WO2012123613A1 - Inhibitors of s1p receptors for the treatment of calcific aortic stenosis - Google Patents

Inhibitors of s1p receptors for the treatment of calcific aortic stenosis Download PDF

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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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PCT/ES2012/070170
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Spanish (es)
French (fr)
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Mª Carmen GARCÍA RODRÍGUEZ
Mariano SÁNCHEZ CRESPO
José Alberto SAN ROMÁN CALVAR
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Consejo Superior De Investigaciones Científicas (70%)
Universidad De Valladolid (30%)
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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
    • 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
    • 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 N.A.

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

The invention relates to the use of at least one inhibitor of sphingosine-1-phosphate (S1 P) receptors, preferably of receptors S1 P1, S1 P3, S1 P4 or S1 P2, for the preparation of a drug for preventing and/or treating calcified aortic stenosis. More preferably, the inhibitor is an antagonist or an interfering RNA.

Description

USO DE INHIBIDORES DE RECEPTORES DE S1 P PARA EL  USE OF S1 P RECEIVER INHIBITORS FOR
TRATAMIENTO DE LA ESTENOSIS AÓRTICA CALCIFICADA  TREATMENT OF CALCIFIED AORTIC STENOSIS
La presente invención se encuentra dentro del campo de la biomedicina. Específicamente, se refiere al uso de al menos un inhibidor de los receptores de esfingosina-1 -fosfato (S1 P), preferiblemente de los receptores S1 Pi , SI P2, SI P3, o SI P4, para la elaboración de un medicamento para la prevención y/o el tratamiento de la estenosis aórtica calcificada. ESTADO DE LA TÉCNICA ANTERIOR 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. STATE OF THE PREVIOUS TECHNIQUE
La estenosis aórtica degenerativa calcificada es la enfermedad valvular más frecuente en los países desarrollados, está asociada a una elevada morbi- mortalidad y se espera que su incidencia aumente dado el progresivo envejecimiento de la población. 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.
La estenosis aórtica calcificada es una enfermedad degenerativa caracterizada por fibrosis y calcificación de la válvula aórtica que puede impedir el desarrollo normal de la vida del paciente; es la valvulopatía más frecuente en los países desarrollados y su prevalencia aumenta con la edad (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). Esta enfermedad evoluciona lentamente, aunque cuando los síntomas aparecen, la progresión es rápida y si no hay sustitución valvular antes de 3 años la tasa de mortalidad supera el 50%. Se conocen varios aspectos de su patogenia y se han descrito varias moléculas pro-inflamatorias y pro-osteogénicas con papel relevante en la enfermedad, aunque aún no se ha encontrado la diana farmacológica de aplicación en la clínica. Se ha descrito la contribución de procesos de inflamación crónica y de calcificación tanto en el inicio como en la progresión de la enfermedad. En lesiones valvulares se ha detectado la infiltración de células inflamatorias, depósitos de lípidos oxidados, y la expresión de citocinas y metaloproteinasas. Las válvulas cardiacas constan de una matriz extracelular y el contenido celular lo forman células endoteliales y células intersticiales, estas últimas con capacidad de diferenciarse a osteoblastos in vitro bajo la influencia de diferentes mediadores, y se piensa que son las candidatas a promover la calcificación característica de la enfermedad (Osman L, et al. 2006, Circulation, 1 14[suppl I]: I-547-I-552). La patogenia de la estenosis aórtica calcificada presenta similitudes etiopatogénicas con el proceso aterosclerótico (Yetkin E, et al. 2009, Int J Cardiol, 135: 4-13; O'Brien KD. 2006, Arteríoscler Thromb Vasc Biol, 26: 1721 - 1728). En un estudio multiétnico de aterosclerosis se ha asociado a slCAM-1 , que es la forma soluble de la molécula de adhesión ICAM-1 , con un aumento de la prevalencia y la severidad de la calcificación de la válvula aórtica (Shavelle et al. 2008, J Heart Valve Dis, 17: 388-395), que es una característica de la estenosis aórtica calcificada (Yetkin E, et al. 2009, Int J Cardiol, 135: 4-13; O'Brien KD. 2006, Arteríoscler Thromb Vasc Biol, 26: 1721 -1728). 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. 2006, Arteríoscler Thromb Vasc Biol, 26: 1721-1728 ). In a multi-ethnic study of atherosclerosis, it has been associated with slCAM-1, which is the soluble form of the ICAM-1 adhesion molecule, with an increase in the prevalence and severity of aortic valve calcification (Shavelle et al. 2008 , J Heart Valve Dis, 17: 388-395), which is a characteristic of calcified aortic stenosis (Yetkin E, et al. 2009, Int J Cardiol, 135: 4-13; O'Brien KD. 2006, Arteríoscler Thromb Vasc Biol, 26: 1721-1728).
En la actualidad el único tratamiento efectivo de la enfermedad es el reemplazamiento quirúrgico de la válvula aórtica. Se han desarrollado terapias experimentales inicialmente prometedoras, como el uso de los inhibidores de la enzima convertidora de angiotensina o el tratamiento con estatinas que inhiben la HMCoA reductasa y reducen los niveles de colesterol. Sin embargo estas estatinas, a pesar del potencial terapéutico mostrado en modelos animales y en un estudio preliminar en pacientes (Yetkin E, et al. 2009, Int J Cardiol, 135: 4- 13, Moura et al. 2007, Am Coll Cardiol, 49: 554-561 ), mostraron escasa eficacia en dos ensayos clínicos de doble ciego en pacientes (CoweII SJ, et al. 2005, N Engl J Med, 352: 2389-2397; Rossebo AB, et al. 2008, N Engl J Med, 359: 1343-1356). Currently, the only effective treatment of the disease is the surgical replacement of the aortic valve. Initially promising experimental therapies have been developed, such as the use of angiotensin-converting enzyme inhibitors or treatment with statins that inhibit HMCoA reductase and reduce cholesterol levels. However, these 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. 2007, Am Coll Cardiol, 49: 554-561) showed poor efficacy in two clinical trials in patients double-blind (CoweII SJ, et al 2005, N Engl J Med, 352: 2389-2397; Rossebo AB, et al 2008, N Engl J.. Med, 359: 1343-1356).
Por otra parte, se sabe que la esfingosina 1 -fosfato (S1 P) es un mediador lipídico inflamatorio con potentes efectos en migración y proliferación celular que juega un papel importante en la regulación de los sistemas cardiovascular e inmune y en la patogenia de enfermedades como la aterosclerosis (Means y Brown, 2009, Cardiovasc Res, 82: 193-200; Fyrst H, et al. 2010, Nat Chem Biol. 6: 489-497). Las funciones de S1 P varían según el tipo celular y el subtipo de receptor expresado, y se sabe que en el corazón juega un papel en la supervivencia celular y es cardioprotector en modelos experimentales de isquemia/reperfusión (Means y Brown, 2009, Cardiovasc Res, 82: 193-200; Alewijnse et al., 2008. Eur J Pharmacol., 585: 292-302). La S1 P se une a receptores de membrana denominados receptores de S1 P, o EDG según la antigua nomenclatura; en mamíferos se han descrito cinco receptores S1 P/EDG: S1 Pi/EDG1 , S1 P2/EDG5, S1 P3/EDG3, SI P4/EDG6, SI P5/EDG8 (Rivera J, et al. 2008, Nat Rev Immunol, 8: 753-763; Chun J, et al. 2010, Pharmacol Rev , 62: 579-587). Estos receptores no han sido relacionados con la prevención y/o tratamiento de la estenosis aórtica calcificada. On the other hand, it is known that sphingosine 1-phosphate (S1 P) is an inflammatory lipid mediator with potent effects on migration and cell proliferation that plays an important role in the regulation of the cardiovascular and immune systems and in the pathogenesis of diseases such as atherosclerosis (Means and Brown, 2009, Cardiovasc Res, 82: 193-200; Fyrst H, et al. 2010, Nat Chem Biol. 6: 489-497). 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; In mammals, five 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). These receptors have not been related to the prevention and / or treatment of calcified aortic stenosis.
