhibition of gene promoters.

Platelet-derived growth factor (PDGF) is a cationic protein, stable against heat, found in a variety of cell types, including circulating platelet granules, vascular smooth muscle cells, endothelial cells, macrophages and keratinocytes , and is known to stimulate in vitro protein synthesis and collagen production by fibroblasts. It is also known to act as an in vitro mitogenic and chemotactic agent for fibroblasts, smooth muscle cells, osteoblasts and glial cells. PDGF was initially described by Ross et al., Proc. Nati Acad. Sci. USA., 71, 1207-1210 (1974), as a factor found in whole blood serum (but not in low platelet serum) that is capable of supporting the growth of fibroblasts in the culture. The PDG F was isolated from the platelets and the serum, the natural non-reduced PDGF being identified as a 27-35 kd molecular weight dimera protein. It was found that PDGF reduction provided two or more smaller bands on gels, in a molecular weight range of 10-18 kd. It was believed that these smaller bands represented two distinct smaller monomeric subunits of approximately 18 kd and 16 kd of molecular weights called, respectively, the "A" and "B" subunits, or alternatively, the PDGF A chain and the B chain of PDGF. The amino acid sequences of the two subunits of the PDGF have already been described, identifying the amino acid sequence of the B-chain of the PDGF homologous in more than 90% to the predicted protein product of v-sis, the oncogene contained in the sarcoma virus of oncogenic ape (SSV); see Doolittle et al., Science, 221, 275-276 (1983), and Waterfield et al., Nature, 304, 2810-2814 (1983). It has been found that chain A is approximately 60% homologous to chain B. It is believed that PDGF is biologically active only in dimera form. These biologically active PDGF dimers can take the form of a PDGF-AB heterodimer, a PDGF-BB homodimer or a PDGF-AA homodimer. Each monomer subunit of the biologically active dimer, regardless of whether it is a chain A monomer or a chain B monomer, contains eight cysteine residues. Some of these cysteine residues form disulfide bonds between chains that hold the dimeros together.

The homologous sequence of 109 amino acid v-sis (PDGF B ₁₀ g), identified by Johnsson et al. 1984. EMBO Journal, 3 (5), 921-928 as the mature form of PDGF B, has been used in recombinant technology using yeast and other eukaryotic host cell systems to obtain recombinant active PDGF (rPDGF) (US 4,766,073) .

It has been shown that a recombinant human PDGF-BB (= rhPDGF-BB) stimulates wound healing and bone regeneration in both animals and humans. It has been approved both in the United States and in Europe for human use in topical applications in order to accelerate the healing of chronic diabetic foot sores. It has also been shown that a recombinant hPDGF-BB is effective, either alone or in combination with other factors of growth, to improve periodontal regeneration, ie the renewed growth of bone, cement and ligament around the teeth (US 5,124,316).

The presence of vascular calcifications alters the structure and vascular function, these being responsible for the occurrence of life-threatening cardiovascular events such as ischemic heart disease, heart failure, strokes and peripheral vascular disease. Clinical studies have shown an association between the presence of intimate and / or arterial mean calcification and increased risk of cardiovascular and global mortality.

Until now, CV has been considered an irreversible process, and the effort has been directed at slowing their progression. Since in the last decade several epidemiological studies have identified vascular calcification as an independent prognostic factor of cardiovascular mortality, both in the general population and in the uremic population, it is necessary to find a suitable treatment for it, which allows the regression of the disease.

DESCRIPTION OF THE INVENTION

The authors of the present invention have demonstrated that the use of platelet-derived growth factor, as well as its pharmaceutical compositions, is useful for the treatment of vascular calcification pathology.

Thus, a first aspect of the invention relates to the use of platelet derived growth factor (PDGF), or any of its biologically active analogues, fragments or variants, in the preparation of a medicament for prevention or treatment of vascular calcification, or alternatively, to the platelet-derived growth factor, or any of its biologically active analogs, fragments or variants, for use in the prevention or treatment of vascular calcification.

Five different isoforms of PDGF are known that activate the cellular response through two receptors. Known ligands include A, B, C and D and an AB heterodimer and alpha (PDGFRA) and beta (PDGFRB) receptors. Unless otherwise specified, the platelet-derived growth factor is any combination of PDGF monomers and / or dimers, preferably, but not limited to, of PDGF B, including its analogues, reduced or not reduced, biologically active, or inactive, recombinant or no. Preferably, the PDGF analogs are biologically active or capable of becoming biologically active by refolding techniques or other similar manipulations. More preferably, it refers to homogeneous, highly expressed rPDGF B analogs, which are biologically active and closely resemble the PDGF B that exists in nature.

The terms "PDGF monomer" and "PDGF monomer" mean a single monomeric PDGF molecule that is not bound by disulfide bonds to any other PDGF molecule. It will be appreciated that "reduced PDGF" will necessarily be monomeric PDGF.

The terms "PDGF dimer" or "PDGF dimer" mean a PDGF molecule comprising two monomeric subunits of PDGF that are linked together by disulfide bonds.

The term "biologically active PDGF dimer" means dimer PDGF that has substantially the same activity as natural PDGF.

The term "biologically active PDGF monomer" means PDGF monomer that has a specific mitogenic activity of at least about one to one thousand of the specific mitogenic activity of the natural dimer PDGF.

The term "biologically active conformation", as used herein, refers to the conformation of a biologically active PDGF dimer or a biologically active PDGF monomer.

Preferably, the term "platelet-derived growth factor" means an amino acid sequence protein of Formula (I):

PR ¹ -CVR ² -R ³ -R ⁴ -RCR ⁵ -GCC

 Formula (I):

Where R ¹ is S or P;

R ² is P, E, T, A, S, L, N;

R ³ is V, L or A;

R ⁴ is M, L, Q, K, F, Y or S;

R ⁵ is G, S, T or A. The term "vascular calcification", herein, refers to alterations in the vascular wall that are characterized by the appearance of calcium salt deposits at the level of the intimate and middle layers. The term vascular calcification includes coronary calcification.

For the quantification of coronary calcification, the most used algorithm is the one created by Agatston (Agatston score), based on studies of electron beam CT (TCHE) obtained with prospective cardiac synchronization.

 Depending on the total amount of calcium observed, the risk of coronary heart disease can be classified into five different categories:

Vitamin D receptor activators or agonists (RVD activators) effectively suppress parathyroid hormone and are routinely used to control the development and progression of secondary hyperparathyroidism related to chronic kidney disease (CKD). Among them is 1, 25- (OH) 2D3 (calcitriol), 19-nor-1 a, 25 (OH) 2D2 (paricalcitol), 1a- (OH) D2 (doxercalciferol). And 22-oxa-1 a, (OH) 2D3 (maxacalcitol). These activators have a calcemic and phosphatic effect, being the last three lower compared to calcitriol.

Therefore, in a preferred embodiment of this aspect of the invention, vascular calcification is produced and / or induced by treatment with at least one vitamin D receptor agonist. In another more preferred embodiment of this aspect of the invention, The vitamin D receptor agonist is selected from the list consisting of calcitriol, paricalcitol, doxercalciferol and maxacalcitol.

Calcitriol (DCI) or 1-alpha, 25-dihydroxycholecalciferol (abevia1, 25- (OH) 2D3) is the active form of vitamin D found in the body (vitamin D3). It is produced by the hydrolyzing kidneys of 25-hydroxycholecalciferol (25-OH D, or calcifediol) which is previously hydrolyzed in the liver and regulates calcium levels, increasing calcium absorption in the gastrointestinal tract. It is a compound of formula (II):

 Formula (II)

By regulating the level of calcium in the blood, it helps maintain calcium in the bones and in the chemical balance in the body. When the kidney cannot properly eliminate toxic substances in the urine, then it also does not expel phosphorus. By not eliminating phosphates in the body automatically lowers calcium; When this happens, the parathyroid gland detects it and begins to secrete more parathormone. As it constantly does, it transforms into a disease called secondary hyperparathyroidism.

It has been observed that the activity of Parathormone (PTH) is closely linked to the presence of Calcitriol, in the absence of this vitamin the action of PTH is substantially lower.

19-nor-1, 25- (OH) 2-vitamin D2 or 19-nor-1, 25-dihydroxyvitamin D2 or paracalcitol, is an analogue of 1,25-dihydroxyergocalciferol, the active form of vitamin D2. It is also used for the treatment of secondary hyperparathyroidism (excessive secretion of parathormone). It is a compound of formula (III):

 Formula (III)

(1 S, 3, 5Z, 7E, 22E) -9, 10-secoergosta-5,7,10,22-tetraeno-1, 3-diol, or doxercalciferol is also used for the treatment of secondary hyperparathyroidism and for Bone metabolic disease treatment. It is a synthetic analogue of ergocalciferol (vitamin D), of formula (IV):

 Formula (IV)

1-alpha, 25-dihydroxy-22-oxacalcitriol or maxacalcitol is an analogue of vitamin D3, of formula (V).

 Formula (V)

In another preferred embodiment of this invention, the platelet-derived growth factor, or any of its biologically active analogs, fragments or variants, can be administered in a substantially pure form to a mammal, and preferably to a human, or can be administered as part. of a more complex composition. When administered as part of a composition, the amount of platelet-derived growth factor, or its analogues, is such that it achieves the desired effect, thus being an effective amount. Examples of mixtures that these compounds may contain are, but are not limited to, solutions or suspensions for administration intradermally, parenterally, and preferably intravenously. Examples of pharmaceutical vehicles or vectors, but not limited to, are aptamers.

The aptamers are single stranded nucleic acids (ssDNA and RNA), isolated from oligonucleotide libraries by in vitro selection, by exponential enrichment in the presence of the ligand (SELEX method), called chemical antibodies. Aptamers capable of vehiculizing the PDGF are known to those skilled in the art, such as, but not limited to, those described in patent application US2012171304.

Therefore, another aspect of the invention relates to the use of a pharmaceutical composition, hereinafter pharmaceutical composition of the invention, comprising growth factor, or any of its biologically active analogs, fragments or variants, and a pharmaceutically acceptable carrier. , in the development of a medicine for the prevention or treatment of vascular calcification. Alternatively, it also refers to a composition comprising the factor of growth derived from platelets, or an analogue, and a pharmaceutically acceptable carrier, for use in the prevention or treatment of vascular calcification.

A "pharmaceutically acceptable carrier" refers to those substances, or combination of substances, known in the pharmaceutical sector, used in the preparation of pharmaceutical forms of administration and includes, but are not limited to, solids, liquids, solvents or surfactants.

Another aspect of the invention relates to a pharmaceutical composition comprising platelet-derived growth factor or any of its biologically active analogs, fragments or variants, and at least one vitamin D receptor agonist, and a pharmaceutically acceptable carrier. In a preferred embodiment of this aspect of the invention, the vitamin D receptor agonist is selected from the list consisting of calcitriol, paricalcitol, doxercalciferol and maxacalcitol. Even more preferably it is calcitriol.

