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WO2012123613A1 - Utilisation d'inhibiteurs de récepteurs s1p pour le traitement de la sténose aortique calcifiée - Google Patents

Utilisation d'inhibiteurs de récepteurs s1p pour le traitement de la sténose aortique calcifiée Download PDF

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Publication number
WO2012123613A1
WO2012123613A1 PCT/ES2012/070170 ES2012070170W WO2012123613A1 WO 2012123613 A1 WO2012123613 A1 WO 2012123613A1 ES 2012070170 W ES2012070170 W ES 2012070170W WO 2012123613 A1 WO2012123613 A1 WO 2012123613A1
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seq
sequence
use according
receptors
inhibitor
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Spanish (es)
Inventor
Mª Carmen GARCÍA RODRÍGUEZ
Mariano SÁNCHEZ CRESPO
José Alberto SAN ROMÁN CALVAR
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UNIVERSIDAD DE VALLADOLID (30%)
Consejo Superior de Investigaciones Cientificas CSIC
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UNIVERSIDAD DE VALLADOLID (30%)
Consejo Superior de Investigaciones Cientificas CSIC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/133—Amines having hydroxy groups, e.g. sphingosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]

Definitions

  • the present invention is within the field of biomedicine. Specifically, it refers to the use of at least one sphingosine-1-phosphate (S1 P) receptor inhibitor, preferably S1 Pi, SI P 2 , SI P3, or SI P 4 receptors, for the preparation of a medicament. for the prevention and / or treatment of calcified aortic stenosis.
  • S1 P sphingosine-1-phosphate
  • Degenerative calcific aortic stenosis is the most common in developed countries valvular disease, is associated with high morbidity and mortality and its incidence is expected to increase due to the progressive aging of the population.
  • Calcified aortic stenosis is a degenerative disease characterized by fibrosis and calcification of the aortic valve that can impede the normal development of the patient's life; it is the most frequent valvulopathy in developed countries and its prevalence increases with age (Blase A, et al. 2009, Lancet, 373: 956-66; Yetkin E, et al. 2009, Int J Cardiol, 135: 4-13 ; O'Brien KD. 2006, Arterioscler Thromb Vasc Biol, 26: 1721-1728; Newby DE, et al. 2006, Heart, 92: 729-734).
  • This disease evolves slowly, although when the symptoms appear, the progression is rapid and if there is no valve replacement before 3 years the mortality rate exceeds 50%.
  • Several aspects of its pathogenesis are known and several pro-inflammatory and pro-osteogenic molecules with relevant role in the disease have been described, although the pharmacological target for application in the clinic has not yet been found.
  • the contribution of chronic inflammation and calcification processes has been described both at the onset and in the progression of the disease. In valvular lesions, infiltration of inflammatory cells, deposits of oxidized lipids, and the expression of cytokines and metalloproteinases have been detected.
  • the heart valves consist of an extracellular matrix and the cellular content is formed by endothelial cells and interstitial cells, the latter with the ability to differentiate in vitro osteoblasts under the influence of different mediators, and are thought to be the candidates to promote the characteristic calcification of the disease (Osman L, et al. 2006, Circulation, 1 14 [suppl I]: I-547-I-552 ).
  • the pathogenesis of calcified aortic stenosis presents etiopathogenic similarities with the atherosclerotic process (Yetkin E, et al. 2009, Int J Cardiol, 135: 4-13; O'Brien KD.
  • statins that inhibit HMCoA reductase and reduce cholesterol levels.
  • statins despite the therapeutic potential shown in animal models and in a preliminary study in patients (Yetkin E, et al. 2009, Int J Cardiol, 135: 4-13, Moura et al.
  • S1 P sphingosine 1-phosphate
  • S1 P The functions of S1 P vary according to the cell type and the receptor subtype expressed, and it is known that in the heart it plays a role in cell survival and is cardioprotective in experimental models of ischemia / reperfusion (Means and Brown, 2009, Cardiovasc Res , 82: 193-200; Alewijnse et al., 2008. Eur J Pharmacol., 585: 292-302).
  • S1 P binds to membrane receptors called S1 P receptors, or EDG according to the old nomenclature;
  • S1 P / EDG receptors have been described: S1 Pi / EDG1, S1 P 2 / EDG5, S1 P 3 / EDG3, SI P 4 / EDG6, SI P 5 / EDG8 (Rivera J, et al. 2008, Nat Rev Immunol, 8: 753-763; Chun J, et al. 2010, Pharmacol Rev, 62: 579-587).
  • S1 Pi / EDG1 membrane receptors
  • S1 P 2 / EDG5 membrane receptors
  • SI P 4 / EDG6 SI P 5 / EDG8
  • the present invention relates to the use of S1 P receptor inhibitors, preferably an antagonist or a siRNA of said receptors for the preparation of a medicament for the prevention and / or treatment of calcified aortic stenosis.
  • S1 P receptor inhibitors preferably an antagonist or a siRNA of said receptors for the preparation of a medicament for the prevention and / or treatment of calcified aortic stenosis.
  • the receivers are S1 Pi, SI P2,
  • the present invention provides a solution to the problem of prevention and / or treatment of this disease through the use of inhibitors of S1 P receptors, preferably of S1 Pi, S1 P 2 , S1 P 3 , or S1 P 4 receivers.
  • S1 P receptors preferably of S1 Pi, S1 P 2 , S1 P 3 , or S1 P 4 receivers.
  • the results provide a new therapeutic target that can be used to prevent and / or reduce both chronic inflammation and calcification associated with calcified aortic stenosis.
  • the present invention has the following advantages over the state of the art: - In the present invention it has been shown that there is a synergistic effect on the activation of the S1 Pi, SI P 2 , SI P3, or S1 P 4 receptors in conjunction with the innate immunity receptor type Toll (TLR) -4, resulting in an exponential increase in cyclooxygenase (COX) -2, recognized anti-inflammatory marker, in interstitial cells of heart valves. Blocking this synergy decreases COX-2 mediated inflammation, and could block the inflammation associated with calcified aortic stenosis.