Por tanto, en la actualidad no existe ningún fármaco para la prevención y/o el tratamiento efectivo de la estenosis aórtica calcificada. Como consecuencia, es necesaria la búsqueda de nuevos blancos farmacológicos con relevancia clínica que puedan usarse tanto para el tratamiento como para la prevención de la estenosis aórtica calcificada. Therefore, there is currently no drug for the prevention and / or effective treatment of calcified aortic stenosis. As a consequence, it is necessary to search for new pharmacological targets with clinical relevance that can be used both for the treatment and for the prevention of calcified aortic stenosis.
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
La presente invención se refiere al uso de inhibidores de receptores de S1 P, preferiblemente un antagonista o un siRNA de dichos receptores para la elaboración de un medicamento para la prevención y/o el tratamiento de la estenosis aórtica calcificada. Preferiblemente los receptores son S1 Pi , SI P2,
Figure imgf000004_0001
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. Preferably the receivers are S1 Pi, SI P2,
Figure imgf000004_0001
Ante la falta de estrategias terapéuticas efectivas para el tratamiento farmacológico de la estenosis aórtica degenerativa calcificada, la presente invención proporciona una solución al problema de prevención y/o tratamiento de esta enfermedad mediante el uso de inhibidores de los receptores de S1 P, preferiblemente de los receptores S1 Pi , S1 P2, S1 P3, o S1 P4. Los resultados proporcionan una nueva diana terapéutica que puede ser usada para prevenir y/o reducir tanto la inflamación crónica como la calcificación asociadas a la estenosis aórtica calcificada. In the absence of effective therapeutic strategies for the pharmacological treatment of calcified degenerative aortic stenosis, 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. 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.
La presente invención presenta las siguientes ventajas respecto del estado de la técnica: - En la presente invención se ha demostrado que hay un efecto sinérgico en la activación de los receptores S1 Pi , SI P2, SI P3, o S1 P4 conjuntamente con el receptor de la inmunidad innata tipo Toll (TLR)-4, resultando en un aumento exponencial de ciclooxigenasa (COX)-2, reconocido marcador anti-inflamatorio, en células intersticiales de válvulas cardiacas. El bloqueo de esta sinergia, disminuye la inflamación mediada por COX-2, y podría bloquear la inflamación asociada a la estenosis aórtica calcificada. 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.
- Además en la presente invención se demuestra un efecto aditivo de los receptores de S1 P junto con el receptor TLR4 en lo que respecta a la calcificación in vitro asociada a estenosis aórtica, con lo que bloqueando los receptores de S1 P, preferiblemente los receptores S1 Pi , SI P2 o SI P3 adicionalmente se disminuye dicha calcificación. - In addition, 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.
- Adicionalmente se demuestra que el aumento de la expresión de ICAM-1 y de su forma soluble slCAM-1 , un biomarcador asociado a la prevalencia y severidad de la calcificación de la válvula aórtica que subyace a la estenosis aórtica calcificada, se produce como consecuencia de la activación conjunta de los receptores de S1 P y TLR4 en células intersticiales de válvulas cardiacas. Como consecuencia, el uso de antagonistas de los receptores de S1 P, preferiblemente de los receptores S1 Pi , SI P2, SI P3 o S1 P4 evitaría la agravación de la estenosis aórtica calcificada. Por tanto, la presente invención proporciona el uso de antagonistas de los receptores de esfingosina 1 -fosfato (S1 P), preferiblemente de los receptores S1 Pi , SI P2, SI P3 o SI P4 para bloquear de forma simultánea la inflamación y calcificación que subyacen a la estenosis aórtica calcificada y como consecuencia, para prevenir y/o tratar dicha enfermedad, para la que actualmente no existe terapia farmacológica efectiva. - Additionally, it is shown that the increase in the expression of ICAM-1 and its soluble form slCAM-1, a biomarker associated with the prevalence and severity of calcification of the aortic valve that underlies calcified aortic stenosis, occurs as a consequence of the joint activation of S1 P and TLR4 receptors in interstitial cells of cardiac valves. As a consequence, the use of S1 P receptor antagonists, preferably S1 Pi, SI P2, SI P3 or S1 P 4 receptors would prevent aggravation of calcified aortic stenosis. Therefore, 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.
Un aspecto de la presente invención se refiere al uso de al menos un inhibidor de un receptor de S1 P (el artículo "un" debe interpretarse como artículo indefinido), es decir, al uso de al menos un inhibidor del receptor S1 Pi , SI P2, SI P3, SI P4 o SI P5, o de cualquier combinación de inhibidores, para la elaboración de un medicamento para la prevención y/o el tratamiento de la estenosis aórtica calcificada. En adelante, para referirnos al uso descrito en este párrafo se puede emplear el término "uso de la presente invención" o "uso de la invención". 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. Hereinafter, the term "use of the present invention" or "use of the invention" may be used to refer to the use described in this paragraph.
En la presente invención no se muestran resultados de inhibición del receptor SI P5. Al igual que ocurre con el receptor S1 P4 por el momento no hay disponibles inhibidores químicos para estos receptores. A modo de ejemplo, en la presente invención se muestran resultados de inhibición del receptor S1 P4 con la tecnología de ARN de interferencia. Teniendo en cuenta la similitud estructural y funcional con respecto al resto de inhibidores S1 P ensayados en la presente invención, es esperable que la inhibición de la función de SI P5 por medios conocidos por el experto en la materia, es decir, por medios que no requieran un paso inventivo, o experimentación indebida, como por ejemplo el diseño de ARN de interferencia capaces de inhibir o disminuir la actividad del mismo, proporcione el mismo efecto técnico descrito para la inhibición del resto de receptores. Los inhibidores pueden ser específicos de un tipo de receptor S1 P, preferiblemente de los receptores S1 Pi , SI P2, SI P3 o S1 P4, pueden inhibir dos, tres o cuatro de ellos. La inhibición puede producirse en un rango de concentración óptimo del inhibidor correspondiente. Es decir, mientras que un rango de concentración puede inhibir a un receptor, es posible que la inhibición de otro receptor se produzca a una concentración que no se encuentre en dicho rango. In the present invention no results of inhibition of the SI P5 receptor are shown. As with the S1 P 4 receptor, no chemical inhibitors are currently available for these receptors. By way of example, the present invention shows results of inhibition of the S1 P 4 receptor with interference RNA technology. Taking into account the structural and functional similarity with respect to the rest of S1 P inhibitors tested in the present invention, it is expected that 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.
El término "inhibidor del receptor de S1 P" tal como se utiliza en la presente invención, se refiere a una molécula que se une a cualquiera de los receptores de S1 P, preferiblemente de los receptores S1 Pi , S1 P2, S1 P3 o S1 P4 y disminuye la expresión y/o actividad del receptor al que se une, y/o su señalización intracelular. El inhibidor se selecciona de la lista que comprende, pero sin que sirva de limitación, antagonistas (preferiblemente químicos), ARN de silenciamiento o anticuerpo específico para el receptor correspondiente (preferiblemente el anticuerpo es monoclonal), en la presente invención este anticuerpo puede denominarse anticuerpo neutralizante del efecto de S1 P. Se ha descrito el posible papel terapéutico de anticuerpos que neutralizan el efecto de S1 P en cáncer (Huwiler A y Pfeilschifter J, 2008. Biochem Pharmacol, 75: 1893-1900; Visentin B et al. 2006 Cáncer Cell, 9: 225-38). En la presente invención se demuestra de forma clara y no ambigua que mediante la inhibición específica de los receptores S1 Pi, SI P2, SI P3 o S1 P4 se consiguen reducir los parámetros indicadores de la inflamación y calcificación asociada a la estenosis aórtica calcificada. En los ejemplos de la presente invención se demuestra que los antagonistas químicos y diferentes ARN de silenciamiento, específicos para dichos receptores, solucionan el problema técnico de la presente invención, suponiendo estos ejemplos una demostración clara de que inhibidores de muy diferente naturaleza tienen el mismo efecto técnico, por tanto la presente invención no debe limitarse al uso de los inhibidores concretos ensayados sino que debe referirse a otros inhibidores conocidos para los receptores S1 Pi, SI P2, SI P3 o S1 P4 en la fecha de presentación de la presente invención. En definitiva, la presente invención aporta al estado de la técnica que la inhibición de dichos receptores, mediante cualquier medio o producto, es útil en la prevención y/o tratamiento de la estenosis aórtica calcificada. The term "S1 P receptor inhibitor" as used in the present invention 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 possible therapeutic role of antibodies that neutralize the effect of S1 P in cancer has been described (Huwiler A and Pfeilschifter J, 2008. Biochem Pharmacol, 75: 1893-1900; Visentin B et al. 2006 Cancer Cell, 9: 225-38). The present invention clearly and unambiguously demonstrates that by specific inhibition of the S1 Pi, SI P2, SI P3 or S1 P 4 receptors, it is possible to reduce the parameters of inflammation and calcification associated with calcified aortic stenosis. In the examples of the present invention it is shown that chemical antagonists and different silencing RNAs, specific for said receptors, solve the technical problem of the present invention, these examples assuming a clear demonstration that inhibitors of very different nature have the same effect. technical, therefore 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. Ultimately, 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.