In another preferred embodiment of this aspect of the invention, the composition further comprises angiotensin, and even more preferably angiotensin II, a compound that could have an activity similar to platelet-derived growth factor.

Another aspect relates to the pharmaceutical composition of the invention, for use in the prevention or treatment of vascular calcification, or alternatively, to the use of the pharmaceutical composition of the invention, in the preparation of a medicament for the prevention or treatment of vascular calcification

Another aspect of the invention relates to a pharmaceutical form, hereinafter pharmaceutical form of the invention, comprising the pharmaceutical composition of the invention. For intradermal, parenteral or intramuscular routes, the pharmaceutical forms are, preferably but not limited to, the solution and the suspension.

In this specification, "pharmaceutical form" means the mixture of one or more active ingredients with or without additives that have physical characteristics for proper dosage, preservation, administration and bioavailability.

A "solution" is a pharmaceutical form consisting of a liquid, transparent and homogeneous preparation, obtained by dissolving it or the active ingredients and additives in water, and that is used for external or internal use. In the case of injectable, ophthalmic and otic solutions they must be sterile solutions.

A "suspension" is a pharmaceutical form consisting of a dispersed system, composed of two phases, which contain the active ingredient or additives. One of the phases, the continuous or the external one, is generally a liquid or a semi-solid and the dispersed or internal phase consists of insoluble solids (active principles), but dispersible in the external phase. In the case of injectables they must be sterile.

The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as the age, weight, sex, tolerance, ... of the mammal. In the sense used in this description, the term "therapeutically effective amount" refers to the amount of growth factor derived from platelets or their salts, prodrugs, derivatives or analogs, or combinations thereof, that produce the desired effect and, in general, it will be determined, among other causes, by the characteristics of said prodrugs, derivatives or analogues and the therapeutic effect to be achieved. The "adjuvants" and "pharmaceutically acceptable carriers" that can be used in said compositions are the vehicles known to those skilled in the art.

As used herein, the term "active substance", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" means any component that potentially provides a pharmacological activity or other different diagnostic effect , cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals. The term includes those components that promote a chemical change in the preparation of the drug and are present therein in a modified form intended to provide the specific activity or effect.

The term "medication", as used herein, refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals. In the context of the present invention, the disease is vascular calcification.

Another aspect of the invention relates to a combined preparation, hereafter combined preparation of the invention, comprising as active ingredients: (i) platelet-derived growth factor or any of its biologically active analogs, fragments or variants, and

 (ii) at least one vitamin D receptor agonist.

In a preferred embodiment of this aspect, the at least one agonist of the vitamin D receptors is selected from the list consisting of calcitriol, paricalcitol, d Ixex, Icif eral eral and maxacalcitol. In an even more preferred embodiment, the at least one vitamin D receptor agonist is calcitriol. In another even more preferred embodiment, the combined preparation of the invention further comprises angiotensin II.

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 application or sequential. In this way, the expression "juxtaposed" implies that it is not necessarily a true combination, in view of the physical separation of the components.

Another aspect concerns the use of the composition of the invention for the preparation of a medicament, or the separate, simultaneous or sequential use of the active ingredients of the combined preparation of the invention for the preparation of a medicament. Alternatively, it also refers to the composition of the invention for use as a medicine, or to the combined preparation of the invention for use as a medicine.

Another aspect relates to the use of the composition of the invention for the preparation of a medicament for the prevention or treatment of vascular calcification, or for the separate, simultaneous or sequential use of the active ingredients of the combined preparation of the invention for the preparation of a medicine for the prevention or treatment of vascular calcification. Alternatively, it also refers to the composition of the invention for use in the prevention or treatment of vascular calcification, or to the combined preparation of the invention for use in the prevention or treatment of vascular calcification.

Another aspect concerns the use of the platelet-derived growth factor of the composition of the invention for the preparation of a medicament for the prevention or the treatment of chronic kidney disease, or the separate, simultaneous or sequential use of the active ingredients of the combined preparation of the invention for the preparation of a medicament for the prevention or treatment of chronic kidney disease. Alternatively, it also refers to the platelet-derived growth factor or to the composition of the invention for use in the prevention or treatment of chronic kidney disease, or to the combined preparation of the invention for use in prevention or Treatment of chronic kidney disease.

Bone metabolic disease associated with chronic kidney disease, CKD (EMO-CKD) is defined as a progressive and multifactorial systemic disorder, which includes the set of biochemical alterations, bone abnormalities and abnormal extra-skeletal calcifications that occur in patients with CKD. Each of these complications results in important clinical consequences that condition, in turn, high morbidity and mortality in uremic patients.

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

DESCRIPTION OF THE FIGURES

 Fig. 1.A) Image of CMLV from human aorta under an inverted microscope B) Culture of Vascular Smooth Muscle Cells

Fig. 2. Neubauer Chamber.

 Fig. 3. Processing of CMLV cultures after the time of the experiment, for their demineralization and quantification of calcium.

Fig. 4. Angiotensin II dose-effect curve (10 ^"11 M-10 ^{" 4} M) on the levels of Calcium in CMLV incubated in the presence of high levels of P (3.3mM) for 9 days. The results are expressed as mean ± SE. n = 1 1. T-test: * p <0.01 vs. P.

Fig. 5. Effect of Angiotensin II (10 ^"5 M) on the calcification process (calcium levels) induced by P (3.3mM) in CMLV, in the presence of calcitriol (10 ^{" 8} M) or paricalcitol (3x10 ^"8 M) for 9 days of treatment. The results are expressed as mean ± SE. N = 12. T-test: a pO.01 vs Control; * p <0.01 vs P; #p <0.01 vs P + CTR 10 ^"8 M. Fig. 6. Dose-effect curve of PDGF (20ng / ml-80ng / ml) on the levels of Calcium in CMLV incubated in the presence of elevated levels of P (3.3mM) for 9 days. The results are expressed as mean ± SE. n = 18. T-test: ap <0.01 vs Control; * p <0.01vs P. Fig. 7. Effect of PDGF (20ng / ml) on the calcification process (calcium levels) induced by P (3.3mM) in CMLV, in presence of calcitriol (10 ^"8 M) or paricalcitol (3x10 ^" 8M) for 9 days of treatment. The results are expressed as mean ± SE. n = 18. T-test: a p <0.01 vs Control; * p <0.01vs P; #p <0.01 vs P + CTR 10-8M.

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which show the effectiveness of the use of platelet-derived growth factor, in the treatment of vascular calcification individually and in the presence of the drugs calcitriol and paricalcitol.

ANGIOTENSIN II

The role of angiotensin II, on the development of vascular calcifications has been evaluated.

In vitro studies were carried out with vascular smooth muscle (aorta) cells of human origin. Vascular Smooth Muscle Cells (CMLV) from human aorta were obtained from a cell line (Clonetics-Lonza Waikersville, Inc., USA). Cells were studied between 5 and 8 ^{or or} pass. The medium used in the growing cells was SmBm (Smooth muscle Basal medium), supplemented with SmGM-2 SingleQuot Kit Suppl. & Growth Factors (Clonetics- Lonza Waikersville, Inc., USA) containing: Bovine Fetal Serum (15%), hEGF (Recombinant epidermal growth factor, human), insulin, hFGF-B (Fibroblast growth factor β, human), gentamicin and amphotericin-B. Growing cells were grown in 75 cm ² bottles (Becton Dickinson Labware, NJ, USA).

Upon reaching 80% confluence, the cells were subcultured as follows: the culture medium was removed and the cells adhered to the bottle were washed with physiological serum. Subsequently, the cells were raised by adding a solution of Trypsin / EDTA (Sigma Aldrich Inc, MO, USA) to the bottle and incubated for 5 minutes. After the incubation period, growth medium (15% FBS) was added to inhibit the action of trypsin. The cell suspension was centrifuged at 1,500 rpm, for 5 minutes, at 20 ° C. The pellet was resuspended in growth medium, and an aliquot was extracted to count the cells. For studies with CMLV, cells were cultured in 6-well plates (Becton Dickinson Labware, NJ, USA) (Fig. 1).

Count and measure of cell viability.

To count the cells in each subculture, the same amount of Trypan Blue (Sigma Aldrich Inc, MO, USA) was added to the cell suspension aliquot, microliters were placed in a Neubauer Chamber (Fig. 2) and the live cells in each quadrant of 1 mm ² were counted, in the microscope.

Tripan Blue is a colored molecule of great molecular weight that is only able to enter cells that have altered membrane. Therefore a living cell in perfect condition will be colorless while a dead or very altered cell will be blue. In this way, cell viability can be determined.

(Colorless cells / total cells) x 100 =% cell viability

The Neubauer chamber (Fig. 2) is a counting chamber adapted to the bright-field or phase contrast microscope. It is a slide with a depression in the center. It has engraved in its central part, two microscopic grids with four 4x4 square areas.

The volume of each zone is 1 x 1 x 0, 1 mL. If we count the cells in each area, we can calculate the concentration of the cell suspension according to the following formula:

Average of counts of the four zones x 104 x 10 = no. cells / mL

When the cells in culture reached 80% confluence, the phosphate treatment was started. For the induction of calcification in the CMLV model, Monobasic and Dibasic Phosphate was used to prepare the medium high phosphorus. Calcification studies were carried out for 9 days, with a phosphorus concentration of 3.3 mM. Calcium quantification.

After the end of the experiment time, the wells medium was removed and the wells were washed twice with PBS (Sigma Aldrich Inc, MO, USA). The cells were then decalcified with 0.6 N HCI (Merck, Darmstadt, Germany) for 24 hours, at 37 ° C in a humid atmosphere with 5% C0 ₂ .

The calcium content in the acid extract obtained was determined colorimetrically by the o-Cresoftalein Complexone method (Calcium C Test, WAKO Chemicals GmbH, Neuss, Germany). After decalcification, the cells were solubilized with 0.1 M NaOH (Panreac Química, SA, Barcelona, Spain) and 0.1% SDS (Sigma Aldrich Inc, MO, USA), and raised by the method of scraped off. The protein content in the cell suspension obtained was quantified by the Bradford method (Bio-Rad Protein Assay, Bio-Rad Laboratories, Munich, Germany). The steps taken after finishing the experiment are schematized in Fig. 3.

The effect of angiotensin II on vascular calcification was evaluated independently, as well as in the presence of calcitriol and paricalcitol, after 9 days of treatment. The student's t-test was used to check if the differences in the degree of vascular calcification were statistically significant.

A dose-effect curve was performed (Figure 4) to optimize the dose to be used in subsequent studies. Thus, vascular smooth muscle cells were maintained for 9 days at different doses of angiotensin II: 10 ^"11 M-10 ^{" 4} M.

The levels of calcification ^ grCa / mgrProt) obtained were (n = 11):

0.35 ± 0.04 (control);

2.70 ± 0.23 (P), p <0.01 vs. control;

1.92 ± 0.15 (P + All 10 ^"11 ); p <0.01 vs. P

1.94 ± 0.10 (P + All 10 ^"9 ); p <0.01 vs. P

1.89 ± 0.20 (P + All 10 ^"7 ); p <0.01 vs. P

1.06 ± 0.13 (P + All 10 ^"5 ) p <0.01 vs. P

1.34 ± 0.20 (P + All 10 ^"4 ); p <0.01 vs. P The addition of none of the angiotensin II concentrations alone produced any effect on the controls. The concentration 10 ^"5 M was chosen, concentration at which a greater inhibition of calcification is obtained.