  • TLR innate immunity receptor type Toll
  • the present invention demonstrates an additive effect of S1 P receptors together with the TLR4 receptor in regard to in vitro calcification associated with aortic stenosis, thereby blocking S1 P receptors, preferably S1 receptors Pi, SI P 2 or SI P3 further decreases said calcification.
  • the present invention provides the use of sphingosine 1-phosphate (S1 P) receptor antagonists, preferably S1 Pi, SI P 2 , SI P3 or SI P4 receptors to simultaneously block inflammation and calcification that they underlie calcified aortic stenosis and as a consequence, to prevent and / or treat said disease, for which there is currently no effective pharmacological therapy.
  • S1 P sphingosine 1-phosphate
  • One aspect of the present invention relates to the use of at least one inhibitor of an S1 P receptor (the article “a” should be construed as an indefinite article), that is, the use of at least one inhibitor of the S1 Pi receptor, SI P 2 , YES P3, YES P4 or YES P5, or any combination of inhibitors, for the preparation of a medicament for the prevention and / or treatment of calcified aortic stenosis.
  • the term "use of the present invention” or “use of the invention” may be used to refer to the use described in this paragraph.
  • the present invention shows results of inhibition of the S1 P 4 receptor with interference RNA technology.
  • the inhibition of the function of SI P5 by means known to the person skilled in the art, that is, by means that are not require an inventive step, or undue experimentation, such as the design of interfering RNA capable of inhibiting or decreasing its activity, provide the same technical effect described for the inhibition of the rest of the receptors.
  • the inhibitors can be specific to one type of S1 P receptor, preferably S1 Pi receptors, SI P 2 , SI P3 or S1 P 4, can inhibit two, three or four of them.
  • the inhibition can occur in an optimum concentration range of the corresponding inhibitor. That is, while a concentration range can inhibit one receptor, it is possible that inhibition of another receptor occurs at a concentration that is not in that range.
  • S1 P receptor inhibitor refers to a molecule that binds to any of the S1 P receptors, preferably of the S1 Pi, S1 P 2 , S1 P 3 receptors. or S1 P 4 and decreases the expression and / or activity of the receptor to which it binds, and / or its intracellular signaling.
  • the inhibitor is selected from the list comprising, but not limited to, antagonists (preferably chemical), silencing RNA or antibody specific for the corresponding receptor (preferably the antibody is monoclonal), in the present invention this antibody may be referred to as an antibody. Neutralizing the effect of S1 P.
  • the present invention should not be limited to the use of the specific inhibitors tested but should refer to other known inhibitors for the S1 Pi, SI P2, SI P3 or S1 P 4 receptors on the date of presentation of the present invention.
  • the present invention contributes to the state of the art that the inhibition of said receptors, by any means or product, is useful in the prevention and / or treatment of calcified aortic stenosis.
  • calcified aortic stenosis refers to a degenerative disease of the aortic valve caused by degenerative calcification of the aortic cusps, that is, calcified aortic stenosis is characterized by thickening and calcification of the aortic valve leaflets and the consequent narrowing of the valvular orifice of the heart, thus hindering the flow of blood from the left ventricle to the aorta with respect to a healthy individual who does not suffer from said disease.
  • treatment as understood in the present invention refers to combating the effects caused as a result of a disease or pathological condition of interest in a subject (preferably mammal, and more preferably a human) that includes:
  • prevention consists in preventing the onset of the disease, that is, preventing the disease or pathological condition from occurring in a subject (preferably mammal, and more preferably a human), in particularly, when said subject has a predisposition for the pathological condition.
  • the medicament referred to in the present invention can be for human or veterinary use.
  • the "medicine for human use” is any substance or combination of substances that is presented as having properties for the treatment or prevention of diseases in humans or that can be used in humans or administered to humans in order to restore, correct or modify physiological functions by exerting a pharmacological, immunological or metabolic action, or establishing a medical diagnosis.
  • the "veterinary drug” is any substance or combination of substances presented for treating or preventing concerning animal disease or may be administered to animals in order to restore, correct or modify physiological functions by exerting a pharmacological, immunological or metabolic action, or to establish a veterinary diagnosis. "Premixes for medicated feed" prepared to be incorporated into a feed will also be considered “veterinary medicinal products”.
  • the medicament of the present invention comprises at least one pharmaceutically acceptable carrier and / or excipient.
  • excipient refers to a substance that aids the absorption of any of the components of the composition of the present invention, stabilizes said components or aids in the preparation of the pharmaceutical composition in the sense of giving it consistency or providing flavors that Make it more enjoyable.
  • the excipients could have the function of keeping the components together such as starches, sugars or cellulose, sweetening function, dye function, drug protection function such as to isolate it from air and / or moisture, function filling a tablet, capsule or any other form of presentation such as dibasic calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph. Therefore, the term "excipient” is defined as that matter which, included in the "galenic forms", is added to the active principles or to their associations to enable their preparation and stability, modify their organoleptic properties or determine the physicochemical properties of the pharmaceutical composition and its bioavailability.
  • the "pharmaceutically acceptable" excipient must allow the activity of the compounds of the pharmaceutical composition, that is, to be compatible with said components.
  • galenic form or pharmaceutical form is the provision to which the active ingredients and excipients are adapted to constitute a medicament. It is defined by the combination of the way in which the pharmaceutical composition is presented by the manufacturer and the way in which it is administered.
  • the "vehicle” or carrier is preferably an inert substance.
  • the function of the vehicle is to facilitate the incorporation of other compounds, allow a better dosage and administration or give consistency and form to the pharmaceutical composition. Therefore, the carrier is a substance that is used in the medicament to dilute any of the components of the pharmaceutical composition of the present invention to a certain volume or weight; or that even without diluting said components it is capable of allowing a better dosage and administration or giving consistency and form to the medicine.