El término "estenosis aórtica calcificada" se refiere a una enfermedad degenerativa de la válvula aórtica causada por la calcificación degenerativa de las cúspides aórticas, es decir, la estenosis aórtica calcificada está caracterizada por el engrosamiento y la calcificación de las valvas de la válvula aórtica y el consiguiente estrechamiento del orificio valvular del corazón, dificultando de esta manera el flujo de sangre desde el ventrículo izquierdo hacia la aorta respecto de un individuo sano que no padece dicha enfermedad. El término "tratamiento" tal como se entiende en la presente invención se refiere a combatir los efectos causados como consecuencia de una enfermedad o condición patológica de interés en un sujeto (preferiblemente mamífero, y más preferiblemente un humano) que incluye: The term "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. The term "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:
(i) inhibir la enfermedad o condición patológica, es decir, detener su desarrollo; (i) inhibit the disease or pathological condition, that is, stop its development;
(ii) aliviar la enfermedad o la condición patológica, es decir, causar la regresión de la enfermedad o la condición patológica o su sintomatología;  (ii) alleviate the disease or the pathological condition, that is, cause the regression of the disease or the pathological condition or its symptomatology;
(iii) estabilizar la enfermedad o la condición patológica.  (iii) stabilize the disease or pathological condition.
El término "prevención" tal como se entiende en la presente invención consiste en evitar la aparición de la enfermedad, es decir, evitar que se produzca la enfermedad o la condición patológica en un sujeto (preferiblemente mamífero, y más preferiblemente un humano), en particular, cuando dicho sujeto tiene predisposición por la condición patológica. The term "prevention" as understood in the present invention 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.
El medicamento al que se refiere la presente invención puede ser de uso humano o veterinario. El "medicamento de uso humano" es toda sustancia o combinación de sustancias que se presente como poseedora de propiedades para el tratamiento o prevención de enfermedades en seres humanos o que pueda usarse en seres humanos o administrarse a seres humanos con el fin de restaurar, corregir o modificar las funciones fisiológicas ejerciendo una acción farmacológica, inmunológica o metabólica, o de establecer un diagnóstico médico. El "medicamento de uso veterinario" es toda sustancia o combinación de sustancias que se presente como poseedora de propiedades curativas o preventivas con respecto a las enfermedades animales o que pueda administrarse al animal con el fin de restablecer, corregir o modificar sus funciones fisiológicas ejerciendo una acción farmacológica, inmunológica o metabólica, o de establecer un diagnóstico veterinario. También se considerarán "medicamentos veterinarios" las "premezclas para piensos medicamentosos" elaboradas para ser incorporadas a un pienso. 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".
Opcionalmente, el medicamento de la presente invención comprende, al menos, un vehículo y/o un excipiente farmacéuticamente aceptables. El término "excipiente" hace referencia a una sustancia que ayuda a la absorción de cualquiera de los componentes de la composición de la presente invención, estabiliza dichos componentes o ayuda a la preparación de la composición farmacéutica en el sentido de darle consistencia o aportar sabores que lo hagan más agradable. Así pues, los excipientes podrían tener la función de mantener los componentes unidos como por ejemplo almidones, azúcares o celulosas, función de endulzar, función de colorante, función de protección del medicamento como por ejemplo para aislarlo del aire y/o la humedad, función de relleno de una pastilla, cápsula o cualquier otra forma de presentación como por ejemplo el fosfato de calcio dibásico, función desintegradora para facilitar la disolución de los componentes y su absorción en el intestino, sin excluir otro tipo de excipientes no mencionados en este párrafo. Por tanto, el término "excipiente" se define como aquella materia que, incluida en las "formas galénicas", se añade a los principios activos o a sus asociaciones para posibilitar su preparación y estabilidad, modificar sus propiedades organolépticas o determinar las propiedades físico-químicas de la composición farmacéutica y su biodisponibilidad. El excipiente "farmacéuticamente aceptable" debe permitir la actividad de los compuestos de la composición farmacéutica, es decir, que sea compatible con dichos componentes. Optionally, the medicament of the present invention comprises at least one pharmaceutically acceptable carrier and / or excipient. The term "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. Thus, 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.
La "forma galénica o forma farmacéutica" es la disposición a que se adaptan los principios activos y excipientes para constituir un medicamento. Se define por la combinación de la forma en la que la composición farmacéutica es presentada por el fabricante y la forma en la que es administrada. The "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.
El "vehículo" o portador, es preferiblemente una sustancia inerte. La función del vehículo es facilitar la incorporación de otros compuestos, permitir una mejor dosificación y administración o dar consistencia y forma a la composición farmacéutica. Por tanto, el vehículo es una sustancia que se emplea en el medicamento para diluir cualquiera de los componentes de la composición farmacéutica de la presente invención hasta un volumen o peso determinado; o bien que aún sin diluir dichos componentes es capaz de permitir una mejor dosificación y administración o dar consistencia y forma al medicamento. Cuando la forma de presentación es líquida, el vehículo farmacéuticamente aceptable es el diluyente. 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. When the form of presentation is liquid, the pharmaceutically acceptable carrier is the diluent.
Además, el excipiente y el vehículo deben ser farmacológicamente aceptables, es decir, que el excipiente y el vehículo esté permitido y evaluado de modo que no cause daño a los organismos a los que se administra. In addition, 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.
Por otra parte, el medicamento descrito, además de dicho vehículo y/o excipiente, comprende opcionalmente otra sustancia activa. Además del requerimiento de la eficacia terapéutica, donde dicha composición farmacéutica puede necesitar el uso de otros agentes terapéuticos, pueden existir razones fundamentales adicionales que obligan o recomiendan en gran medida el uso de una combinación de un compuesto de la invención y otro agente terapéutico. El término "principio activo" es toda materia, cualquiera que sea su origen, humano, animal, vegetal, químico o de otro tipo, a la que se atribuye una actividad apropiada para constituir un medicamento. Moreover, the disclosed medicament in addition to said vehicle and / or excipient, optionally comprising another active substance. In addition to the requirement of therapeutic efficacy, where 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. The term "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.
En cada caso la forma de presentación del medicamento se adaptará al tipo de administración utilizada, por ello, la composición de la presente invención se puede presentar bajo la forma de soluciones o cualquier otra forma de administración clínicamente permitida y en una cantidad terapéuticamente efectiva. El medicamento descrito en la invención se puede formular en formas sólidas, semisólidas, líquidas o gaseosas, tales como comprimido, cápsula, polvo, gránulo, ungüento, solución, supositorio, inyección, inhalante, gel, microesfera o aerosol. El medicamento se puede presentar en una forma adaptada a la administración oral, sublingual, nasal, intracatecal, bronquial, linfática, rectal, transdérmica o inhalada, pero sin limitarse a estas formas. Por otra parte, el inhibidor de los receptores de S1 P de la presente invención, preferiblemente de los receptores S1 Pi , SI P2, SI P3 o S1 P4, puede ir asociado, por ejemplo, pero sin limitarse, con liposomas o micelas. In each case the form of presentation of the medicament will be adapted to the type of administration used, therefore, 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. On the other hand, 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.
Una realización preferida de la presente invención se refiere al uso de la invención, donde el inhibidor es un antagonista, o cualquiera de sus sales, isómeros o solvatos farmacéuticamente aceptables. El antagonista de la presente invención es de tipo químico. El antagonista es un compuesto que se une a un receptor de S1 P, preferiblemente a los receptores S1 Pi , SI P2, SI P3 o SI P4, y actúa como un antagonista funcional bloqueando parcial o totalmente su activación por los agonistas. 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.