The next step was to evaluate the effect of angiotensin II on P-induced calcification in the presence of 10 ^"8 M calcitriol (calcification inducer) and 3x10 ^{" 8} M paricalcitol (partial inhibitor thereof).

The levels of calcification ^ grCa / mgrProt) obtained, after 9 days of treatment are represented in Figure 5 and in absolute values were (n = 12):

0.55 ± 0.05 (control);

 3.20 ± 0.38 (P), p <0.01 vs. control;

0.69 ± 0.02 (All 10 ^"5 );

1.86 ± 0.38 (P + All 10 ^"5 ); pO.01 vs P

5.26 ± 0.61 (P + CTR 10 ^"8 ); p <0.01 vs. P.

3.66 ± 0.69 (P + CTR 10 ^"8 + All 10 ^{" 5} ); p <0.01 vs P + CTR.

2.13 ± 0.38 (P + PC 10 ^"8 ); p <0.01 vs P

1.45 ± 0.46 (P + PC 10 ^"8 + All 10-5); pO.01 vs P

These data suggest that angiotensin II reduces calcification induced by elevated levels of P as well as by treatment with calcitriol; however, it did not produce an additional reduction in calcification to that produced by paricalcitol.

PDGF

The role of platelet-derived growth factor on the development of vascular calcifications has been evaluated.

In vitro studies were carried out with vascular smooth muscle (aorta) cells of human origin. Vascular Smooth Muscle Cells (CMLV) from human aorta were obtained from a cell line (Clonetics-Lonza Waikersville, Inc., USA). Cells were studied between 5 and 8 ^{or or} pass. The medium used in the growing cells was SmBm (Smooth muscle Basal medium), supplemented with SmGM-2 SingleQuot Kit Suppl. & Growth Factors (Clonetics- Lonza Waikersville, Inc., USA) that Contains: Fetal Bovine Serum (15%), hEGF (Recombinant epidermal growth factor, human), insulin, hFGF-B (Fibroblast growth factor β, human), gentamicin and amphotericin-B. Growing cells were grown in 75 cm ² bottles (Becton Dickinson Labware, NJ, USA).

Upon reaching 80% confluence, the cells were subcultured as follows: the culture medium was removed and the cells adhered to the bottle were washed with physiological serum. Subsequently, the cells were lifted by adding a solution of Trypsin / EDTA (Sigma Aldrich Inc, MO, USA) to the bottle and incubated for 5 minutes. After the incubation period, growth medium (15% FBS) was added to inhibit the action of trypsin. The cell suspension was centrifuged at 1,500 rpm, for 5 minutes, at 20 ° C. The pellet was resuspended in growth medium, and an aliquot was extracted to count the cells. For studies with CMLV, cells were grown in 6-well plates (Becton Dickinson Labware, NJ, USA).

Count and measure of cell viability.

To count the cells in each subculture, the same amount of Trypan Blue (Sigma Aldrich Inc, MO, USA) was added to the cell suspension aliquot, microliters were placed in a Neubauer Chamber (Fig. 2) and the live cells in each quadrant of 1 mm ² were counted, in the microscope.

Tripan Blue is a colored molecule of great molecular weight that is only able to enter cells that have altered membrane. Therefore a living cell in perfect condition will be colorless while a dead or very altered cell will be blue. In this way, cell viability can be determined.

(Colorless cells / total cells) x 100 =% cell viability

The Neubauer chamber (Fig. 2) is a counting chamber adapted to the bright-field or phase contrast microscope. It is a slide with a depression in the center. It has engraved in its central part, two microscopic grids with four 4x4 square areas.

The volume of each zone is 1 x 1 x 0, 1 mL. If we count the cells in each area, we can calculate the concentration of the cell suspension according to the following formula: Average of counts of the four zones x 104 x 10 = no. cells / mL

When the cells in culture reached 80% confluence, the phosphate treatment was started. For the induction of calcification in the CMLV model, Monobasic and Dibasic Phosphate was used to prepare the medium high phosphorus. Calcification studies were carried out for 9 days, with a phosphorus concentration of 3.3 mM.

Calcium quantification.

After the end of the experiment time, the wells medium was removed and the wells were washed twice with PBS (Sigma Aldrich Inc, MO, USA). The cells were then decalcified with 0.6 N HCI (Merck, Darmstadt, Germany) for 24 hours, at 37 ° C in a humid atmosphere with 5% C0 ₂ .

The calcium content in the acid extract obtained was determined colorimetrically by the o-Cresoftalein Complexone method (Calcium C Test, WAKO Chemicals GmbH, Neuss, Germany). After decalcification, the cells were solubilized with 0.1 M NaOH (Panreac Química, SA, Barcelona, Spain) and 0.1% SDS (Sigma Aldrich Inc, MO, USA), and raised by the method of scraped off. The protein content in the cell suspension obtained was quantified by the Bradford method (Bio-Rad Protein Assay, Bio-Rad Laboratories, Munich, Germany). The steps taken after finishing the experiment are schematized in Fig. 3.

The effect of platelet-derived growth factor on vascular calcification was observed independently, as well as in the presence of calcitriol and paricalcitol after 9 days of treatment. The student's t-test was used to check if the differences in the degree of vascular calcification were statistically significant.

A dose-effect curve was performed (Fig. 6) to optimize the dose to be used in subsequent studies. Thus, vascular smooth muscle cells were maintained for 9 days at different doses of platelet-derived growth factor:

 - 20 ng / ml.

 40 ng / ml

 - 80 ng / ml.

The calcification levels ^ grCa / mgrProt) obtained were (n = 18):

0.52 ± 0.04 (control); 3.34 ± 0.08 (P), p <0.01 vs. control;

1.15 ± 0.21 (P + PDGF 20), p <0.01 vs. P;

1.83 ± 0.13 (P + PDGF 40), p <0.01 vs P;

1.61 ± 0.08 (P + PDGF 80) p <0.01 vs. P.

The addition of none of the platelet-derived growth factor concentrations alone produced any effect on the controls. The concentration 20 ng / ml was chosen, concentration at which a greater inhibition of calcification is obtained. The next step was to evaluate the effect of platelet-derived growth factor on P-induced calcification in the presence of 10 ^"8 M calcitriol (calcification inducer) and 10 ^{" 8} M paricalcitol (partial inhibitor thereof).

The levels of calcification ^ grCa / mgrProt) obtained, represented in Fig. 7, were (n = 18):

 0.33 ± 0.05 (control);

 2.88 ± 0.3 (P), pO.01 vs. control;

 0.41 ± 0.04 (PDGF 20);

 0.66 ± 0.18 (P + PDGF 20); p <0.01 vs P

4.32 ± 0.6 (P + CTR 10 ^"8 ); p <0.01 vs P

1.92 ± 0.18 (P + CTR 10 ^"8 + PDGF 20); p <0.01 vs P + CTR

1.90 ± 0.3 (P + PC 3 x 10 ^"8 ); p <0.01 vs P

1.5 ± 0.18 (P + PC 3 x 10 ^"8 + PDGF 20); p <0.01 vs P

These data suggest that platelet-derived growth factor reduces calcification induced by elevated levels of P as well as by treatment with calcitriol; however, it did not produce an additional reduction in calcification to that produced by paricalcitol. CLAUSES

1. - Use of platelet-derived growth factor (PDGF), or any of its biologically active analogues, fragments or variants, in the preparation of a medicament for the prevention or treatment of vascular calcification.

2. - The use of platelet-derived growth factor or any of its biologically active analogues, fragments or variants according to the preceding claim, wherein vascular calcification is induced by treatment with at least one vitamin D receptor agonist.

3. - The use of the platelet derived growth factor according to claim 2, wherein the vitamin D receptor agonist is selected from the list consisting of calcitriol, paricalcitol, doxercalciferol and maxacalcitol.

4. - The use of the platelet derived growth factor or any of its biologically active analogs, fragments or variants according to any of claims 1-3, wherein the platelet derived growth factor or any of its biologically analogs, fragments or variants active is attached to an aptamer.

5. - The use of the platelet-derived growth factor according to any of claims 1-4, wherein the platelet-derived growth factor or any of its biologically active analogues, fragments or variants is in a solution or suspension, and the route of administration is the parenteral route.

6. - The use of the platelet derived growth factor according to the preceding claim, wherein the route of administration is the intravenous route.

7. - The use of a pharmaceutical composition comprising the platelet-derived growth factor or any of its biologically active analogs, fragments or variants as described in claims 1-6, and a pharmaceutically acceptable carrier, in the preparation of a medicine for the prevention or treatment of vascular calcification.

8. - A pharmaceutical composition comprising the platelet-derived growth factor, or any of its biologically active analogs, fragments or variants, and at least one vitamin D receptor agonist, and a pharmaceutically acceptable carrier.

9. - The pharmaceutical composition according to the preceding claim, wherein the agonist of the vitamin D receptors is selected from the list consisting of calcitriol, paricalcitol, d Ixex, Icif eral eral and maxacalcitol.

10. - The pharmaceutical composition according to any of claims 8-9, wherein the vitamin D receptor agonist is calcitriol.

1 1. - The pharmaceutical composition according to any of claims 8-10, further comprising angiotensin II.

12. - Pharmaceutical form comprising a composition according to any of claims 7-11.

13. - A combined preparation comprising as active ingredients the platelet-derived growth factor or any of its biologically active analogs, fragments or variants, and at least one vitamin D receptor agonist.

14. - The combined preparation according to claim 13, wherein the agonist of the vitamin D receptors is selected from the list consisting of calcitriol, paricalcitol, d Ixex, Icif eral eral and maxacalcitol.

15. The combined preparation according to any of claims 13-14, wherein the vitamin D receptor agonist is calcitriol.

16. - The combined preparation according to any of claims 13-15, further comprising angiotensin II.

17. - The separate, simultaneous or sequential use of the active ingredients of the combined preparation according to any of claims 13-16, in the preparation of a medicament.

18. - The use, separately, simultaneously or sequentially, of the active ingredients of the combined preparation according to any of claims 13-16, in the preparation of a medicament for the prevention or treatment of vascular calcification. INVENTION 2

USE OF AN AT2 RECEIVER SELECTIVE AGONIST IN THE TREATMENT OF

 VASCULAR CALCIFICATIONS.

The present invention is within the field of medicine and pharmacy, and refers to the use of a selective AT2 receptor agonist, preferably of angiotensin, and even more preferably of angiotensin II, for the preparation of a medicament or composition. pharmaceutical aimed at combating the calcification of blood vessels, and especially the treatment of vascular calcifications associated with treatment with vitamin D receptor agonists.