  • the pharmaceutically acceptable carrier is the diluent.
  • the excipient and the vehicle must be pharmacologically acceptable, that is, the excipient and the vehicle are allowed and evaluated so as not to cause damage to the organisms to which it is administered.
  • the disclosed medicament in addition to said vehicle and / or excipient, optionally comprising another active substance.
  • said pharmaceutical composition may require the use of other therapeutic agents, there may be additional fundamental reasons that compel or strongly recommend the use of a combination of a compound of the invention and another therapeutic agent.
  • active principle is any matter, whatever its origin, human, animal, plant, chemical or other, to which an appropriate activity is attributed to constitute a medicine.
  • the composition of the present invention can be presented in the form of solutions or any other form of clinically permitted administration and in a therapeutically effective amount.
  • the medicament described in the invention can be formulated in solid, semi-solid, liquid or gaseous forms, such as tablet, capsule, powder, granule, ointment, solution, suppository, injection, inhalant, gel, microsphere or aerosol.
  • the medicine can be presented in a form adapted to oral, sublingual, nasal, intracatecal, bronchial, lymphatic, rectal, transdermal or inhaled administration, but not limited to these forms.
  • the inhibitor of the S1 P receptors of the present invention preferably of the S1 Pi, SI P2, SI P3 or S1 P 4 receptors, can be associated, for example, but not limited, with liposomes or micelles.
  • a preferred embodiment of the present invention relates to the use of the invention, wherein the inhibitor is an antagonist, or any of its pharmaceutically acceptable salts, isomers or solvates.
  • the antagonist of the The present invention is of the chemical type.
  • the antagonist is a compound that binds to an S1 P receptor, preferably to the S1 Pi, SI P2, SI P3 or SI P4 receptors, and acts as a functional antagonist by partially or totally blocking its activation by the agonists.
  • the antagonist of the present invention may exist in the form of enantiomers or diastereomers.
  • the present invention also contemplates the use of antagonist solvates (such as, but not limited to, hydrates), prodrugs (synonymous with prodrugs), or clathrates.
  • Pharmaceutically acceptable salts are selected from chloride, iodide bromide or any other pharmaceutically acceptable salt.
  • the S1 P receptor antagonists of the present invention may include isomers, depending on the presence of multiple bonds (e.g., Z, E), including optical isomers or enantiomers, depending on the presence of chiral centers.
  • the individual isomers, enantiomers or diastereoisomers and mixtures thereof fall within the scope of the present invention.
  • the individual enantiomers or diastereoisomers, as well as mixtures thereof, can be separated by conventional techniques.
  • the term "pharmaceutically acceptable salts" of the antagonist compounds refers to salts prepared of pharmaceutically acceptable non-toxic acids, including organic or inorganic acids.
  • Non-toxic organic or inorganic acids are selected from the list comprising, but not limited to: acetic, alginic, anthranilic, benzenesulfonic, benzoic, camforsulfonic, citric, ethanesulfonic, formic, fumaric, furoic, gluconic, glutamic, glucorhenic, galacturonic, glycidic , hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phenylacetic, propionic, phosphoric, salicylic, stearic, succinic, sulfanyl, sulfuric, tartaric or p-toluenesulfonic
  • the compound of the invention may be in crystalline form as free compounds or as solvates.
  • the term "solvate”, as used herein, includes both pharmaceutically and pharmacologically acceptable solvates, that is, solvates of the S1 P receptor antagonist, preferably of the S1 Pi, S1 P 2 , S1 P receptors 3 or S1 P 4, which may be used in the manufacture of a medicament, such as pharmaceutically acceptable solvates, which may be useful in the preparation of pharmaceutically and pharmacologically acceptable solvates or salts.
  • the nature of the pharmaceutically acceptable solvate is not critical as long as it is pharmaceutically acceptable.
  • the solvate is a hydrate. Solvates can be obtained by conventional solvation methods known to those skilled in the art.
  • prodrugs or prodrugs of S1 P receptor antagonists preferably S1 Pi, SI P 2 , SI P3 or S1 P 4 receptors .
  • the term "prodrug” or “prodrug. "as used herein includes any compound derived from the antagonist, for example and not limited to: esters (including carboxylic acid esters, amino acid esters, phosphate esters, metal salt sulphonate esters, etc.), carbamates, amides, biohydrolysable amides, biohydrolysable esters, biohydrolysable carbamates, biohydrolysable ureides, biohydrolysable phosphates.
  • prodrugs include compounds comprising groups -NO, -NO 2 , -ONO, or -ONO 2 - which when administered to an individual can be transformed directly or indirectly into said antagonist in said individual.
  • said derivative is a compound that increases the bioavailability of the antagonist when administered to an individual or that enhances the release of the antagonist 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 antagonist of S1 P receptors, preferably S1 Pi, SI P 2 , SI P3 or S1 P 4 receptors , in a compartment Biological of the same.
  • the preparation of said prodrug can be carried out by conventional methods known to those skilled in the art.
  • biohydrolyzable amides refer to carbamate, carbonate, ureido and phosphate, respectively, of a compound that: 1) does not interfere with the biological activity of the complex but which gives the compound advantageous properties in vivo, as absorption , duration of effect, or onset of effect; or 2) it is biologically inactive, but it is converted in vivo to a biologically active compound.
  • the antagonist is selected from the list comprising FTY720, VPC23019, VPC25239, VPC01091, VPC 23153, Ascotricin A, Ascotricin B, JTE013, W146, W123, BML-241, Suramine or pertussis toxin, or any of its salts, isomers or pharmaceutically acceptable solvates.
  • the antagonist is selected from the list comprising VPC23019, JTE013, Suramine, W146, FTY720 or pertussis toxin, or any of its pharmaceutically acceptable salts, isomers or solvates.