El antagonista de la presente invención, puede existir en forma de enantiómeros o diastereómeros. En la presente invención también se contempla el uso de solvatos del antagonista (como por ejemplo, pero sin limitarse, hidratos), prodrogas (sinónimo de profármacos), o clatratos. Las sales farmacéuticamente aceptables se seleccionan de entre cloruro, bromuro ioduro o cualquier otra sal farmacéuticamente aceptable. 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.
Los antagonistas de los receptores de S1 P de la presente invención, preferiblemente de los receptores S1 Pi, SI P2, SI P3 o S1 P4, pueden incluir isómeros, dependiendo de la presencia de enlaces múltiples (por ejemplo, Z, E), incluyendo isómeros ópticos o enantiómeros, dependiendo de la presencia de centros quirales. Los isómeros, enantiómeros o diastereoisómeros individuales y las mezclas de los mismos caen dentro del alcance de la presente invención. Los enantiómeros o diastereoisómeros individuales, así como sus mezclas, pueden separarse mediante técnicas convencionales. The S1 P receptor antagonists of the present invention, preferably of the S1 Pi, SI P2, SI P3 or S1 P 4 receptors, 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.
En la presente invención, el término "sales farmacéuticamente aceptables" de los compuestos antagonistas, se refiere a sales preparadas de ácidos no tóxicos farmacéuticamente aceptables, incluyendo ácidos orgánicos o inorgánicos. Los ácidos orgánicos o inorgánicos no tóxicos se seleccionan de la lista que comprende, pero sin limitarse: acético, algínico, antranílico, bencenosulfónico, benzoico, camforsulfónico, cítrico, etanosulfónico, fórmico, fumárico, furoico, glucónico, glutámico, glucorénico, galacturónico, glicídico, hidrobrómico, hidroclórico, isetiónico, láctico, maleico, málico, mandélico, metanesulfónico, múcico, nítrico, pamoico, pantoténico, fenilacético, propiónico, fosfórico, salicílico, esteárico, succínico, sulfanílico, sulfúrico, tartárico o p- toluenosulfónico. El compuesto de la invención puede estar en forma cristalina como compuestos libres o como solvatos. En este sentido, el término "solvato", tal como aquí se utiliza, incluye tanto solvatos farmacéuticamente y farmacológicamente aceptables, es decir, solvatos del antagonista de los receptores de S1 P, preferiblemente de los receptores S1 Pi , S1 P2, S1 P3 o S1 P4, que pueden ser utilizados en la elaboración de un medicamento, como solvatos farmacéuticamente no aceptables, los cuales pueden ser útiles en la preparación de solvatos o sales farmacéuticamente y farmacológicamente aceptables. La naturaleza del solvato farmacéuticamente aceptable no es crítica siempre y cuando sea farmacéuticamente aceptable. En una realización particular, el solvato es un hidrato. Los solvatos pueden obtenerse por métodos convencionales de solvatación conocidos por los expertos en la materia. In the present invention, 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. In this sense, 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. In a particular embodiment, the solvate is a hydrate. Solvates can be obtained by conventional solvation methods known to those skilled in the art.
Asimismo, dentro del alcance de esta invención se encuentran las prodrogas o profármacos de los antagonistas de los receptores de S1 P, preferiblemente de los receptores S1 Pi , SI P2, SI P3 o S1 P4. El término "profármaco" o "prodroga" tal como aquí se utiliza incluye cualquier compuesto derivado del antagonista, por ejemplo y no de forma limitativa: ésteres (incluyendo ésteres de ácidos carboxílicos, ésteres de aminoácidos, ésteres de fosfato, ésteres de sulfonato de sales metálicas, etc.), carbamatos, amidas, amidas biohidrolizables, ésteres biohidrolizables, carbamatos biohidrolizables, ureidos biohidrolizables, fosfatos biohidrolizables. Otros ejemplos de prodrogas incluyen compuestos que comprenden grupos -NO, -NO2, -ONO, o -ONO2- que al ser administrado a un individuo puede ser transformado directa o indirectamente en dicho antagonista en el mencionado individuo. Ventajosamente, dicho derivado es un compuesto que aumenta la biodisponibilidad del antagonista cuando se administra a un individuo o que potencia la liberación del antagonista en un compartimento biológico. La naturaleza de dicho derivado no es crítica, siempre y cuando pueda ser administrado a un individuo y proporcione el antagonista de los receptores de S1 P, preferiblemente de los receptores S1 Pi , SI P2, SI P3 o S1 P4, en un compartimento biológico del mismo. La preparación de dicho profármaco puede llevarse a cabo mediante métodos convencionales conocidos por los expertos en la materia. Los términos "amidas biohidrolizables", "ésteres biohidrolizables", "carbamatos biohidrolizables", "ureidos biohidrolizables", "fosfatos biohidrolizables", se refieren a carbamato, carbonato, ureido y fosfato, respectivamente, de un compuesto que: 1 ) no interfiera con la actividad biológica del complejo pero que confiere al compuesto propiedades ventajosas in vivo, como absorción, duración del efecto, o del inicio del efecto; o 2) es biológicamente inactivo, pero se convierte in vivo a compuesto biológicamente activo. Also, within the scope of this invention are 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. Other examples of 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. Advantageously, 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. The terms "biohydrolyzable amides", "biohydrolysable esters", "biohydrolysable carbamates", "biohydrolysable ureides", "biohydrolysable phosphates", 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.
Más preferiblemente el antagonista se selecciona de la lista que comprende FTY720, VPC23019, VPC25239, VPC01091 , VPC 23153, Ascotricina A, Ascotricina B, JTE013, W146, W123, BML-241 , Suramina o toxina pertussis, o cualquiera de sus sales, isómeros o solvatos farmacéuticamente aceptables. Según realizaciones aún más preferidas del uso de la presente invención, el antagonista se selecciona de la lista que comprende VPC23019, JTE013, Suramina, W146, FTY720 o toxina pertussis, o cualquiera de sus sales, isómeros o solvatos farmacéuticamente aceptables. More preferably 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. According to even more preferred embodiments of the use of the present invention, 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.
A continuación se describen los antagonistas de la lista anterior: The antagonists listed above are described below:
- FTY720 (2-amino-2-[2-(4-octilfenil)etil]-propano-1 ,3-diol) (sales: hidrocloruro, fosfato), también denominado "fingolimod" es un agente con efecto inmunosupresor que regula la migración de linfocitos mediante su interacción con S1 Pi , aunque puede unirse a todos los receptores de S1 P/EDG con excepción de S1 P2 (Takabe K et al. 2008, Pharmacol Rev, 60: 181 -195; Dong- Soon I, 2010, Acta Pharmacol Sínica, 31 : 1213-1222). Además de su efecto agonista, FTY720 se comporta como un antagonista funcional de S1 Pi , pues el tratamiento con FTY720 in vivo induce la internalización y degradación de la expresión de S1 Pi , con lo que actuaría como un inhibidor no competitivo selectivo de receptores de S1 Pi (M.H. Gráler y E.J. Goetzl, 2004. FASEB J, 18: 551-553; Matloubian et al., 2004. Nature, 427: 355-360; Dong-Soom I, 2010. Acta Pharmacol Sínica, 31 : 1213-1222) - 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 Sínica, 31: 1213-1222). In addition to its agonist effect, 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 Sínica, 31: 1213-1222 )
La estructura química de la sal de ácido clorhídrico de FTY720 es la siguiente:
Figure imgf000014_0001
The chemical structure of the hydrochloric acid salt of FTY720 is as follows:
Figure imgf000014_0001
- VPC23019 ((R)-ácido fosfórico mono-[2-amino-2-(3-octil-fenilcarbamoil)-etil] ester) es un antagonista selectivo no competitivo de S1 Pi y SI P3 (Takabe K et al. 2008. Pharmacol Rev, 60: 181 -195; Dong-Soon I, 2010. Acta Pharmacol Sínica, 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 Sínica, 31: 1213-1222).