STATE OF THE PREVIOUS TECHNIQUE

Cardiovascular disease is the leading cause of death in patients with chronic renal failure (CRF). Hemodialysis patients present with atherosclerosis and arteriosclerosis. These vascular diseases begin to develop in the early stages of kidney disease, and as the kidney disease progresses, progressive worsening of cardiovascular disease occurs. Cardiovascular disease of patients with advanced renal failure is characterized by calcifications in the area of the intimate and middle layers of the arterial wall. Calcifications of the arteries cause decreased elasticity of the vessels and the degree of calcification correlates with mortality.

For many years, vascular calcifications of patients with renal insufficiency were considered to be due to passive deposition of calcium salts, the result of an oversaturation due to the increase in plasma concentrations of calcium and phosphorus (increase in Ca x P product) . Undoubtedly, hyperphosphatemia and the increase in the Ca x P product contribute significantly to vascular calcification. Hyperphosphatemia is a predictor of mortality, possibly due to its role in the pathogenesis of ratings. To prevent vascular calcifications in renal patients, it is essential to control plasma phosphate without providing an excess of calcium, that is, avoiding using intestinal phosphorus chelants that contain calcium.

However, at present, it is known that vascular calcification is an active process regulated in the cellular field and by certain components of the plasma, such as certain proteins and other pathogenic factors. Therefore, there are proteins that regulate vascular calcification, favoring or inhibiting it. It is evident that in patients with renal pathologies the inhibitory mechanisms are overcome by the other mechanisms that promote vascular calcification, which results in an imbalance.

Among the proteins that modulate extraosseous calcification are: Matrix gla-protein

It is an extracellular protein that has an affinity for hydroxyapatite. In bone, it promotes bone formation and, in the artery, inhibits calcification. The matrix gla-protein (MGP) is dependent on vitamin K and its function is affected by warfarin or acenocoumarol. MGP inhibits the activity of bone matrix protein 2a (BMP2a), which is a procalcifying protein. It has been proven that a mouse deficient in MGP shows diffuse arterial calcifications.

Fetish

It is a2-Heremans-Schmid (Ahsg) glycoprotein or fetuin A. This serum protein is an important inhibitor of extraosseous calcifications. The concentration of fetuin decreases in situations of inflammation (immune cell response). This protein decreases the precipitation of calcium and phosphorus in vitro. It has been seen that a mouse deficient in fetuin shows diffuse arterial calcifications. The clinical importance of fetuin was illustrated in a study that demonstrated an association between cardiovascular mortality and low fetuin values in hemodialysis patients. It has been described that the fetuin / MGP protein complex inhibits the precipitation of calcium salts and the formation of hydroxyapatite. It also seems that the inhibition of calcification by fetuin depends on the formation of stable protein complexes that incorporate the mineral nuclei.

Osteoprotegerin

In bone, the interaction of osteoprotegerin (OPG-L) with its RANK receptor (the English receptor-activator of NF-κΒ) regulates the differentiation, activation and apoptosis of osteoclasts. It has been seen that in OPG-deficient mice, osteoporosis and arterial calcification occur. Osteopontin

It is a potent inhibitor of hydroxyapatite deposition in vascular smooth muscle cell (VSMC) cultures. In addition, it protects endothelial cells from apoptosis that is caused by deprivation of growth factors. The most important proteins that favor calcification are alkaline phosphatase, osteonectin, osteocalcin and BMP2. These proteins are expressed in abundance in calcified vascular wall and in vascular smooth muscle cell cultures in which calcifications are induced.

Among the pathogenic factors are: Hyperiosphatemia

Increasing the concentration of phosphorus in the medium in VSMC cultures induces the calcification of these cells. In order for the phosphorus to induce the calcification of the VSMC, it needs to have an active transport from outside to the inside of the cell (Pit-1), and that subsequently Cbfa-1, an osteoblast-specific transcription factor that regulates the expression, is induced of procalcifying proteins. It has been observed that using phosphonorphic acid (PFA) can prevent phosphorus cell transport. The calcifying effect of phosphorus requires a prior alteration of the vascular wall that facilitates or promotes the phenotypic transformation of VSMC into osteoblast.

Uremia

In vitro studies with VSMC show that uremic serum is capable of inducing its calcification, independently of phosphorus concentration. In the calcified arteries of uremic patients, there is an increase in Cbfa-1 expression in the middle and intimate layers.

Hypercalcemia

It has also been shown that an increase in calcium concentration exerts a direct stimulating effect on calcification. In VSMC cultures, the high concentration induces calcification even when the phosphorus is not increased. Like phosphorus, elevated calcium also stimulates the expression of the Na-P transporter (Pit-1), and inhibition of this transporter inhibits calcium-induced calcification. Too elevated calcium induces Cbfa-1 expression. Hyperphosphoremia and hypercalcemia induce vascular calcifications independently, but together they exert a synergistic effect that favors vascular calcifications. It is important to note that, in the presence of a high phosphorus, a small increase in calcium means an exaggerated calcification.

Microvesicles

The first step in the calcification process is the accumulation of calcium and phosphorus in vesicles that come from the apoptosis of VSMC (apoptotic bodies), or that are released by cells of the osteoblast line vascular wall that express Cbfa-1 (matrix vesicles)

In addition to these factors, there are drugs that induce vascular calcifications. Thus, Calcitriol (DCI) or 1-alpha, 25-dihydroxycholecalciferol (abevia1, 25- (OH) 2D3) is the active form of vitamin D found in the body (vitamin D3). It is used to treat secondary hyperparathyroidism in uremic patients. But calcitriol increases the intestinal absorption of calcium and phosphorus, and produces hypercalcemia and hyperphosphatemia, which can favor vascular calcification. The danger of vitamin D calcification is greater if it is administered to patients with hyperphosphatemia who are being treated simultaneously with phosphorus chelators containing calcium. In addition, some authors have found that calcitriol stimulates vascular calcification by direct action on the vascular wall.

Currently, other vitamin D analogues, such as paricalcitol or 22-oxacalcitriol (OCT), can be used in the treatment of secondary hyperparathyroidism, which, while continuing to inhibit PTH, appear to have less effect on calcium intestinal absorption and phosphorus In a recent study, lower mortality has been shown in patients on hemodialysis who are being treated with paricalcitol, compared with those treated with calcitriol. One of the possible reasons that explain the beneficial effect of paricalcitol on mortality may be a lower hypercalcemic and hyperphosphatemic effect.

An alternative to calcitriol treatment is the use of calcimimetics, allosteric calcium receptor activators (RCa), which has been used for a few years to control secondary hyperparathyroidism in uremic patients. The effect of calcitriol and calcimimetic on vascular calcifications in uremic rats with secondary hyperparathyroidism has been analyzed (López et al., 2006. J Am Soc Nephrol 17 (3): 795-804). The administration of calcitriol in doses necessary to control hyperparathyroidism, produced severe calcifications of the vessel wall; Calcimimetic administration controlled hyperparathyroidism and did not cause calcifications. In addition, the most important thing is that in rats that received the combination of calcitriol and calcimimetics calcifications were not observed, that is, that calcimimetic is able to avoid calcifications induced by calcitriol. A possible explanation would be that in animals treated with calcimimetics there is a moderate decrease in calcium and phosphorus compared to animals treated with calcitriol, however rats treated with calcitriol and calcimimetics had high values of calcium and phosphorus. The fact that rats treated with calcimimetics have an increase in MGP (anticalcifying protein) suggests a direct effect of calcimimetic on the vascular wall. In bovine VSMC (BVSMC) cultures the presence of RCa has been demonstrated and calcification is associated with a decrease in RCa expression levels, which could be inherent to osteoblastic transformation (Block et al., 2004. N Engl J Med; 350: 1516-25).

In recent years the implication of the Wnt ^ -catenin pathway has been characterized in the calcification processes. Activation of Wnt ^ -catenin leads to increased proliferation and osteogenesis (Dahlóf 2010. Am J Cardiol. 4; 105 (1 Suppl): SAGA). Its activation has also been related to methylation and inhibition of gene promoters.

Almost a century ago, in 1898, it was discovered that kidney extract produces a strong vasopressor effect; This principle was given the name Renina. Later it was discovered that renin was not vasopressor by itself, but that it was an enzyme that converted an inactive plasma product, angiotensinogen, into an active one, angiotensin. Angiotensin is a hormone that Dr. Braun-Menéndez and his group discovered in Argentina around 1939. Almost at the same time, Page's group made the same discovery. Each of these groups gave a name to the compound generated in the plasma, the first called "hypertensin" and the second "angiotonin". It was necessary for almost 20 years to pass, so that the field researchers could agree on the appropriate name for the hormone, and in 1957 it was given the hybrid name of angiotensin.

Currently it is known that there are three angiotensins: I, II and III, which are increasingly smaller products; that is, the angiotensinogen forms the angiotensin I, of her angiotensin II and of this in turn angiotensin III; the most active is angiotensin II.

From angiotensinogen, a peptide of 14 amino acids and by action of a proteolytic enzyme (renin) angiotensin II is formed by the action of the angiotensin converting enzyme (ACE), this octapeptide stimulates thirst, fluid intake, increased blood pressure and aldosterone secretion.

Platelet-derived growth factor (PDGF) is a cationic protein, stable against heat, found in a variety of cell types, including circulating platelet granules, vascular smooth muscle cells, endothelial cells, macrophages and keratinocytes , and is known to stimulate in vitro protein synthesis and collagen production by fibroblasts. It is also known to act as an in vitro mitogenic and chemotactic agent for fibroblasts, smooth muscle cells, osteoblasts and glial cells.

PDGF was initially described by Ross et al., Proc. Nati Acad. Sci. USA., 71, 1207-1210 (1974), as a factor found in whole blood serum (but not in low platelet serum) that is capable of supporting the growth of fibroblasts in the culture. The PDG F was isolated from the platelets and the serum, the natural non-reduced PDGF being identified as a 27-35 kd molecular weight dimera protein. It was found that PDGF reduction provided two or more smaller bands on gels, in a molecular weight range of 10-18 kd. It was believed that these smaller bands represented two distinct smaller monomeric subunits of approximately 18 kd and 16 kd of molecular weights called, respectively, the "A" and "B" subunits, or alternatively, the PDGF A chain and the B chain of PDGF. The amino acid sequences of the two subunits of the PDGF have already been described, identifying the amino acid sequence of the B-chain of the PDGF homologous in more than 90% to the predicted protein product of v-sis, the oncogene contained in the sarcoma virus of oncogenic ape (SSV); see Doolittle et al., Science, 221, 275-276 (1983), and Waterfield et al., Nature, 304, 2810-2814 (1983). It has been found that chain A is approximately 60% homologous to chain B. It is believed that PDGF is biologically active only in dimera form. These biologically active PDGF dimers can take the form of a PDGF-AB heterodimer, a PDGF-BB homodimer or a PDGF-AA homodimer. Each monomer subunit of the biologically active dimer, regardless of whether it is a chain A monomer or a chain B monomer, It contains eight cysteine residues. Some of these cysteine residues form disulfide bonds between chains that hold the dimers together.