  • FTY720 (2-amino-2- [2- (4-octylphenyl) ethyl] -propane-1,3-diol) (salts: hydrochloride, phosphate), also called "fingolimod” is an immunosuppressive agent that regulates the lymphocyte migration through its interaction with S1 Pi, although it can bind to all S1 P / EDG receptors with the exception of S1 P 2 (Takabe K et al. 2008, Pharmacol Rev, 60: 181-195; Dong-Soon I, 2010, Acta Pharmacol S ⁇ nica, 31: 1213-1222).
  • FTY720 behaves as a functional antagonist of S1 Pi, since treatment with FTY720 in vivo induces internalization and degradation of S1 Pi expression, thereby acting as a selective non-competitive inhibitor of S1 receptors Pi (MH Gráler and EJ Goetzl, 2004. FASEB J, 18: 551-553; Matloubian et al., 2004. Nature, 427: 355-360; Dong-Soom I, 2010. Acta Pharmacol S ⁇ nica, 31: 1213-1222 )
  • VPC23019 ((R) -phosphoric acid mono- [2-amino-2- (3-octyl-phenylcarbamoyl) -ethyl] ester) is a non-competitive selective antagonist of S1 Pi and SI P3 (Takabe K et al. 2008. Pharmacol Rev, 60: 181-195; Dong-Soon I, 2010. Acta Pharmacol S ⁇ nica, 31: 1213-1222).
  • VPC25239 is a selective antagonist S1 Pi and S1 P 3 (Takabe K et al. 2008. Pharmacol Rev, 60: 181-195; Dong-Soon I, 2010. Acta Pharmacol S ⁇ nica, 31: 1213-1222).
  • VPC-01091 -P.1 - [1-Amino-3- (4-octylphenyl) cyclopentyl] methanol-phosphate is an SI P3 receptor antagonist; Dong-Soon I, 2010. Acta Pharmacol
  • VPC 23153 ((R) -phosphoric acid mono- [2-amino-2- (6-octyl-1 H-benzoimiazol-2- yl) ethyl] ester), is a competitive agonist of the S1 P 4 receptor (Skoura A , Hla T. 2009. J Lipid Res 50: S293-298)
  • Ascotricin A and B are inhibitors of S1 Pi receptors (Ascotricin A and Ascotricin B, the term is more widespread in English than in Spanish, which is why it is mentioned in this application) (Yonesu K, et al. J Antibiot , 2009, 62: 359-64; Dong-Soon I, 2010. Acta Pharmacol S ⁇ nica, 31: 1213-1222).
  • JTE-013 may not be such a selective compound, at least in rodent models, since its inhibitory effect of vascular contraction is observed in both mice deficient in SI P 2 and in those not deficient (Salomone S et al. 2008. Br J Pharmacol, 153: 140-7).
  • W146 (3-amino-4- (3-hexylphenylamino) -4-oxobutyl phosphonic acid) is a specific antagonist of S1 P1.
  • the structure of the trifluoroacetate salt is:
  • BML-241 (2-undecyl-thiazolidine-4-carboxylic acid), also referred to as CAY10444. It is a selective SI P3 receptor antagonist, blocking the increase in calcium in HeLa cells by 40% at a concentration of 10 ⁇ . Its chemical structure is as follows:
  • Toxin pertussis This toxin is an exotoxin based on AB5 type proteins produced by Bordetella pertussis bacteria.
  • B. pertussis toxin catalyzes the ADP-ribosylation of the alpha chains of heterotrimeric G proteins, preventing their interaction with receptors, and therefore blocking the intracellular signaling of G-protein coupled receptors, including S1 P receptors (Goodemote KA et al. J Biol Chem, 1995. 270: 10272-7.). It has been described that in the cardiovascular system the pertussis toxin inhibits the signaling of the S1 Pi and SI P3 receptors and partially that of SI P 2 (Takuwa And et al.
  • the inhibitor is a silencing RNA or what is the same, an interfering RNA (siRNA).
  • the siRNA is a double stranded RNA whose sequence is SEQ ID NO: 1 1 (5 ' -GCCCACUUUAUCUAAAUGA- 3 ' ).
  • double stranded RNA is formed by a sense chain and an antisense chain, wherein said chains are complementary sequences to each other.
  • the siRNA is a double stranded RNA sequence whose antisense chain is SEQ ID NO: 12 (5 ' -CGGGUGAAAUAGAUUUACU-3 ' ), which is fully hybridized with SEQ ID NO: 1 1.
  • this preferred embodiment of the present invention relates to the use of the double-stranded siRNA sequence SEQ ID NO: 11 / SEQ ID NO: 12 for the preparation of a medicament for the prevention and / or treatment of aortic stenosis calcified
  • the siRNA is a double stranded RNA whose sequence is SEQ ID NO: 13 (5 ' -GGUGGCCAACCACAACAAC-3 ' ).
  • the siRNA is a double stranded RNA sequence whose antisense chain is SEQ ID NO: 14 (5 ' -CCACCGGUUGGUGUUGUUG-3 ' ), which is fully hybridized with SEQ ID NO: 13. That is, this preferred embodiment of the present invention relates to the use of the double-stranded siRNA sequence SEQ ID NO: 13 / SEQ ID NO: 14 for the preparation of a medicament for the prevention and / or treatment of calcified aortic stenosis.
  • the siRNA is a double stranded RNA whose sequence is SEQ ID NO: 15 (5 ' -GCCGCGUCUACGGCUUCAU-3 ' ).
  • the siRNA is a double stranded RNA sequence whose chain antisense is SEQ ID NO: 16 (5 ' -CGGCGCAGAUGCCGAAGUA-3 ' ), which is fully hybridized with SEQ ID NO: 15. That is, this preferred embodiment of the present invention relates to the use of the double-stranded siRNA sequence.