Figure imgf000014_0002
- VPC25239 es un antagonista selectivo S1 Pi y S1 P3 (Takabe K et al. 2008. Pharmacol Rev, 60: 181 -195; Dong-Soon I, 2010. Acta Pharmacol Sínica, 31 : 1213-1222).
Figure imgf000014_0002
- 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 Sínica, 31: 1213-1222).
- VPC-01091 -P.1 -[1 -Amino-3-(4-octilfenil)ciclopentil]metanol-fosfato, es un antagonista de los receptores SI P3; Dong-Soon I, 2010. Acta Pharmacol- VPC-01091 -P.1 - [1-Amino-3- (4-octylphenyl) cyclopentyl] methanol-phosphate, is an SI P3 receptor antagonist; Dong-Soon I, 2010. Acta Pharmacol
Sínica, 31 : 1213-1222). Sinic, 31: 1213-1222).
- VPC 23153 ((R)-ácido fosfórico mono-[2-amino-2-(6-octil-1 H-benzoimiazol-2- il) etil] ester), es un agonista competitivo del receptor S1 P4 (Skoura A, Hla T. 2009. J Lipid Res 50:S293-298) - 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)
- Ascotricina A y B son inhibidores de receptores de S1 Pi (Ascotricin A y Ascotricin B, está más extendido el término en inglés que en español, es por ello que se menciona en la presente solicitud) (Yonesu K, et al. J Antibiot, 2009, 62: 359-64; Dong-Soon I, 2010. Acta Pharmacol Sínica, 31 : 1213-1222). - 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 Sínica, 31: 1213-1222).
- JTE013 (N-(3,5-dicloropiridina-4-il)-2-(4-isopropil-1 ,3-dimetil-1 H-pirazolo[3,4- b]piridina-6-il)-hidrazinacarboxamida), es capaz de unirse a los receptores S1 Pi , SI P2 , SI P3 , S1 P4 y SI P5. Es un antagonista potente de SI P2. Sin embargo, el fabricante comenta que usado 1000 veces más concentrado inhibe un 4.5% de SI P3 y no antagoniza S1 Pi . JTE-013 puede no ser un compuesto tan selectivo, al menos en modelos de roedores, pues su efecto inhibitorio de la contracción vascular se observa tanto en ratones deficientes en SI P2 como en los no deficientes (Salomone S et al. 2008. Br J Pharmacol, 153: 140-7). - JTE013 (N- (3,5-dichloropyridine-4-yl) -2- (4-isopropyl-1,3-dimethyl-1 H -pyrazolo [3,4- b] pyridine-6-yl) -hydrazinecarboxamide) , is able to bind to S1 Pi receptors , YES P2, YES P3, S1 P 4 and YES P5. It is a potent antagonist of SI P2. However, the manufacturer says that used 1000 times more concentrate inhibits 4.5% of SI P3 and does not antagonize S1 Pi. 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).
La estructura química del compuesto JTE013 es: The chemical structure of compound JTE013 is:
Figure imgf000015_0001
Figure imgf000015_0001
- W146 (3-amino-4-(3-hexilfenilamino)-4-oxobutil ácido fosfónico) es un antagonista específico de S1 P1 . - W146 (3-amino-4- (3-hexylphenylamino) -4-oxobutyl phosphonic acid) is a specific antagonist of S1 P1.
La estructura de la sal de trifluoroacetato es:  The structure of the trifluoroacetate salt is:
Figure imgf000015_0002
Figure imgf000015_0002
- W123 (3-(2-(3-hexilfenilamino)-2-oxoetilamino) ácido propanoico) antagonista específico de S1 Pi . - W123 (3- (2- (3-hexylphenylamino) -2-oxoethylamino) propanoic acid) S1 Pi specific antagonist.
Figure imgf000015_0003
- BML-241 (2-undecil-tiazolidina-4-ácidocarboxílico), también denominado como CAY10444. Es un antagonista selectivo del receptor SI P3, bloqueando el aumento de calcio en células HeLa en un 40% a una concentración de 10 μΜ. Su estructura química es la siguiente:
Figure imgf000015_0003
- 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 μΜ. Its chemical structure is as follows:
Figure imgf000016_0001
Figure imgf000016_0001
- Suramina (8-[(4-metil-3-{[3-({[3-({2-metil-5-[(4,6,8-trisulfo-1 -naftil)carbamoil] fenil}carbamoil)fenil]carbamoil}amino)benzoil]amino}benzoil)amino]naftaleno- 1 ,3,5-ácido trisulfónico), es un antagonista específico de la señalización intracelular de SI P3 por inhibir de modo competitivo y reversible las fosfatasas de tirosina y desacoplar las proteínas G. - Suramine (8 - [(4-methyl-3 - {[3 - ({[3 - ({2-methyl-5 - [(4,6,8-trisulfo-1-naphthyl) carbamoyl] phenyl} carbamoyl) phenyl] carbamoyl} amino) benzoyl] amino} benzoyl) amino] naphthalene-1,3,5-trisulfonic acid), is a specific antagonist of the intracellular signaling of SI P3 by competitively and reversibly inhibiting tyrosine phosphatases and decoupling G. proteins
Figure imgf000016_0002
Figure imgf000016_0002
- Toxina pertussis. Esta toxina es una exotoxina a base de proteínas tipo AB5 producida por la bacteria Bordetella pertussis. La toxina de B. pertussis cataliza la ADP-ribosilación de las cadenas alfa de proteínas G heterotriméricas impidiendo su interacción a receptores, y por tanto bloqueando la señalización intracelular de receptores acoplados a proteínas G, entre ellos los receptores de S1 P (Goodemote KA et al. J Biol Chem, 1995. 270: 10272-7.). Se ha descrito que en el sistema cardiovascular la toxina pertussis inhibe la señalización de los receptores S1 Pi y SI P3 y parcialmente la de SI P2 (Takuwa Y et al. 2008, Biochim Biophys Acta,, 1781 :483-488) aunque podría inhibir la señalización de S1 P4 y S1 P5 , que se sabe que está acoplada a Gi/o (Takabe K et al. 2008, Pharmacol Rev, 60:181 -195) Según otra realización preferida, el inhibidor es un ARN de silenciamiento o lo que es lo mismo, un ARN de interferencia (siRNA). - 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. 2008, Biochim Biophys Acta ,, 1781: 483-488) although it could inhibit the signaling of S1 P 4 and S1 P 5 , which is known to be coupled to Gi / o (Takabe K et al. 2008, Pharmacol Rev, 60: 181-195) According to another preferred embodiment, the inhibitor is a silencing RNA or what is the same, an interfering RNA (siRNA).
Según varias realizaciones más preferidas del uso de la presente invención: - En el caso de que el receptor sea S1 Pi, el siRNA es un ARN de doble cadena cuya secuencia sentido es SEQ ID NO: 1 1 (5'-GCCCACUUUAUCUAAAUGA- 3'). En la presente invención se entiende que el ARN de doble cadena está formado por una cadena sentido y una cadena antisentido, donde dichas cadenas son secuencias complementarias entre sí. En este caso, el siRNA es una secuencia de ARN de doble cadena cuya cadena antisentido es SEQ ID NO: 12 (5'-CGGGUGAAAUAGAUUUACU-3'), que está hibridada completamente con SEQ ID NO: 1 1 . Es decir, esta realización preferida de la presente invención se refiere al uso de la secuencia de siRNA de doble cadena SEQ ID NO: 11/SEQ ID NO: 12 para la elaboración de un medicamento para la prevención y/o el tratamiento de estenosis aórtica calcificada. According to several more preferred embodiments of the use of the present invention: - In the case that the receiver is S1 Pi, the siRNA is a double stranded RNA whose sequence is SEQ ID NO: 1 1 (5 ' -GCCCACUUUAUCUAAAUGA- 3 ' ). In the present invention it is understood that double stranded RNA is formed by a sense chain and an antisense chain, wherein said chains are complementary sequences to each other. In this case, 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. That is, 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
- Si el receptor es S1 P3, el siRNA es un ARN de doble cadena cuya secuencia sentido es SEQ ID NO: 13 (5'-GGUGGCCAACCACAACAAC-3'). En este caso, el siRNA es una secuencia de ARN de doble cadena cuya cadena antisentido es SEQ ID NO: 14 (5'-CCACCGGUUGGUGUUGUUG-3'), que está hibridada completamente con SEQ ID NO: 13. Es decir, esta realización preferida de la presente invención se refiere al uso de la secuencia de siRNA de doble cadena SEQ ID NO: 13/SEQ ID NO: 14 para la elaboración de un medicamento para la prevención y/o el tratamiento de estenosis aórtica calcificada. - If the receiver is S1 P3, the siRNA is a double stranded RNA whose sequence is SEQ ID NO: 13 (5 ' -GGUGGCCAACCACAACAAC-3 ' ). In this case, 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.