The homologous sequence of 109 amino acid v-sis (PDGF B ₁₀ g), identified by Johnsson et al. 1984. EMBO Journal, 3 (5), 921-928 as the mature form of PDGF B, has been used in recombinant technology using yeast and other eukaryotic host cell systems to obtain recombinant active PDGF (rPDGF) (US 4,766,073) .

It has been shown that a recombinant human PDGF-BB (= rhPDGF-BB) stimulates wound healing and bone regeneration in both animals and humans. It has been approved both in the United States and in Europe for human use in topical applications in order to accelerate the healing of chronic diabetic foot sores. It has also been shown that a recombinant hPDGF-BB is effective, either alone or in combination with other growth factors, to improve periodontal regeneration, ie the renewed growth of bone, cement and ligament around the teeth (US 5,124 .316).

The presence of vascular calcifications alters the structure and vascular function, if these are responsible for the occurrence of life-threatening cardiac ovarian events such as ischemic heart disease, heart failure, strokes and peripheral vascular disease. Clinical studies have shown an association between the presence of intimate and / or arterial mean calcification and increased risk of cardiovascular and global mortality.

Until now, vascular calcification has been considered an irreversible process, and the effort has been directed at slowing their progression. Since in the last decade several epidemiological studies have identified vascular calcification as an independent prognostic factor of cardiovascular mortality, both in the general population and in the uremic population, it is necessary to find a suitable treatment for it, which allows the regression of the disease.

DESCRIPTION OF THE INVENTION

The authors of the present invention have demonstrated that the use of a selective AT2 receptor agonist, and in particular of angiotensin II, as well as its pharmaceutical compositions, is useful for the treatment of vascular calcification. Thus, a first aspect of the invention relates to the use of a selective AT2 receptor agonist in the preparation of a medicament for the prevention or treatment of vascular calcification, or alternatively, to a selective AT2 receptor agonist for its use in the prevention or treatment of vascular calcification.

In a preferred embodiment of this aspect of the invention, the selective agonist of the AT2 receptor is angiotensin, an angiotensin analog, a biologically active variant or fragment of angiotensin or the angiotensin analog. In an even more preferred embodiment, it is angiotensin II (A II), a biologically active variant or fragment of angiotensin II.

Angiotensin analogs may be peptide agents or non-peptide agents (eg, peptidomimetics) that have the requirement of the agonist activity of AT2.

The term "selective" in this specification refers to the agonist preferring to act against AT2 type receptors over others, although this preference does not imply absolute exclusion, and may also act on other receptors, although to a lesser extent.

A n g i ote n s i n a e s u n a h o rm o n a peptide that causes vasoconstriction and subsequently increases blood pressure. It is part of the renin-angiotensin system. Angiotensin also stimulates the release of aldosterone, another hormone, from the adrenal cortex. Aldosterone promotes sodium retention in the distal nephron, in the kidney, which also causes an increase in blood pressure. There are several types of angiotensin (I, II, III, IV). Angiotensin I appears to have no biological activity, and exists only as a precursor to angiotensin II.

Angiotensin I is converted into angiotensin II (Al I) through the elimination of C-terminal residues by the angiotensin-converting enzyme (ACE).

Angiotensin II is an octapeptide that acts as an endocrine, autocrine / paracrine, and intracrine hormone and has the sequence SEQ ID NO: 1.

Asp-Arg-Val-Tyr-lle-His-Pro-Phe (SEQ ID NO: 1)

The active analogs of Al l, the fragments of Al l and the analogs thereof of particular interest according to the present invention comprise a sequence consisting of at least three contiguous amino acids of the groups R ¹ to R ⁸ in the sequence of formula general (I):

R ¹ -R ² -R ³ -R ⁴ -R ⁵ -R ⁶ -R ⁷ -R ⁸

Formula (I) in which R ¹ is suitably selected from H, Asp, Glu, Asn, Acpc (1-aminocyclopentanecarboxylic acid), Ala, Me ² Gly, Pro, Bet, Glu (NH ₂ ), Gly, Asp (NH ₂ ) and Suc, R ² is suitably selected from Arg, Lys, Ala, Orn, Ser (Ac), Sar, D-Arg and D-Lys,

R ³ is selected from the group consisting of Val, Ala, Leu, norLeu, lie, Gly, Pro, Aib, Acpc and Tyr, while Lys has been found to be effective in this remainder;

R ⁴ is selected from the group consisting of Tyr, Tyr (P0 ₃ ) ₂ , Thr, Ser, homoSer, azaTyr, and Ala;

R ⁵ is selected from the group consisting of lie, Ala, Leu, norLeu, Val and Gly;

R ⁶ is His, Arg or 6-NH ₂ -Phe;

R ⁷ is Pro or Ala; Y

R ⁸ is absent or selected from the group consisting of Phe, Phe (Br), lie and Tyr, excluding sequences that include R ⁴ as a terminal Tyr group.

Compounds within the category of AT2 agonists useful in the practice of the invention include the Al 1 analogs indicated above subject to the restriction that R ⁶ is p-NH ₂ -Phe.

Preferred sequences of analogs include the following: AHI or AII (2-8), Arg-Val-Tyr-lle-His-Pro-Phe; AII (3-8), also known as-AIII or AIV, Val Tyr-lle-Pro-Phe; All (1- 7), Asp-Arg-Val-Tyr-lle-His-Pro; AII (2-7), Arg-Val-Tyr-lle His-Pro; AII (3-7), Val-Tyr-lle-His-Pro; AII (5-8), lle-His-Pro-Phe; AII (1-6), Asp Arg-Val-Tyr-lle-His; AII (1-5), Asp-Arg-Val-Tyrle; At 1 (1-4), Asp-Arg-Val-Tyr; and Al 1 (1-3), Asp-Arg-Val. Other preferred embodiments include: Arg-norLeu-Tyr-lle-His-Pro-Phe and Arg-Val-Tyr-norLeu-His-Pro Phe. Still another preferred embodiment within the scope of the invention is a peptide having the sequence Asp-Arg-Pro Tyr-lle-His-Pro-Phe. AII (6-8), His-Pro-Phe and AII (4-8), Tyr-lle His-Pro-Phe.

Another class of especially preferred compounds according to the present invention consists of those having the following general structure:

Asp-Arg-R ¹ -Tyr-lle-His-Pro in which R ¹ is selected from the group consisting of Lys, Leu, norLeu, Val, lie and Ala.

 Another class of compounds of special interest according to the present invention is that of the general formula (II):

R ² -R ³ -R ⁴ -R ⁵ -R ⁶ -R ⁷ -R ⁸

 Formula (II)

in which

R ² is selected from the group consisting of H, Arg, Lys, Ala, Orn, Ser (Ac), Sar, D-Arg and D-Lys;

R ³ is selected from the group consisting of Val, Ala, Leu, norLeu, lie, Gly, Pro, Aib, Acpc and Tyr;

R ⁴ is selected from the group consisting of Tyr, Tyr (P0 ₃ ) ₂ , Thr, Ser, homoSer, azaTyr, and Ala;

R ⁵ is selected from the group consisting of lie, Ala, Leu, norLeu, Val and Gly;

R ⁶ is His, Arg or 6-NH ₂ -Phe;

R ⁷ is Pro or Ala; Y

R ⁸ is absent or selected from the group consisting of Phe, Phe (Br), lie and Tyr.

An especially preferred subclass of compounds of general formula (II) has Formula (III):

R ² - R ³ -Tyr- R ⁵ -His-Pro-Phe

Formula (III) in which R ² , R ³ and R ⁵ are as defined above. Especially preferred is angiotensin III of the formula Arg-Val-Tyr-lle-His-Pro-Phe. Other preferred compounds include peptides having the structures Arg-Val-Tyr-Gly-His-Pro-Phe and Arg-Val-Tyr-Ala-His Pro-Phe.

Other especially preferred embodiments include: 1 GD Ala4-AII- (1-7) DRVAIHP

 2GD Pro3-AII- (1-7) DRPYIHP

 5GD Lys3-AII- (1-7) DRKYIHP

 9GD Norl_eu-AII- (1-7) DR- (nor) -YIHP

GSD 28 lle ^a -AII DRVYIHPI

 Ala3aminoPhe6 All DRVYIF * PF

 Ala3-AIII RVAIHPF

 Gly1-AII GRVYIHPF

 NorLeu4-AIII -RVYnLHPF

 Acpc3-AIII DR- (Acpc) -YIHPF

 GSD3 37B Om2-AII D- (Om) -VYIHPF

 GSD38B Citron2-AII D- (Citron) -VYIHPF

3GD Pro3Ala4-AII- (1-7) DRPAIHP

In the above formulas, the usual three-letter abbreviations for amino acid residues are used. In the absence of an indication to the contrary, the L-form of the amino acid is proposed. Other remains are abbreviated as follows:

Also, within the scope of this invention are prodrugs and angiotensin II derivatives. Advantageously, said derivative is a compound that increases the bioavailability of angiotensin II, when administered to an individual or that enhances the release of the compound in a biological compartment. The nature of said derivative is not critical as long as it can be administered to an individual and provides the compound in an individual's biological compartment. The preparation of said prodrug can be carried out by conventional methods known to those skilled in the art.

By vascular calcification, herein, we refer to alterations in the vascular wall that are characterized by the appearance of calcium salt deposits at the level of the intimate and middle layers. The term vascular calcification includes coronary calcification.

For the quantification of coronary calcification, the most used algorithm is the one created by Agatston (Agatston score), based on studies of electron beam CT (TCHE) obtained with prospective cardiac synchronization.

Depending on the total amount of calcium observed, the risk of coronary heart disease can be classified into five different categories:

Vitamin D receptor agonists or activators (RVD activators) effectively suppress parathyroid hormone and are routinely used to control the development and progression of secondary hyperparathyroidism related to chronic kidney disease (CKD). Among them is 1, 25- (OH) 2D3 (calcitriol), 19-nor-1 a, 25 (OH) 2D2 (paricalcitol), 1a- (OH) D2 (doxercalciferol). And 22-oxa-1 a ^' (OH) 2D3 (maxacalcitol). These activators have a calcemic and phosphatic effect, being the last three lower compared to calcitriol.

Therefore, in a preferred embodiment of this aspect of the invention, vascular calcification is produced or induced by treatment with at least one vitamin D receptor agonist or activator. In another more preferred embodiment of this aspect of the invention. , the vitamin D receptor agonist or activator is selected from the list consisting of calcitriol, paricalcitol, doxercalciferol and maxacalcitol.

Calcitriol (DCI) or 1-alpha, 25-dihydroxycholecalciferol (abevia1, 25- (OH) 2D3) is the active form of vitamin D found in the body (vitamin D3). It is produced by the kidneys that hydrolyze 25-hydroxycholecalciferol (25-OH D, or calcifediol) which is previously hydrolyzed in the liver and regulates calcium levels, increasing calcium absorption in the gastrointestinal tract. It is a compound of formula (IV):

 Formula (IV)

By regulating the level of calcium in the blood, it helps maintain calcium in the bones and in the chemical balance in the body. When the kidney cannot properly eliminate toxic substances in the urine, then it also does not expel phosphorus. By not eliminating phosphates in the body automatically lowers calcium; When this happens, the parathyroid gland detects it and begins to secrete more parathormone. As it constantly does, it transforms into a disease called secondary hyperparathyroidism.