  • SEQ ID NO: 15 / SEQ ID NO: 16 for the preparation of a medicament for the prevention and / or treatment of calcified aortic stenosis.
  • the siRNA is a double stranded RNA whose sequence is SEQ ID NO: 35 (5 ' -GCAAGUUCCACUCGGCAAU-3 ' ).
  • the siRNA is a double stranded RNA sequence whose antisense chain is SEQ ID NO: 36 (5 ' -CGUUCAAGGUGAGCCGUUA-3 ' ), which is fully hybridized with SEQ ID NO: 35. That is, this preferred embodiment of the present invention relates to the use of the double-stranded siRNA sequence SEQ ID NO: 35 / SEQ ID NO: 36 for the preparation of a medicament for the prevention and / or treatment of calcified aortic stenosis.
  • the nucleotides (tt) or (tg) can be added at the 3 ' end of the sense chain or at the 5 ' end of the antisense chain of the siRNA of the present invention. In this way these nucleotides are outgoing, not hybridizing with any other nucleotide of the complementary chain. The addition of these nucleotides at the 3 ' or 5 ' ends does not affect the recognition of the corresponding messenger RNA.
  • the siRNAs resulting from said addition are: - SEQ ID NO: 1 1 -tt (SEQ ID NO: 26): That is, SEQ ID NO: 26 has the sequence 5 ' -GCCCACUUUAUCUAAAUGAtt-3 ' / SEQ ID NO: 12-tt (SEQ ID NO: 27): That is, SEQ ID NO: 27 has the sequence 5 '- ttCGGGUGAAAUAGAUUUACU-3'.
  • SEQ ID NO: 28 has the sequence 5 ' -GGUGGCCAACCACAACAAC-tt-3 ' / tt-SEQ ID NO: 14 (SEQ ID NO:
  • SEQ ID NO: 29 That is, SEQ ID NO: 29 has the sequence 5 '- ttCCACCGGUUGGUGUUGUUG-3'.
  • SEQ ID NO: 30 has the sequence 5 ' -GCCGCGUCUACGGCUUCAUtt-3 ' / gt-SEQ ID NO: 16 (SEQ ID NO: 31): That is, SEQ ID NO: 31 has the sequence 5 '- gtCGGCGCAGAUGCCGAAGUA-3'.
  • SEQ ID NO: 40 has the sequence 5 ' -GCAAGUUCCACUCGGCAAUtt-3 ' / gt-SEQ ID NO: 36 (SEQ ID NO: 41): That is, SEQ ID NO: 41 has the sequence 5 '- gtCGUUCAAGGUGAGCCGUUA-3'.
  • An even more preferred embodiment relates to the use of the invention in which the double-stranded siRNA sequence is encoded in a DNA sequence formed by two sequences, A and B, where sequence A is linked by the 3 ' end to the 5 ' end of sequence B, forming an AB sequence. That is, this embodiment of the present invention relates to the use of the AB sequence or preferably to the use of the expression vector comprising said AB sequence, for the preparation of a medicament for the prevention and / or treatment of calcified aortic stenosis. In this sense several possibilities are contemplated:
  • A is SEQ ID NO: 17 (5 ' - GCCCACTTTATCTAAATGA-3 ' ) and B is SEQ ID NO: 18 (5 ' - TCATTTAGATAAAGTGGGC-3 ' ).
  • sequence SEQ ID NO: 19 would be formed; 5 ' -GCCCACTTTATCTAAATGA-TCATTTAGATAAAGTGGGC-3 ' (SEQ ID NO: 17-SEQ ID NO: 18).
  • sequences A and B have nucleotides (tt) attached to the 3 ' end, resulting in sequence SEQ ID NO: 32; 5 ' -GCCCACTTTATCTAAATGAtt- TCATTTAGATAAAGTGGGCtt-3 ' .
  • A is SEQ ID NO: 20 (5 ' - GGTGGCCAACCACAACAAC-3 ' ) and B is SEQ ID NO: 21 (5 ' - GTTGTTGTGGTTGGCCACC-3 ' ).
  • sequence SEQ ID NO: 22 would be formed; 5 ' -GGTGGCCAACCACAACAAC-GTTGTTGTGGTTGGCCACC-3 ' (SEQ ID NO: 20-SEQ ID NO: 21).
  • sequences A and B have nucleotides (tt) attached to the 3 ' end, resulting in sequence SEQ ID NO: 33; 5 ' -GGTGGCCAACCACAACAACtt-
  • A is SEQ ID NO: 23 (5 ' - GCCGCGTCTACGGCTTCAT-3 ' ) and B is SEQ ID NO: 24 (5 ' - ATGAAGCCGTAGACGCGGC-3 ' ).
  • sequence SEQ ID NO: 25 would be formed; 5 ' -GCCGCGTCTACGGCTTCAT-ATGAAGCCGTAGACGCGGC-3 ' (SEQ ID NO: 23-SEQ ID NO: 24).
  • sequence A has the nucleotides (tt) attached to the 3 ' end and the sequence B has the nucleotides (tg) attached to the 3 ' end, resulting in the sequence SEQ ID NO: 34; 5 ' - GCCGCGTCTACGGCTTCATtt-ATGAAGCCGTAGACGCGGCtg-3 ' .
  • A is SEQ ID NO: 37 (5 ' - GCAAGTTCCACTCGGCAAT-3 ' ) and B is SEQ ID NO: 38 (5 ' - ATTGCCGAGTGGAACTTGC-3 ' ).
  • sequence SEQ ID NO: 39 would be formed; 5 ' -GCAAGTTCCACTCGGCAAT-ATTGCCGAGTGGAACTTGC-3 ' (SEQ ID NO: 37-SEQ ID NO: 38).