- Si el receptor es S1 P4, el siRNA es un ARN de doble cadena cuya secuencia sentido es SEQ ID NO: 15 (5'-GCCGCGUCUACGGCUUCAU-3'). En este caso, el siRNA es una secuencia de ARN de doble cadena cuya cadena antisentido es SEQ ID NO: 16 (5'-CGGCGCAGAUGCCGAAGUA-3'), que está hibridada completamente con SEQ ID NO: 15. Es decir, esta realización preferida de la presente invención se refiere al uso de la secuencia de siRNA de doble cadena SEQ ID NO: 15/SEQ ID NO: 16 para la elaboración de un medicamento para la prevención y/o el tratamiento de estenosis aórtica calcificada. - If the receiver is S1 P 4 , the siRNA is a double stranded RNA whose sequence is SEQ ID NO: 15 (5 ' -GCCGCGUCUACGGCUUCAU-3 ' ). In this case, 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.
- Si el receptor es SI P2, el siRNA es un ARN de doble cadena cuya secuencia sentido es SEQ ID NO: 35 (5'-GCAAGUUCCACUCGGCAAU-3'). En este caso, el siRNA es una secuencia de ARN de doble cadena cuya cadena antisentido es SEQ ID NO: 36 (5'-CGUUCAAGGUGAGCCGUUA-3'), que está hibridada completamente con SEQ ID NO: 35. Es decir, esta realización preferida de la presente invención se refiere al uso de la secuencia de siRNA de doble cadena SEQ ID NO: 35/SEQ ID NO: 36 para la elaboración de un medicamento para la prevención y/o el tratamiento de estenosis aórtica calcificada. - If the receiver is SI P2, the siRNA is a double stranded RNA whose sequence is SEQ ID NO: 35 (5 ' -GCAAGUUCCACUCGGCAAU-3 ' ). In this case, 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.
Para mejorar la función de los siRNA se pueden añadir los nucleótidos (tt) o (tg) en el extremo 3' de la cadena sentido o en el extremo 5' de la cadena antisentido del siRNA de la presente invención. De esta forma estos nucleótidos son salientes, no hibridando con ningún otro nucleótido de la cadena complementaria. La adición de estos nucleótidos en los extremos 3' o 5' no afecta al reconocimiento del ARN mensajero correspondiente. Preferiblemente, los siRNA resultado de dicha adición son: - SEQ ID NO: 1 1 -tt (SEQ ID NO: 26): Es decir, SEQ ID NO: 26 tiene la secuencia 5'-GCCCACUUUAUCUAAAUGAtt-3' / SEQ ID NO: 12-tt (SEQ ID NO: 27): Es decir, SEQ ID NO: 27 tiene la secuencia 5'- ttCGGGUGAAAUAGAUUUACU-3'. To improve the function of the siRNA, 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. Preferably, 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: 13-tt (SEQ ID NO: 28): Es decir, SEQ ID NO: 28 tiene la secuencia 5'-GGUGGCCAACCACAACAAC-tt-3' / tt-SEQ ID NO: 14 (SEQ ID- SEQ ID NO: 13-tt (SEQ ID NO: 28): That is, SEQ ID NO: 28 has the sequence 5 ' -GGUGGCCAACCACAACAAC-tt-3 ' / tt-SEQ ID NO: 14 (SEQ ID
NO: 29): Es decir, SEQ ID NO: 29 tiene la secuencia 5'- ttCCACCGGUUGGUGUUGUUG-3'. NO: 29): That is, SEQ ID NO: 29 has the sequence 5 '- ttCCACCGGUUGGUGUUGUUG-3'.
- SEQ ID NO: 15-tt (SEQ ID NO: 30): Es decir, SEQ ID NO: 30 tiene la secuencia 5'-GCCGCGUCUACGGCUUCAUtt-3' / gt-SEQ ID NO: 16 (SEQ ID NO: 31 ): Es decir, SEQ ID NO: 31 tiene la secuencia 5'- gtCGGCGCAGAUGCCGAAGUA-3'. - SEQ ID NO: 15-tt (SEQ ID NO: 30): That is, 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: 35-tt (SEQ ID NO: 40): Es decir, SEQ ID NO: 40 tiene la secuencia 5'-GCAAGUUCCACUCGGCAAUtt-3' / gt-SEQ ID NO: 36 (SEQ ID NO: 41 ): Es decir, SEQ ID NO: 41 tiene la secuencia 5'- gtCGUUCAAGGUGAGCCGUUA-3'. - SEQ ID NO: 35-tt (SEQ ID NO: 40): That is, 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'.
Una realización aún más preferida se refiere al uso de la invención en el que la secuencia de siRNA de doble cadena está codificada en una secuencia de ADN formada por dos secuencias, A y B, donde la secuencia A está unida por el extremo 3' al extremo 5' de la secuencia B, formando una secuencia A-B. Es decir, esta realización de la presente invención se refiere al uso de la secuencia A-B o preferiblemente al uso del vector de expresión que comprende dicha secuencia A-B, para la elaboración de un medicamento para la prevención y/o el tratamiento de estenosis aórtica calcificada. En este sentido se contemplan varias posibilidades: 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:
- En el caso de que el receptor sea S1 Pi , A es SEQ ID NO: 17 (5'- GCCCACTTTATCTAAATGA-3') y B es SEQ ID NO: 18 (5'- TCATTTAGATAAAGTGGGC-3'). En este caso se formaría la secuencia SEQ ID NO: 19; 5'-GCCCACTTTATCTAAATGA-TCATTTAGATAAAGTGGGC-3' (SEQ ID NO: 17-SEQ ID NO: 18). Preferiblemente las secuencias A y B tienen los nucleótidos (tt) unidos al extremo 3', dando como resultado la secuencia SEQ ID NO: 32; 5'-GCCCACTTTATCTAAATGAtt- TCATTTAGATAAAGTGGGCtt-3'. - In the event that the receiver is S1 Pi, A is SEQ ID NO: 17 (5 ' - GCCCACTTTATCTAAATGA-3 ' ) and B is SEQ ID NO: 18 (5 ' - TCATTTAGATAAAGTGGGC-3 ' ). In this case the sequence SEQ ID NO: 19 would be formed; 5 ' -GCCCACTTTATCTAAATGA-TCATTTAGATAAAGTGGGC-3 ' (SEQ ID NO: 17-SEQ ID NO: 18). Preferably sequences A and B have nucleotides (tt) attached to the 3 ' end, resulting in sequence SEQ ID NO: 32; 5 ' -GCCCACTTTATCTAAATGAtt- TCATTTAGATAAAGTGGGCtt-3 ' .