It has been observed that the activity of Parathormone (PTH) is closely linked to the presence of Calcitriol, in the absence of this vitamin the action of PTH is substantially lower.

19-nor-1, 25- (OH) 2-vitamin D2 or 19-nor-1, 25-dihydroxyvitamin D2 or paracalcitol, is an analogue of 1,25-dihydroxyergocalciferol, the active form of vitamin D2. It is also used for the treatment of secondary hyperparathyroidism (excessive secretion of parathormone). It is a compound of formula (V):

 Formula (V)

(1 S, 3, 5Z, 7E, 22E) -9, 10-secoergosta-5,7,10,22-tetraeno-1, 3-diol, or doxercalciferol is also used for the treatment of secondary hyperparathyroidism and for Bone metabolic disease treatment. It is a synthetic analogue of ergocalciferol (vitamin D), of formula (VI):

 Formula (VI)

1-alpha, 25-dihydroxy-22-oxacalcitriol or maxacalcitol is an analogue of vitamin D3, of formula (VII).

 Formula (VII)

In another preferred embodiment of this invention, angiotensin II, or its salts, prodrugs, derivatives or analogs, variants or biologically active fragments may be administered in a substantially pure form to a mammal, and preferably a human, or may be administered as part of a composition. more complex When administered as part of a composition, the amount of angiotensin II, or its salts, prodrugs, derivatives or the like, is such that it achieves the desired effect, thus being an effective amount. Examples of mixtures that these compounds may contain are, but are not limited to, solutions or suspensions for administration intradermally, parenterally, and more preferably, intravenously.

Therefore, another aspect of the invention relates to the use of a pharmaceutical composition, hereafter referred to as a pharmaceutical composition of the invention, comprising a selective agonist of the AT2 receptor, or any of its salts, prodrugs, derivatives or analogs, or any of its combinations, and a pharmaceutically acceptable vehicle, in the preparation of a medicament for the prevention or treatment of vascular calcification. Alternatively, it also refers to a composition comprising a selective AT2 receptor agonist, or any of its salts, prodrugs, derivatives or analogs, or any combination thereof, and a pharmaceutically acceptable carrier, for use in prevention or treatment. of vascular calcification. In a preferred embodiment of this aspect of the invention, the selective agonist of the AT2 receptor is angiotensin, an analogue or a biologically active variant thereof. More preferably, the selective agonist of the AT2 receptor is angiotensin II, or an analogue or a biologically active variant thereof. Preferably, the selective agonist of the AT2 receptor is in a solution or suspension, and the route of administration is the parenteral route. More preferably, the route of administration is the intravenous route.

A "pharmaceutically acceptable carrier" refers to that substances, or combination of substances, known in the pharmaceutical sector, used in the preparation of pharmaceutical forms of administration and includes, but is not limited to, solids, liquids, solvents or surfactants.

Another aspect of the invention relates to a pharmaceutical composition comprising a selective AT2 receptor agonist, at least one vitamin D receptor agonist, and a pharmaceutically acceptable carrier. In a preferred embodiment of this aspect of the invention, the vitamin D receptor agonist is selected from the list consisting of calcitriol, paricalcitol, doxercalciferol and maxacalcitol. In another even more preferred embodiment, the vitamin D receptor agonist or activator is calcitriol.

In another preferred embodiment of this aspect of the invention, the composition further comprises platelet-derived growth factor, an analogue, a variant, or a biologically active fragment thereof.

Another aspect relates to the pharmaceutical composition of the invention, for use in the prevention or treatment of vascular calcification, or alternatively, to the use of the pharmaceutical composition of the invention, in the preparation of a medicament for the prevention or treatment of vascular calcification

Another aspect of the invention relates to a pharmaceutical form, hereafter referred to as the pharmaceutical form of the invention, which comprises the pharmaceutical composition of the invention. For intradermal, parenteral or intramuscular routes, the pharmaceutical forms are, but are not limited to, the solution and the suspension.

In this specification, "pharmaceutical form" means the mixture of one or more active ingredients with or without additives that have physical characteristics for proper dosage, preservation, administration and bioavailability.

A "solution" is a pharmaceutical form consisting of a liquid, transparent and homogeneous preparation, obtained by dissolving it or the active ingredients and additives in water, and that is used for external or internal use. In the case of injectable, ophthalmic and otic solutions they must be sterile solutions.

A "suspension" is a pharmaceutical form consisting of a dispersed system, composed of two phases, which contain the active ingredient or additives. One of the phases, the continuous or the external one, is generally a liquid or a semi-solid and the dispersed or internal phase consists of insoluble solids (active principles), but dispersible in the external phase. In the case of injectables they must be sterile.

The dosage to obtain a therapeutically effective amount depends on a variety of factors, such as the age, weight, sex, tolerance, ... of the mammal. In the sense used in this description, the term "therapeutically effective amount" refers to the amount of selective agonist of the AT2 receptor, or of its salts, prodrugs, derivatives or analogs, or combinations thereof, that produce the desired effect and, in general, it will be determined, among other causes, by the characteristics of said prodrugs, derivatives or analogues and the therapeutic effect to be achieved. The "adjuvants" and "pharmaceutically acceptable carriers" that can be used in said compositions are the vehicles known to those skilled in the art.

As used herein, the term "active ingredient", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" means any component that potentially provides a pharmacological activity or other different diagnostic effect, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals. The term includes those components that promote a chemical change in the preparation of the drug and are present therein in a modified form intended to provide the specific activity or effect.

The term "medication", as used herein, refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals. In the context of the present invention, the disease is vascular calcification.

Another aspect of the invention relates to a combined preparation, hereafter combined preparation of the invention, comprising as active ingredients a selective AT2 receptor agonist, and a vitamin D receptor activator. In a preferred embodiment of this aspect, the selective agonist of the AT2 receptor is the angiotensin, an analogue, a variant or a biologically active fragment thereof. More preferably, the selective agonist of the AT2 receptor is angiotensin II, an analogue, a variant or a biologically active fragment thereof. In another preferred embodiment of this aspect, the vitamin D receptor activator is selected from the list consisting of calcitriol, paricalcitol, doxercalciferol and maxacalcitol. In an even more preferred embodiment, the vitamin D receptor activator is calcitriol. In another even more preferred embodiment, the combined preparation of the invention further comprises the platelet-derived growth factor, an analogue, a variant or a biologically active fragment 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 application or sequential In this way, the expression "juxtaposed" implies that it is not necessarily a true combination, in view of the physical separation of the components.

Another aspect concerns the use of the composition of the invention for the preparation of a medicament, or the separate, simultaneous or sequential use of the active ingredients of the combined preparation of the invention for the preparation of a medicament. Alternatively, it also refers to the composition of the invention for use as a medicine, or to the combined preparation of the invention for use as a medicine.

Another aspect relates to the use of the composition of the invention for the preparation of a medicament for the prevention or treatment of vascular calcification, or for the separate, simultaneous or sequential use of the active ingredients of the combined preparation of the invention for the preparation of a medicine for the prevention or treatment of vascular calcification. Alternatively, it also refers to the composition of the invention for use in the prevention or treatment of vascular calcification, or to the combined preparation of the invention for use in the prevention or treatment of vascular calcification.

Another aspect relates to the use of the composition of the invention for the preparation of a medicament for the prevention or treatment of chronic kidney disease, or for the separate, simultaneous or sequential use of the active ingredients of the preparation. combination of the invention for the preparation of a medicament for the prevention or treatment of chronic kidney disease. Alternatively, it also refers to the selective agonist of the AT2 receptor, an analogue, a variant or a biologically active fragment thereof, or the composition of the invention for use in the prevention or treatment of chronic kidney disease, or to the combined preparation of the invention for use in the prevention or treatment of chronic kidney disease.

Bone metabolic disease associated with chronic kidney disease, CKD (EMO-CKD) is defined as a progressive and multifactorial systemic disorder, which includes the set of biochemical alterations, bone abnormalities and abnormal extra-skeletal calcifications that occur in patients with CKD. Each of these complications results in important clinical consequences that condition, in turn, high morbidity and mortality in uremic patients.

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

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which show the effectiveness of the use of platelet-derived growth factor, in the treatment of vascular calcification individually and in the presence of the drugs calcitriol and paricalcitol.

ANGIOTENSIN II

The role of angiotensin II, on the development of vascular calcifications has been evaluated.

In vitro studies were carried out with vascular smooth muscle (aorta) cells of human origin. Vascular Smooth Muscle Cells (CMLV) from human aorta were obtained from a cell line (Clonetics-Lonza Walkersville, Inc., USA). Cells were studied between 5 and 8 ^{or or} pass. The medium used in the cells in growth was SmBm (Smooth muscle Basal medium), supplemented with SmGM-2 SingleQuot Kit Suppl. & Growth Factors (Clonetics- Lonza Walkersville, Inc., USA) containing: Bovine Fetal Serum (15%), hEGF (Recombinant epidermal growth factor, human), insulin, hFGF-B (Fibroblast growth factor β, human), gentamicin and amphotericin-B. Growing cells were grown in 75 cm ² bottles (Becton Dickinson Labware, NJ, USA).

 Upon reaching 80% confluence, the cells were subcultured as follows: the culture medium was removed and the cells adhered to the bottle were washed with physiological serum. Subsequently, the cells were raised by adding a solution of Trypsin / EDTA (Sigma Aldrich Inc, MO, U. U. U.) to the bottle and incubated for 5 minutes. After the incubation period, growth medium (15% FBS) was added to inhibit the action of trypsin. The cell suspension was centrifuged at 1,500 rpm, for 5 minutes, at 20 ° C. The pellet was resuspended in growth medium, and an aliquot was extracted to count the cells. For studies with CMLV, cells were cultured in 6-well plates (Becton Dickinson Labware, NJ, USA) (Fig. 1).

Count and measure of cell viability.

To count the cells in each subculture, the same amount of Trypan Blue (Sigma Aldrich Inc, MO, USA) was added to the cell suspension aliquot, microliters were placed in a Neubauer Chamber (Fig. 2) and the live cells in each quadrant of 1 mm ² were counted, in the microscope.

Tripan Blue is a colored molecule of great molecular weight that is only able to enter cells that have altered membrane. Therefore a living cell in perfect condition will be colorless while a dead or very altered cell will be blue. In this way, cell viability can be determined.

 (Colorless cells / total cells) x 100 =% cell viability

The Neubauer chamber (Fig. 2) is a counting chamber adapted to the bright-field or phase contrast microscope. It is a slide with a depression in the center. It has engraved in its central part, two microscopic grids with four 4x4 square areas.