  • sequence A has the nucleotides (tt) attached to the 3 ' end and the sequence B has the nucleotides (tg) attached to the 3 ' end, resulting in the sequence SEQ ID NO: 42; 5 ' - GCAAGTTCCACTCGGCAATtt-ATTGCCGAGTGGAACTTGCtg-3 ' .
  • sequences A and B are complementary sequences and as a consequence, the RNA transcribed from it will hybridize with itself forming a hairpin, that is, forming a hairpinRNA (hpRNA).
  • the AB DNA sequence encoding the transcribed RNA must be integrated into a suitable expression system for the transcription to take place from the AB DNA sequence.
  • this expression system can be an expression vector in which the DNA AB sequence is inserted between a gene expression regulatory sequence (such as but not limited to, a promoter) and a transcription terminator sequence, so that the transcription that takes place yields any of the siRNA fragments of interest, described in the present invention.
  • expression vector refers to a DNA fragment that has the ability to replicate in a given host and can serve as a vehicle to carry out the transcription of a sequence of interest that has been inserted therein.
  • the vector can be a plasmid, a cosmid, a bacteriophage or a viral vector, without excluding other types of vectors that correspond to the definition made of vector.
  • siRNA fragments of the present invention transcribed by the described expression system are a type of double-stranded RNA (dsRNA) that is recognized and cut by an endoribonuclease, the Dicer endoribonuclease, resulting in fragments.
  • dsRNA double-stranded RNA
  • siRNAs that cause post-transcriptional silencing of the target nucleotide sequences, leading to the degradation of the corresponding messenger RNA, so that the protein resulting from the expression of the messenger RNA sequences is not obtained.
  • siRNAs double-stranded RNA
  • thermodynamic characteristics of the 5 'end of the siRNA determine which of the two strands is incorporated into the RISC complex. Normally, the one with less stability at the 5 'end is incorporated as a guide thread.
  • the guide strand For post-transcriptional silencing to occur, the guide strand must be complementary to the messenger RNA that is intended to be silenced.
  • the RISC complex binds to the complementary RNA of the siRNA guide strand present in the complex and the messenger RNA is cut.
  • the spacer sequence is a non-coding sequence.
  • the spacer sequence may be part of an intron sequence of a gene or the complete sequence of said intron.
  • the function of the spacer sequence is to act as a hinge of the described sequence pairs so that the pairing or hybridization of the RNA sequences encoded by the polynucleotide can occur.
  • Another preferred embodiment relates to the use of any of the S1 P receptor inhibitors of the present invention, in combination with at least one Toll 4 (TLR-4) innate immunity receptor inhibitor for the preparation of a medicament for the prevention and / or treatment of calcified aortic stenosis.
  • TLR-4 Toll 4
  • TLR-4 receptor inhibitor refers to a molecule that binds to any of the TLR-4 receptors, preferably of the TLR-4 type receptors and decreases the expression y / or activity of the receptor to which it binds, and / or its intracellular signaling.
  • the inhibitor is selected from the list comprising, but not limited to, antagonists (preferably chemical), silencing RNA or antibody specific for the corresponding receptor (preferably the antibody is monoclonal).
  • the TLR-4 inhibitor is an antagonist, or any of its pharmaceutically acceptable salts, isomers or solvates.
  • the antagonist of the present invention is of the chemical type.
  • the antagonist is a compound which binds to a TLR-4 receptor, and acts as a functional antagonist by partially or totally blocking its activation by agonists.
  • the TLR-4 antagonist may exist in the form of enantiomers or diastereomers.
  • the present invention also contemplates the use of antagonist solvates (such as, but not limited to, hydrates), prodrugs (synonymous with prodrugs), or clathrates.
  • Pharmaceutically acceptable salts are selected from chloride, iodide bromide or any other pharmaceutically acceptable salt.
  • the TLR-4 receptor antagonists of the present invention may include isomers, depending on the presence of multiple bonds (eg, Z, E), including optical or enantiomeric isomers, depending on the presence of chiral centers.
  • the individual isomers, enantiomers or diastereoisomers and mixtures thereof fall within the scope of the present invention.
  • the individual enantiomers or diastereomers, and mixtures thereof, may be separated by conventional techniques.
  • the TLR-4 antagonist may be in crystalline form as free compounds or as solvate.
  • prodrugs or prodrugs of TLR-4 receptor antagonists are also contemplated.
  • the TLR-4 receptor inhibitor is selected from the list comprising CRX-526, E5531, E5564 or TAK-242 antagonists and NI-0101 and 1A6 antibodies.
  • a pharmaceutical composition comprising at least one inhibitor of an S1 P receptor and at least one inhibitor of the TLR-4 receptor, or any of the pharmaceutically acceptable salts, isomers or solvates of said inhibitors.
  • the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier and / or excipient.
  • pharmaceutical composition is the same as the definition of "medicament” that has been made in the present description. Therefore, the terms "pharmaceutical composition” and “medicament” are used as synonyms.
  • FIG. 1 Shows the expression of messenger RNA transcripts of S1 P receptors in human aortic valve interstitial cells.
  • the results of the quantitative PCR, once normalized with the actin reference gene, indicate that the cells express all S1 P receptors, with SI P2 being the most abundant, followed by SI P3 and S1 P 4 .
  • the results are representative of more than 5 independent experiments.
  • MRNA refers to messenger RNA.
  • FIG. 2. Shows the activation of signaling cascades by S1 P and TLR4.
  • Photographic image that illustrates that both S1 P receptors and TLR4 receptors are functional as the cellular treatment with their ligands, S1 P and LPS respectively, promotes the activation of intracellular signaling cascades such as MAP kinase p38 in interstitial cells of control valve (A) and stenotic valve (B).
  • the gel loading uniformity was confirmed with an antibody that recognizes the ERK MAP kinase, kinase regulated by extracellular signals.
  • R rest (cells treated with the vehicle); LPS, lipopolysaccharide; S1 P, sphingosine 1-phosphate.