- En el caso de que el receptor sea S1 P3, A es SEQ ID NO: 20 (5'- GGTGGCCAACCACAACAAC-3') y B es SEQ ID NO: 21 (5'- GTTGTTGTGGTTGGCCACC-3'). En este caso se formaría la secuencia SEQ ID NO: 22; 5'-GGTGGCCAACCACAACAAC-GTTGTTGTGGTTGGCCACC-3' (SEQ ID NO: 20-SEQ ID NO: 21 ). Preferiblemente las secuencias A y B tienen los nucleótidos (tt) unidos al extremo 3', dando como resultado la secuencia SEQ ID NO: 33; 5'-GGTGGCCAACCACAACAACtt-- In the event that the receiver is S1 P 3 , A is SEQ ID NO: 20 (5 ' - GGTGGCCAACCACAACAAC-3 ' ) and B is SEQ ID NO: 21 (5 ' - GTTGTTGTGGTTGGCCACC-3 ' ). In this case the sequence SEQ ID NO: 22 would be formed; 5 ' -GGTGGCCAACCACAACAAC-GTTGTTGTGGTTGGCCACC-3 ' (SEQ ID NO: 20-SEQ ID NO: 21). Preferably sequences A and B have nucleotides (tt) attached to the 3 ' end, resulting in sequence SEQ ID NO: 33; 5 ' -GGTGGCCAACCACAACAACtt-
GTTGTTGTGGTTGGCCACCtt-3'. - En el caso de que el receptor sea S1 P4, A es SEQ ID NO: 23 (5'- GCCGCGTCTACGGCTTCAT-3') y B es SEQ ID NO: 24 (5'- ATGAAGCCGTAGACGCGGC-3'). En este caso se formaría la secuencia SEQ ID NO: 25; 5'-GCCGCGTCTACGGCTTCAT-ATGAAGCCGTAGACGCGGC-3' (SEQ ID NO: 23-SEQ ID NO: 24). Preferiblemente la secuencia A tiene los nucleótidos (tt) unidos al extremo 3' y la secuencia B tiene los nucleótidos (tg) unidos al extremo 3', dando como resultado la secuencia SEQ ID NO: 34; 5'- GCCGCGTCTACGGCTTCATtt-ATGAAGCCGTAGACGCGGCtg-3'. GTTGTTGTGGTTGGCCACCtt-3 ' . - In the event that the receiver is S1 P 4 , A is SEQ ID NO: 23 (5 ' - GCCGCGTCTACGGCTTCAT-3 ' ) and B is SEQ ID NO: 24 (5 ' - ATGAAGCCGTAGACGCGGC-3 ' ). In this case the sequence SEQ ID NO: 25 would be formed; 5 ' -GCCGCGTCTACGGCTTCAT-ATGAAGCCGTAGACGCGGC-3 ' (SEQ ID NO: 23-SEQ ID NO: 24). Preferably the 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 ' .
- En el caso de que el receptor sea SI P2, A es SEQ ID NO: 37 (5'- GCAAGTTCCACTCGGCAAT-3') y B es SEQ ID NO: 38 (5'- ATTGCCGAGTGGAACTTGC-3'). En este caso se formaría la secuencia SEQ ID NO: 39; 5'-GCAAGTTCCACTCGGCAAT-ATTGCCGAGTGGAACTTGC-3' (SEQ ID NO: 37-SEQ ID NO: 38). Preferiblemente la secuencia A tiene los nucleótidos (tt) unidos al extremo 3' y la secuencia B tiene los nucleótidos (tg) unidos al extremo 3', dando como resultado la secuencia SEQ ID NO: 42; 5'- GCAAGTTCCACTCGGCAATtt-ATTGCCGAGTGGAACTTGCtg-3'. Como se puede observar, las secuencias A y B son secuencias complementarias y como consecuencia, el ARN transcrito a partir de la misma hibridará consigo mismo formando una horquilla, es decir, formando un hairpinRNA (hpRNA). En estos casos, la secuencia A-B de ADN que codifica el ARN transcrito tiene que estar integrada en un sistema de expresión adecuado para que la transcripción tenga lugar a partir de la secuencia A-B de ADN. Por ejemplo, pero sin que sirva de limitación, este sistema de expresión puede ser un vector de expresión en el que la secuencia A-B de ADN esté insertada entre una secuencia reguladora de la expresión génica (como por ejemplo pero sin limitarse, un promotor) y una secuencia terminadora de la transcripción, de modo que la transcripción que tenga lugar rinda cualquiera de los fragmentos de siRNA de interés, descritos en la presente invención. El término "vector de expresión" se refiere a un fragmento de ADN que tiene la capacidad de replicarse en un determinado huésped y puede servir de vehículo para llevar a cabo la transcripción de una secuencia de interés que haya sido insertada en el mismo. El vector puede ser un plásmido, un cósmido, un bacteriófago o un vector viral, sin excluir otro tipo de vectores que se correspondan con la definición realizada de vector. - In the case that the receiver is YES P2, A is SEQ ID NO: 37 (5 ' - GCAAGTTCCACTCGGCAAT-3 ' ) and B is SEQ ID NO: 38 (5 ' - ATTGCCGAGTGGAACTTGC-3 ' ). In this case the sequence SEQ ID NO: 39 would be formed; 5 ' -GCAAGTTCCACTCGGCAAT-ATTGCCGAGTGGAACTTGC-3 ' (SEQ ID NO: 37-SEQ ID NO: 38). Preferably the 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 ' . As can be seen, 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). In these cases, 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. For example, but without limitation, 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. The term "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.
Los fragmentos de siRNA de la presente invención transcritos por el sistema de expresión descrito, es decir, los hpRNA formados son un tipo de ARN de doble hebra (dsRNA) que es reconocido y cortado por una endoribonucleasa, la endoribonucleasa Dicer, dando como resultado fragmentos de siRNA que provocan el silenciamiento post-transcripcional de las secuencias nucleotídicas diana, llevando a la degradación del ARN mensajero correspondiente, de forma que no se obtiene la proteína resultante de la expresión de las secuencias de ARN mensajero. De las dos cadenas o hebras de siRNA formadas a partir del corte con Dicer, sólo una de ellas, denominada hebra guía, se incorpora en el complejo enzimático RISC mientras que la otra hebra se degrada. Las características termodinámicas del extremo 5' del siRNA determinan cual de las dos hebras se incorpora al complejo RISC. Normalmente se incorpora como hebra guía aquella con menor estabilidad en el extremo 5'. Para que se produzca el silenciamiento post-transcripcional, la hebra guía debe ser complementaria al ARN mensajero que se pretende silenciar. A continuación, el complejo RISC se une al ARN complementario de la hebra guía del siRNA presente en el complejo y se produce el corte del ARN mensajero. The siRNA fragments of the present invention transcribed by the described expression system, that is, the hpRNA formed are a type of double-stranded RNA (dsRNA) that is recognized and cut by an endoribonuclease, the Dicer endoribonuclease, resulting in fragments. of 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. Of the two chains or strands of siRNA formed from the cut with Dicer, only one of them, called a guide strand, is incorporated into the RISC enzyme complex while the other strand is degraded. The 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. For post-transcriptional silencing to occur, the guide strand must be complementary to the messenger RNA that is intended to be silenced. Next, the RISC complex binds to the complementary RNA of the siRNA guide strand present in the complex and the messenger RNA is cut.
Según una realización aún más preferida del uso de la presente invención, entre la secuencia A y B hay una secuencia nucleotídica espaciadora de al menos un nucleótido de longitud. Más preferiblemente, la secuencia espaciadora es una secuencia no codificante. La secuencia espaciadora puede ser parte de una secuencia de un intrón de un gen o la secuencia completa de dicho intrón. La función de la secuencia espaciadora es actuar de bisagra de los pares de secuencias descritos para que se pueda producir el apareamiento o hibridación de las secuencias de ARN codificadas por el polinucleótido. Otra realización preferida se refiere al uso de cualquiera de los inhibidores del receptor de S1 P de la presente invención, en combinación con al menos un inhibidor del receptor de la inmunidad innata tipo Toll 4 (TLR-4) para la elaboración de un medicamento para la prevención y/o el tratamiento de estenosis aórtica calcificada. According to an even more preferred embodiment of the use of the present invention, between the sequence A and B there is a spacer nucleotide sequence of at least one nucleotide in length. More preferably, 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.
En la presente invención se ha demostrado que hay un efecto sinérgico en la activación de los receptores S1 Pi , SI P2, SI P3, o S1 P4 conjuntamente con el receptor de la inmunidad innata tipo Toll (TLR)-4. Tal como queda demostrado en los ejemplos de la presente invención, el bloqueo de esta sinergia, disminuye la inflamación mediada por COX-2 e ICAM-1 , y podría bloquear la inflamación asociada a la estenosis aórtica calcificada. Dicha inflamación es el paso previo a la aparición de calcificación en dicha válvula aórtica. Por tanto, de acuerdo con la enseñanza técnica de la presente invención, el experto en la materia podría usar inhibidores del receptor de S1 P en combinación con al menos un inhibidor del receptor TLR-4 y esperar que el resultado de prevención y/o tratamiento de la estenosis aórtica calcificada se viese incrementada de forma inesperada. 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. As demonstrated in the examples of the present invention, blocking this synergy decreases COX-2 and ICAM-1 mediated inflammation, and could block the inflammation associated with calcified aortic stenosis. Said inflammation is the previous step to the appearance of calcification in said aortic valve. Therefore, according to the technical teaching of the present invention, the person skilled in the art could use S1 P receptor inhibitors in combination with at least one TLR-4 receptor inhibitor and expect the result of prevention and / or treatment. Calcified aortic stenosis was unexpectedly increased.