The volume of each zone is 1 x 1 x 0, 1 mL. If we count the cells in each area, we can calculate the concentration of the cell suspension according to the following formula: Average of counts of the four zones x 104 x 10 = no. cells / mL

When the cells in culture reached 80% confluence, the phosphate treatment was started. For the induction of calcification in the CMLV model, Monobasic and Dibasic Phosphate was used to prepare the medium high phosphorus. Calcification studies were carried out for 9 days, with a phosphorus concentration of 3.3 mM.

Calcium quantification.

After the end of the experiment time, the wells medium was removed and the wells were washed twice with PBS (Sigma Aldrich Inc, MO, USA). The cells were then decalcified with 0.6 N HCI (Merck, Darmstadt, Germany) for 24 hours, at 37 ° C in a humid atmosphere with 5% C0 ₂ .

The calcium content in the acid extract obtained was determined colorimetrically by the o-Cresoftalein Complexone method (Calcium C Test, WAKO Chemicals GmbH, Neuss, Germany). After decalcification, the cells were solubilized with 0.1 M NaOH (Panreac Química, SA, Barcelona, Spain) and 0.1% SDS (Sigma Aldrich Inc, MO, USA), and raised by the method of scraped off. The protein content in the cell suspension obtained was quantified by the Bradford method (Bio-Rad Protein Assay, Bio-Rad Laboratories, Munich, Germany). The steps taken after finishing the experiment are schematized in Fig. 3.

The effect of angiotensin II on vascular calcification was evaluated independently, as well as in the presence of calcitriol and paricalcitol, after 9 days of treatment. The student's t-test was used to check if the differences in the degree of vascular calcification were statistically significant.

A dose-effect curve was performed (Figure 4) to optimize the dose to be used in subsequent studies. Thus, vascular smooth muscle cells were maintained for 9 days at different doses of angiotensin II: 10 ^"11 M-10 ^{" 4} M.

The levels of calcification ^ grCa / mgrProt) obtained were (n = 11):

0.35 ± 0.04 (control);

 2.70 ± 0.23 (P), p <0.01 vs. control;

1.92 ± 0.15 (P + All 10 ^"11 ); p <0.01 vs. P 1.94 ± 0.10 (P + All 1CP); p <0.01 vs. P

1.89 ± 0.20 (P + All 10 ^"7 ); p <0.01 vs. P

1.06 ± 0.13 (P + All 10 ^"5 ) p <0.01 vs. P

1.34 ± 0.20 (P + All 10 ^"4 ); p <0.01 vs. P

The addition of none of the angiotensin II concentrations alone produced any effect on the controls. The concentration 10 ^"5 M was chosen, concentration at which a greater inhibition of calcification is obtained.

The next step was to evaluate the effect of angiotensin II on P-induced calcification in the presence of 10 ^"8 M calcitriol (calcification inducer) and 3x10 ^{" 8} M paricalcitol (partial inhibitor thereof).

The levels of calcification ^ grCa / mgrProt) obtained, after 9 days of treatment are represented in Figure 5 and in absolute values were (n = 12):

0.55 ± 0.05 (control);

 3.20 ± 0.38 (P), p <0.01 vs. control;

0.69 ± 0.02 (All 10 ^"5 );

1.86 ± 0.38 (P + All 10 ^"5 ); p <0.01 vs P

5.26 ± 0.61 (P + CTR 10 ^"8 ); p <0.01 vs. P.

3.66 ± 0.69 (P + CTR 10 ^"8 + All 10 ^{" 5} ); p <0.01 vs P + CTR.

2.13 ± 0.38 (P + PC 10 ^"8 ); p <0.01 vs P

1.45 ± 0.46 (P + PC 10 ^"8 + All 10-5); pO.01 vs P

These data suggest that angiotensin II reduces calcification induced by elevated levels of P as well as by treatment with calcitriol; however, it did not produce an additional reduction in calcification to that produced by paricalcitol.

PDGF

The role of platelet-derived growth factor on the development of vascular calcifications has been evaluated.

In vitro studies were carried out with vascular smooth muscle (aorta) cells of human origin. Vascular Smooth Muscle Cells (CMLV) from human aorta were obtained from a cell line (Clonetics-Lonza Walkersville, Inc., USA). Cells were studied between 5 and 8 ^{or or} pass. The medium used in the growing cells was SmBm (Smooth muscle Basal medium), supplemented with SmGM-2 SingleQuot Kit Suppl. & Growth Factors (Clonetics- Lonza Walkersville, Inc., USA) containing: Bovine Fetal Serum (15%), hEGF (Recombinant epidermal growth factor, human), insulin, hFGF-B (Fibroblast growth factor β, human), gentamicin and amphotericin-B. Growing cells were grown in 75 cm ² bottles (Becton Dickinson Labware, NJ, USA).

 Upon reaching 80% confluence, the cells were subcultured as follows: the culture medium was removed and the cells adhered to the bottle were washed with physiological serum. Subsequently, the cells were lifted by adding a solution of Trypsin / EDTA (Sigma Aldrich Inc, MO, USA) to the bottle and incubated for 5 minutes. After the incubation period, growth medium (15% FBS) was added to inhibit the action of trypsin. The cell suspension was centrifuged at 1,500 rpm, for 5 minutes, at 20 ° C. The pellet was resuspended in growth medium, and an aliquot was extracted to count the cells. For studies with CMLV, cells were grown in 6-well plates (Becton Dickinson Labware, NJ, USA).

Count and measure of cell viability.

To count the cells in each subculture, the same amount of Trypan Blue (Sigma Aldrich Inc, MO, USA) was added to the cell suspension aliquot, microliters were placed in a Neubauer Chamber (Fig. 2) and the live cells in each quadrant of 1 mm ² were counted, in the microscope.

Tripan Blue is a colored molecule of great molecular weight that is only able to enter cells that have altered membrane. Therefore a living cell in perfect condition will be colorless while a dead or very altered cell will be blue. In this way, cell viability can be determined.

 (Colorless cells / total cells) x 100 =% cell viability

The Neubauer chamber (Fig. 2) is a counting chamber adapted to the bright-field or phase contrast microscope. It is a slide with a depression in the center. It has engraved in its central part, two microscopic grids with four 4x4 square areas.

The volume of each zone is 1 x 1 x 0, 1 mL. If we count the cells in each area, we can calculate the concentration of the cell suspension according to the following formula: Average of counts of the four zones x 104 x 10 = no. cells / mL

When the cells in culture reached 80% confluence, the phosphate treatment was started. For the induction of calcification in the CMLV model, Monobasic and Dibasic Phosphate was used to prepare the medium high phosphorus. Calcification studies were carried out for 9 days, with a phosphorus concentration of 3.3 mM.

Calcium quantification.

After the end of the experiment time, the wells medium was removed and the wells were washed twice with PBS (Sigma Aldrich Inc, MO, USA). The cells were then decalcified with 0.6 N HCI (Merck, Darmstadt, Germany) for 24 hours, at 37 ° C in a humid atmosphere with 5% C02.

The calcium content in the acid extract obtained was determined colorimetrically by the o-Cresoftalein Complexone method (Calcium C Test, WAKO Chemicals GmbH, Neuss, Germany). After decalcification, the cells were solubilized with 0.1 M NaOH (Panreac Química, SA, Barcelona, Spain) and 0.1% SDS (Sigma Aldrich Inc, MO, USA), and raised by the method of scraped off. The protein content in the cell suspension obtained was quantified by the Bradford method (Bio-Rad Protein Assay, Bio-Rad Laboratories, Munich, Germany). The steps taken after finishing the experiment are schematized in Fig. 3.

The effect of platelet-derived growth factor on vascular calcification was observed independently, as well as in the presence of calcitriol and paricalcitol after 9 days of treatment. The student's t-test was used to check if the differences in the degree of vascular calcification were statistically significant.

A dose-effect curve was performed (Fig. 6) to optimize the dose to be used in subsequent studies. Thus, vascular smooth muscle cells were maintained for 9 days at different doses of platelet-derived growth factor:

 - 20 ng / ml.

 40 ng / ml

 - 80 ng / ml.

The calcification levels ^ grCa / mgrProt) obtained were (n = 18): 0.52 ± 0.04 (control);

3.34 ± 0.08 (P), p <0.01 vs. control;

1.15 ± 0.21 (P + PDGF 20), p <0.01 vs. P;

1.83 ± 0.13 (P + PDGF 40), p <0.01 vs P;

1.61 ± 0.08 (P + PDGF 80) p <0.01 vs. P.

The addition of none of the platelet-derived growth factor concentrations alone produced any effect on the controls. The concentration 20 ng / ml was chosen, concentration at which a greater inhibition of calcification is obtained. The next step was to evaluate the effect of platelet-derived growth factor on P-induced calcification in the presence of 10 ^"8 M calcitriol (calcification inducer) and 10 ^{" 8} M paricalcitol (partial inhibitor thereof).

The levels of calcification ^ grCa / mgrProt) obtained, represented in Fig. 7, were (n = 18):

0.33 ± 0.05 (control);

 2.88 ± 0.3 (P), pO.01 vs. control;

 0.41 ± 0.04 (PDGF 20);

 0.66 ± 0.18 (P + PDGF 20); p <0.01 vs P

4.32 ± 0.6 (P + CTR 10 ^"8 ); p <0.01 vs P

1.92 ± 0.18 (P + CTR 10 ^"8 + PDGF 20); p <0.01 vs P + CTR

1.90 ± 0.3 (P + PC 3 x 10 ^"8 ); p <0.01 vs P

1.5 ± 0.18 (P + PC 3 x 10 ^"8 + PDGF 20); p <0.01 vs P

These data suggest that platelet-derived growth factor reduces calcification induced by elevated levels of P as well as by treatment with calcitriol; however, it did not produce an additional reduction in calcification to that produced by paricalcitol. CLAUSES

1. - Use of a selective AT2 receptor agonist in the preparation of a medicine, for the prevention or treatment of vascular calcification.

2. - The use of a selective AT2 receptor agonist according to the preceding claim, wherein the selective AT2 receptor agonist is angiotensin, or any of its biologically active analogues, variants or fragments, or any combination thereof.

3. - The use of a selective AT2 receptor agonist according to any of claims 1-2, wherein the selective AT2 receptor agonist is angiotensin II (A II), or any of its biologically active analogues, variants or fragments, or Any of your combinations.

4. - The use of a selective agonist of the AT2 receptor according to any of claims 1-2, wherein the selective agonist of the AT2 receptor is a peptide whose sequence is constituted by at least three contiguous amino acids of the groups R ¹ to R ⁸ in the sequence of general formula (I):

R ¹ -R ² -R ³ -R ⁴ -R ⁵ -R ⁶ -R ⁷ -R ⁸

Formula (I) in which R ¹ is suitably selected from H, Asp, Glu, Asn, Acpc (1-aminocyclopentanecarboxylic acid), Ala, Me ² Gly, Pro, Bet, Glu (NH ₂ ), Gly, Asp (NH ₂ ) and Suc,

R ² is suitably selected from Arg, Lys, Ala, Orn, Ser (Ac), Sar, D-Arg and D-Lys,

R ³ is selected from the group consisting of Val, Ala, Leu, norLeu, lie, Gly, Pro, Aib, Acpc and Tyr, while Lys has been found to be effective in this remainder;

R ⁴ is selected from the group consisting of Tyr, Tyr (P0 ₃ ) ₂ , Thr, Ser, homoSer, azaTyr, and Ala;

R ⁵ is selected from the group consisting of lie, Ala, Leu, norLeu, Val and Gly;

R ⁶ is His, Arg or 6-NH ₂ -Phe;

R ⁷ is Pro or Ala; Y R is absent or selected from the group consisting of Phe, Phe (Br), lie and Tyr, excluding sequences that include R ⁴ as a terminal Tyr group.