  • FIG. 3. It shows the synergy of the S1 P and TLR4 receptors in the induction of pro-inflammatory molecules.
  • Control valve interstitial cells were treated with sphingosine-1-phosphate, S1 P (ligand of SI Pi-s receptors) and / or LPS (TLR4 ligand), and induction of expression of the expression was analyzed.
  • a synergistic effect was observed, because the combined treatment with S1 P and LPS induces the expression of COX-2 and ICAM-1 to a greater extent than the sum of the effect of the agonists separately.
  • the synergistic effect was observed at physiological doses of S1 P, between 0.01-1 ⁇ , and when treated in combination with a TLR4 ligand but not with a TLR2 ligand, Pam3CSK4.
  • Induction of inhibitor expression is inferior in pulmonary valve cells.
  • B Kinetics in interstitial cells of stenotic valve. The synergy effect was observed at times after cell stimulation of 8, 12 and 24h. An anti-p-tubulin antibody was used to check load uniformity. The results are representative of more than 5 independent experiments.
  • C The concentration of slCAM-1 secreted into the extracellular medium of treated cells as in panel A was quantified by ELISA assays. The results are representative of 3 independent experiments.
  • AVIC interstitial aortic valve cells
  • ERK kinase regulated by extracellular signals
  • LPS lipopolysaccharide
  • Pam or also indicated with the abbreviation P
  • Pam3CSK4 agonist of TLR2 / TLR1
  • ICP interstitial lung valve cells
  • p-NF-KB phosphorylated form of the transcription factor NF-kappaB
  • R rest (cells treated with the vehicle);
  • S1 P or also indicated with the abbreviation S
  • S sphingosine 1-phosphate.
  • FIG. 4. It shows the effect of the S1 P and TLR4 receptors on the induction of the alkaline phosphatase enzyme activity in interstitial cells from the control valve.
  • Crtl indicates medium control; MC indicates conditioned medium of calcification.
  • FIG. 5 It shows the effect of inhibition of synergy with some S1 P receptor antagonists.
  • AD The cells were pre-treated with pharmacological antagonists of S1 P receptors, subsequently activated with S1 P and LPS, and the analysis was performed as in Figure 3.
  • Pre-treatment with suramin, SI P3 antagonist blocks the synergistic effect of the combined treatment with S1 P and LPS in the induction of the expression of COX-2 (5A), of ICAM-1 (5A); PTX treatment inhibits to a lesser extent than suramin (5A).
  • New experiments reveal that pre-treatment with FTY720, a functional antagonist of S1 Pi, blocks the synergistic effect of the combined treatment with S1 P and LPS on the induction of ICAM-1 expression (5B).
  • FTY indicates FTY720; L, LPS; PTX, pertussis toxin (Gi inhibitor, inhibits signaling of several S1 P receptors); S, S1 P; Sura, Suramina.
  • VPC solvent refers to the solvent used to reconstitute VPC 23019, and is dimethyl sulfoxide: 1N hydrochloric acid: bovine serum serum albumin (in proportion 0.95: 0.05: 20, v / v / v).
  • the interference RNA assay was performed on cells transfected with duplex of interference RNA specific for S1 Pi, SI P2, SI P3 and SI P4 receptors in order to block its expression.
  • S1 P receivers can be named under the old nomenclature in which S1 P X equals EDG as follows: S1 Pi / EDG1, S1 P 2 / EDG5, S1 P 3 / EDG3 and SI P4 / EDG6.
  • FIG. 6 It shows the effect of inhibition of the additive effect in the induction of the enzymatic activity of alkaline phosphatase with some S1 P receptor antagonists.
  • (A) The figure reflects the enzymatic activity of alkaline phosphatase expressed as product picomoles generated by ⁇ grams of protein and hour, and is Representative of 3 independent experiments in duplicate.
  • the pretreatment of interstitial cells of control aortic valves with VPC23019 inhibits the additive effect in the induction of the enzymatic activity of alkaline phosphatase induced by S1 P and LPS observed in Figure 4A.
  • Pretreatment with JTE013, SI P 2 inhibitor inhibits the additive effect, although to a lesser extent than VPC23019.
  • (B) The figure includes data from new experiments performed.
  • the figure shows the% inhibition (mean value ⁇ standard error) of the alkaline phosphatase enzyme activity induced by the combined treatment with S1 P and LPS (value 100), and is representative of 5-7 independent experiments.
  • the new experiments demonstrate that pretreatment of interstitial cells of aortic valve control with suramin, inhibitor of SI P3, and with pertussis toxin, significantly blocks the additive effect in the induction of the enzymatic activity of alkaline phosphatase induced by S1 P and LPS observed in Figure 4A.
  • the S1 P receptors can be used They can be called EDG receivers with the following equivalence: S1 Pi / EDG1, S1 P 2 / EDG5, S1 P 3 / EDG3, SI P4 / EDG6, SI P5 / EDG8.
  • Interstitial cells from aortic valves were isolated from patients with calcified aortic stenosis who were surgically replaced the aortic valve (stenotic), and from aortic and pulmonary valves from patients undergoing heart transplantation without known valvular disease (non-stenotic control).
  • the samples used in the present invention come from patients who are in the advanced stage of aortic stenosis and are calcified; In the early stages, aortic stenosis is asymptomatic.
  • Cellular isolation was performed according to a recently described method based on sequential treatment with collagenase type I I (Meng et al.
  • RNA extraction and quantitative PCR assays RNA extraction and quantitative PCR assays.
  • Messenger RNA was analyzed by quantitative PCR (Real Time Quantitative Reverse Transcription). For this, the total RNA was isolated by the method of Trizol, and by "retro-transcription" using a reverse transcriptase, cDNA was obtained, thereby providing the stable polymerase template or substrate for the PCR reaction.