5. - The use of a selective AT2 receptor agonist according to any of claims 1-4, wherein vascular calcification is induced by treatment with at least one vitamin D receptor agonist.

6. - The use of a selective AT2 receptor agonist according to claim 5, wherein the vitamin D receptor agonist is selected from the list consisting of calcitriol, paricalcitol, doxercalciferol and maxacalcitol.

7. - The use of a selective AT2 receptor agonist according to any of claims 1-6, wherein the selective AT2 receptor agonist is in a solution or suspension, and the route of administration is the parenteral route.

8. - The use of a selective AT2 receptor agonist according to the preceding claim, wherein the route of administration is the intravenous route.

9. - The use of a pharmaceutical composition comprising an selective AT2 receptor agonist as described in claims 1-8, and a pharmaceutically acceptable method, in the preparation of a medicament for the prevention or treatment of calcification. vascular.

10. - A pharmaceutical composition comprising a selective agonist of the AT2 receptor, and at least one of the vitamin D receptor, and a pharmaceutically acceptable carrier.

1. The pharmaceutical composition according to the preceding claim, wherein the vitamin D receptor agonist is selected from the list consisting of calcitriol, paricalcitol, doxercalciferol and maxacalcitol.

12. - The pharmaceutical composition according to any of claims 10-11, wherein the vitamin D receptor agonist is calcitriol.

13. - The pharmaceutical composition according to any of claims 10-12, further comprising the platelet-derived growth factor, an analogue, a variant, or a biologically active fragment thereof. 14. - Pharmaceutical form comprising a composition according to any of claims 9-13.

15. - A combined preparation comprising as active ingredients a selective AT2 receptor agonist, a vitamin D receptor agonist.

16. - The combined preparation according to claim 15, wherein the selective agonist of the AT2 receptor is angiotensin, an analogue, a variant or a biologically active fragment thereof.

17. - The combined preparation according to any of claims 15-16, wherein the selective agonist of the AT2 receptor is angiotensin II, an analogue, a variant or a biologically active fragment thereof.

18. - The combined preparation according to any of claims 15-17, wherein the vitamin D receptor agonist is selected from the list consisting of calcitriol, paricalcitol, doxercalciferol and maxacalcitol.

19. The combined preparation according to any of claims 15-18, wherein the vitamin D receptor agonist is calcitriol.

20. - The combined preparation according to any of claims 15-19, further comprising the platelet-derived growth factor, an analogue, a variant or a biologically active fragment thereof.

21. - The separate, simultaneous or sequential use of the active ingredients of the combined preparation according to any of claims 15-20, in the preparation of a medicament.

22. - The separate, simultaneous or sequential use of the active ingredients of the combined preparation according to any of claims 15-20, in the preparation of a medicament for the prevention or treatment of vascular calcification.

Claims

1. - Use of platelet-derived growth factor (PDGF), or any of its biologically active analogues, fragments or variants, in the preparation of a medicament for the prevention or treatment of vascular calcification. 2. - The use of platelet-derived growth factor or any of its biologically active analogues, fragments or variants according to the preceding claim, wherein vascular calcification is induced by treatment with at least one vitamin D receptor agonist. 3. - The use of the platelet derived growth factor according to claim 2, wherein the vitamin D receptor agonist is selected from the list consisting of calcitriol, paricalcitol, doxercalciferol and maxacalcitol. 4. - The use of the platelet derived growth factor or any of its biologically active analogs, fragments or variants according to any of claims 1-3, wherein the platelet derived growth factor or any of its biologically analogs, fragments or variants active is attached to an aptamer. 5. - The use of the platelet-derived growth factor according to any of claims 1-4, wherein the platelet-derived growth factor or any of its biologically active analogues, fragments or variants is in a solution or suspension, and the route of administration is the parenteral route. 6. - The use of the platelet derived growth factor according to the preceding claim, wherein the route of administration is the intravenous route. 7. - The use of a pharmaceutical composition comprising the growth factor derived from plans or any of its biologically active analogs, fragments or vari-like as described in claims 1-6, and a pharmaceutically acceptable carrier, in the development of a medicine for the prevention or treatment of vascular calcification. 8. - A pharmaceutical composition comprising the platelet-derived growth factor, or any of its biologically active analogs, fragments or variants, and at least one vitamin D receptor agonist, and a pharmaceutically acceptable carrier. 9. - The pharmaceutical composition according to the preceding claim, wherein the agonist of the vitamin D receptors is selected from the list consisting of calcitriol, paricalcitol, d Ixex, Icif eral eral and maxacalcitol. 10. - The pharmaceutical composition according to any of claims 8-9, wherein the vitamin D receptor agonist is calcitriol. 1 1. - The pharmaceutical composition according to any of claims 8-10, further comprising angiotensin II. 12. - Pharmaceutical form comprising a composition according to any of claims 8-11. 13. - A combined preparation comprising as active ingredients the platelet-derived growth factor or any of its biologically active analogs, fragments or variants, and at least one vitamin D receptor agonist. 14. - The combined preparation according to claim 13, wherein the agonist of the vitamin D receptors is selected from the list consisting of calcitriol, paricalcitol, d Ixex, Icif eral eral and maxacalcitol. 15. The combined preparation according to any of claims 13-14, wherein the vitamin D receptor agonist is calcitriol. 16. - The combined preparation according to any of claims 13-15, further comprising angiotensin II. 17. - The separate, simultaneous or sequential use of the active ingredients of the combined preparation according to any of claims 13-16, in the preparation of a medicament. 18. - The use, separately, simultaneously or sequentially, of the active ingredients of the combined preparation according to any of claims 13-16, in the preparation of a medicament for the prevention or treatment of vascular calcification. 19. - Use of a selective agonist of the AT2 receptor in the preparation of a drug, for the prevention or treatment of vascular calcification. 20. - The use of a selective AT2 receptor agonist according to the preceding claim, wherein the selective AT2 receptor agonist is angiotensin, or any of its biologically active analogues, variants or fragments, or any combination thereof. 21. - The use of a selective AT2 receptor agonist according to any of claims 19-20, wherein the selective AT2 receptor agonist is angiotensin II (A II), or any of its biologically active analogues, variants or fragments, or Any of your combinations. 22. - The use of a selective AT2 receptor agonist according to any of claims 19-21, wherein the selective AT2 receptor agonist is a peptide whose sequence is constituted by at least three contiguous amino acids of groups R ¹ to R ⁸ in the sequence of general formula (I): R ¹ -R ² -R ³ -R ⁴ -R ⁵ -R ⁶ -R ⁷ -R ⁸ Formula (I) in which R ¹ is suitably selected from H, Asp, Glu, Asn, Acpc (1-aminocyclopentanecarboxylic acid), Ala, Me ² Gly, Pro, Bet, Glu (NH ₂ ), Gly, Asp (NH ₂ ) and Suc, R ² is suitably selected from Arg, Lys, Ala, Orn, Ser (Ac), Sar, D-Arg and D-Lys, R ³ is selected from the group consisting of Val, Ala, Leu, norLeu, lie, Gly, Pro, Aib, Acpc and Tyr, while Lys has been found to be effective in this remainder; R ⁴ is selected from the group consisting of Tyr, Tyr (P0 ₃ ) ₂ , Thr, Ser, homoSer, azaTyr, and Ala; R ⁵ is selected from the group consisting of lie, Ala, Leu, norLeu, Val and Gly; R ⁶ is His, Arg or 6-NH ₂ -Phe; R ⁷ is Pro or Ala; Y R ⁸ is absent or selected from the group consisting of Phe, Phe (Br), lie and Tyr, excluding sequences that include R ⁴ as a terminal Tyr group. 23. - The use of a selective AT2 receptor agonist according to any of claims 19-22, wherein vascular calcification is induced by treatment with at least one vitamin D receptor agonist. 24. - The use of a selective AT2 receptor agonist according to claim 23, wherein the vitamin D receptor agonist is selected from the list consisting of calcitriol, paricalcitol, doxercalciferol and maxacalcitol. 25. - The use of a selective AT2 receptor agonist according to any of claims 19-24, wherein the selective AT2 receptor agonist is in a solution or suspension, and the route of administration is the parenteral route. 26. - The use of a selective AT2 receptor agonist according to the preceding claim, wherein the route of administration is the intravenous route. 27. - The use of a pharmaceutical composition comprising a selective AT2 receptor agonist and a pharmaceutically acceptable carrier, in the preparation of a medicament for the prevention or treatment of vascular calcification. 28. - A pharmaceutical composition comprising a selective AT2 receptor agonist, and at least one vitamin D receptor agonist, and a pharmaceutically acceptable carrier. 29. The pharmaceutical composition according to the previous claim, wherein the vitamin D receptor agonist is selected from the list consisting of calcitriol, paricalcitol, doxercalciferol and maxacalcitol. 30. - The pharmaceutical composition according to any of claims 28-29, wherein the vitamin D receptor agonist is calcitriol. 31. The pharmaceutical composition according to any of claims 28-30, which further comprises the platelet-derived growth factor, an analogue, a variant, or a biologically active fragment thereof. 32. Pharmaceutical form comprising a composition according to any of claims 28-31. 33. - A combined preparation comprising as active ingredients a selective AT2 receptor agonist, a vitamin D receptor agonist. 34. - The combined preparation according to claim 33, wherein the selective agonist of the AT2 receptor is angiotensin, an analogue, a variant or a biologically active fragment thereof. 35. - The combined preparation according to any of claims 33-34, wherein the selective agonist of the AT2 receptor is angiotensin II, an analogue, a variant or a biologically active fragment thereof. 36. - The combined preparation according to any of claims 33-35, wherein the vitamin D receptor agonist is selected from the list consisting of calcitriol, paricalcitol, doxercalciferol and maxacalcitol. 37. The combined preparation according to any of claims 33-36, wherein the vitamin D receptor agonist is calcitriol. 38. - The combined preparation according to any of claims 33-37, further comprising the platelet-derived growth factor, an analogue, a variant or a biologically active fragment thereof. 39. - The separate, simultaneous or sequential use of the active ingredients of the combined preparation according to any of claims 33-38, in the preparation of a medicament. 40. - The separate, simultaneous or sequential use of the active ingredients of the combined preparation according to any of claims 33-39, in the preparation of a medicament for the prevention or treatment of vascular calcification.