  • PCR was performed using specific primers of S1 P and SYBR-Green receptors following the manufacturer's instructions (MJ Research). The values obtained were normalized with respect to an internal control to avoid variability in the initial concentration of RNA.
  • the specificity of the reaction was determined by performing the denaturation curve of each DNA population.
  • CT threshold cycle
  • the primers used are: human S1 Pi, forward primer 5'-TATCAGCGCGGACAAGGAGAACAG-3 '(SEQ ID NO: 1); ATAGGCAGGCCACCCAGGATGAG-3 ' reverse primer (SEQ ID NO: 2).
  • S1 P 2 human forward primer 5'-TCGGCCTTCATCGTCATCCTCT-3 '(SEQ ID NO: 3); 5 ' -CCTCCCGGGCAAACCACTG-3 ' reverse primer (SEQ ID NO: 4).
  • Human SI P4 forward primer 5'-GAGAGCGGGGCCACCAAGAC- 3 '(SEQ ID NO: 7); 5 ' -GGTTGACCGCCGAGTTGAGGAC-3 ' reverse primer (SEQ ID NO: 8).
  • Human S1P 5 forward primer 5 '- ACAACTACACCGGCAAGCTC-3' (SEQ ID NO: 9); 5 ' reverse primer - GCCCCGACAGTAGGATGTT-3 ' (SEQ ID NO: 10).
  • the "Digital object identifier" (DOI) system can be used to locate the scientific article since it does not change over time, even if the article is relocated to a different web address since it carries the information incorporated in the form of metadata.
  • DOI Digital object identifier
  • interstitial cells with the corresponding agonists, S1 P and LPS respectively, induces a synergistic effect on the expression of (i) COX-2, inducible enzyme and precursor of inflammatory lipid mediators such as prostaglandins and prostacyclines, (ii) ICAM -1, membrane-associated adhesion molecule, iii) the soluble form of ICAM-1, which has been associated with increased prevalence and severity of aortic valve calcification (Shavelle et al. 2008. J.Heart Valve Dis., 17: 388-95) (Figure 3A).
  • the synergistic effect is observed at physiologically relevant doses of S1 P ( Figure 3A), and is maintained for many hours (Figure 3B).
  • EXAMPLE 4 IN VITRO CALCIFICATION: ADDITIVE EFFECT OF S1 PY TLR4 RECEPTORS Technique: Measurement of the expression and activity of an early calcification marker, alkaline phosphatase (ALP). The method described above was used (Yang X, et al. 2009, J Am Coll Cardiol, 53: 491-500; Osman L et al. 2006, Circulation, 1 14 [suppl l]: l-547-l-552) .
  • 20,000 interstitial cells of control valves were seeded per well in a 24-well plate, and cultured in conditioned medium (M199 medium supplemented with 10 mM glycerophosphate, 10 nM vitamin D3, 10 nM dexamethasone) alone or in the presence of 0.1 ⁇ S1 P, ⁇ g / ml LPS, or a combination of both.
  • conditioned medium M199 medium supplemented with 10 mM glycerophosphate, 10 nM vitamin D3, 10 nM dexamethasone
  • the cells were grown in the M199 medium commonly used for culture (control medium, Ctrl). Fresh medium was added twice a week. At 15-21 days of culture, the activity of ALP was measured.
  • ALP enzymatic activity (quantitative method).
  • the cells were lysed in a buffer composed of 20 mM Tris-HCI, 150 mM NaCI, 0.2% NP40, and 10% glycerol, pH 8).
  • Enzymatic activity was measured in cell lysates ( ⁇ 5 ⁇ gr protein) using a very sensitive fluorometric kit, in which the substrate is coupled to a fluorescent marker, methylumbiliferone phosphate (MUP).
  • MUP methylumbiliferone phosphate
  • Alkaline phosphatase activity was calculated using a standard curve and normalizing against protein concentration. The results were expressed as pmol ⁇ g protein per hour of reaction. Results: The induction of the enzymatic activity of alkaline phosphatase (ALP), an early marker of calcification (Figure 4A-B), was studied. The results indicate that S1 P has a pro-osteogenic effect, since it induces ALP activity with respect to the values obtained with the conditioned medium and the control medium ( Figure 4B). The effect of S1 P is similar to that observed with LPS, which has recently been described as an activator of ALP expression and activity (Yang X, et al. 2009.
  • the toxin and antagonists were pre-incubated for 1 h and then incubated with S1 P 0.1 ⁇ L + LPS ⁇ g / ml for 12 hours and then cell lysates were analyzed as described in example 3 (COX-2 expression and ICAM-1, and the secretion to the extracellular medium of slCAM-1) and as in example 4 (enzymatic activity of ALP).
  • siRNA RNA oligonucleotides
  • RNA interference assays of the S1 P 4 receptor demonstrate that the S1 P 4 receptor (for which there is no specific commercial chemical antagonist, that is why the interference RNA is used), is also involved in the process of synergy of S1 P and TLR4 receptors in the induction of pro-inflammatory molecules.
  • silencing the S1 Pi receptor or SI P 2 receptor gene blocks the synergy effect ( Figure 5F); and the silencing of the expression of the SI P3 receptor also inhibits the synergistic effect, which is in agreement with the results observed in the pharmacological study.

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Abstract

La présente invention concerne l'utilisation d'au moins un inhibiteur des récepteurs de sphingosine-1-phosphate (S1P), de préférence des récepteurs S1P1, S1 P3, SI P4 ou S1 P2, pour la préparation d'un médicament destiné à la prévention et/ou au traitement de la sténose aortique calcifiée. Plus préférentiellement, l'inhibiteur est un antagoniste ou un ARN d'inerférence.
PCT/ES2012/070170 2011-03-16 2012-03-15 Utilisation d'inhibiteurs de récepteurs s1p pour le traitement de la sténose aortique calcifiée Ceased WO2012123613A1 (fr)

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