El término "inhibidor del receptor TLR-4" tal como se utiliza en la presente invención, se refiere a una molécula que se une a cualquiera de los receptores TLR-4, preferiblemente de los receptores tipo TLR-4 y disminuye la expresión y/o actividad del receptor al que se une, y/o su señalización intracelular. The term "TLR-4 receptor inhibitor" as used in the present invention, 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.
El inhibidor se selecciona de la lista que comprende, pero sin que sirva de limitación, antagonistas (preferiblemente químicos), ARN de silenciamiento o anticuerpo específico para el receptor correspondiente (preferiblemente el anticuerpo es monoclonal). 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).
Más preferiblemente el inhibidor de TLR-4 es un antagonista, o cualquiera de sus sales, isómeros o solvatos farmacéuticamente aceptables. El antagonista de la presente invención es de tipo químico. El antagonista es un compuesto que se une a un receptor TLR-4, y actúa como un antagonista funcional bloqueando parcial o totalmente su activación por los agonistas. More preferably 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.
El antagonista de TLR-4 puede existir en forma de enantiómeros o diastereómeros. En la presente invención también se contempla el uso de solvatos del antagonista (como por ejemplo, pero sin limitarse, hidratos), prodrogas (sinónimo de profármacos), o clatratos. Las sales farmacéuticamente aceptables se seleccionan de entre cloruro, bromuro ioduro o cualquier otra sal farmacéuticamente aceptable. 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.
Los antagonistas de los receptores TLR-4 de la presente invención, pueden incluir isómeros, dependiendo de la presencia de enlaces múltiples (por ejemplo, Z, E), incluyendo isómeros ópticos o enantiómeros, dependiendo de la presencia de centros quirales. Los isómeros, enantiómeros o diastereoisómeros individuales y las mezclas de los mismos caen dentro del alcance de la presente invención. Los enantiómeros o diastereoisómeros individuales, así como sus mezclas, pueden separarse mediante técnicas convencionales. El antagonista de TLR-4 puede estar en forma cristalina como compuestos libres o como solvato. 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.
Asimismo, dentro del alcance de esta invención se encuentran las prodrogas o profármacos de los antagonistas de los receptores TLR-4. Also, within the scope of this invention are prodrugs or prodrugs of TLR-4 receptor antagonists.
Más preferiblemente el inhibidor del receptor TLR-4 se selecciona de la lista que comprende los antagonistas CRX-526, E5531 , E5564 o TAK-242 y los anticuerpos NI-0101 y 1A6. Otro aspecto de la presente invención se refiere a una composición farmacéutica que comprende al menos un inhibidor de un receptor de S1 P y al menos un inhibidor del receptor TLR-4, o cualquiera de las sales, isómeros o solvatos farmacéuticamente aceptables de dichos inhibidores. Según una realización preferida, la composición farmacéutica comprende, al menos, un vehículo y/o un excipiente farmacéuticamente aceptables. En la presente invención la definición de "composición farmacéutica" es la misma que la definición de "medicamento" que se ha efectuado en la presente descripción. Por tanto, los términos "composición farmacéutica" y "medicamento" se emplean como sinónimos. More preferably, 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. Another aspect of the present invention relates to 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. According to a preferred embodiment, the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier and / or excipient. In the present invention the definition of "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.
Los inhibidores de los receptores, tipos preferidos de los mismos y los propios receptores y preferencias han quedado explicados en la presente invención en párrafos precedentes. Receptor inhibitors, preferred types thereof and the receptors themselves and preferences have been explained in the present invention in preceding paragraphs.
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos y dibujos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. DESCRIPCIÓN DE LAS FIGURAS Throughout the description and 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. Muestra la expresión de transcritos de ARN mensajero de receptores de S1 P en células intersticiales de válvula aórtica humana. Los resultados de la PCR cuantitativa, una vez normalizados con el gen de referencia actina, indican que las células expresan todos los receptores de S1 P, siendo SI P2 el más abundante, seguido de SI P3 y S1 P4. Los resultados son representativos de más de 5 experimentos independientes. RNAm se refiere al ARN mensajero. 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. Muestra la activación de cascadas de señalización por S1 P y TLR4. FIG. 2. Shows the activation of signaling cascades by S1 P and TLR4.
Imagen fotográfica que ilustra que tanto los receptores de S1 P como los receptores TLR4 son funcionales pues el tratamiento celular con sus ligandos, S1 P y LPS respectivamente, promueve la activación de cascadas de señalización intracelulares como la MAP quinasa p38 en células intersticiales de válvula control (A) y válvula estenótica (B). La uniformidad de carga del gel se confirmó con un anticuerpo que reconoce la MAP quinasa ERK, quinasa regulada por señales extracelulares. R, reposo (células tratadas con el vehículo); LPS, lipopolisacárido; S1 P, esfingosina 1 -fosfato. 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. Muestra la sinergia de los receptores de S1 P y TLR4 en la inducción de moléculas pro-inflamatorias. FIG. 3. It shows the synergy of the S1 P and TLR4 receptors in the induction of pro-inflammatory molecules.
(A) Las células intersticiales de válvula control se trataron con esfingosina-1 - fosfato, S1 P (ligando de los receptores SI Pi-s) y/o LPS (ligando de TLR4), y se analizó la inducción de la expresión de la enzima COX-2 y la molécula de adhesión ICAM-1 usando anticuerpos específicos. Se observó un efecto sinérgico, pues el tratamiento combinado con S1 P y LPS induce la expresión de COX-2 e ICAM-1 en mayor medida que la suma del efecto de los agonistas por separado. El efecto sinérgico se observó a dosis fisiológicas de S1 P, entre 0,01 -1 μΜ, y cuando se trata en combinación con un ligando de TLR4 pero no con un ligando de TLR2, Pam3CSK4. La inducción de la expresión de inhibidores es inferior en células de válvula pulmonar. (B) Cinética en células intersticiales de válvula estenótica. El efecto de sinergia se observó a tiempos después de la estimulación celular de 8, 12 y 24h. Un anticuerpo anti-p-tubulina se usó para comprobar la uniformidad de carga. Los resultados son representativos de más de 5 experimentos independientes. (C) Mediante ensayos de ELISA se cuantificó la concentración de slCAM-1 secretado al medio extracelular de células tratadas como en el panel A. Los resultados son representativos de 3 experimentos independientes. (A) 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. COX-2 enzyme and the ICAM-1 adhesion molecule using specific antibodies. 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 μΜ, 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, células intersticiales de válvula aórtica; ERK, quinasa regulada por señales extracelulares; LPS (o también indicado con la abreviatura L), lipopolisacárido; Pam (o también indicado con la abreviatura P), Pam3CSK4, agonista de TLR2/TLR1 ; PIC, células intersticiales de válvula pulmonar; p-NF- KB, forma fosforilada del factor de transcripción NF-kappaB,; R, reposo (células tratadas con el vehículo); S1 P (o también indicado con la abreviatura S), esfingosina 1 -fosfato. AVIC, interstitial aortic valve cells; ERK, kinase regulated by extracellular signals; LPS (or also indicated with the abbreviation L), 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), sphingosine 1-phosphate.
FIG. 4. Muestra el efecto de los receptores de S1 P y TLR4 en la inducción de la actividad enzimática de fosfatasa alcalina en células intersticiales procedentes de válvula control. 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.
(A) La figura refleja la actividad enzimática de fosfatasa alcalina expresada como picomoles de producto generados por μgramos de proteína y hora, y es representativa de 4 experimentos independientes en duplicado. (B) Con objeto de comparar experimentos independientes, en la figura se representa el aumento de actividad enzimática inducido con respecto a la actividad enzimática en presencia de medio control. Las barras corresponden a la desviación estándar. *Asterisco indica p = 0,0160 cuando se compara con el efecto de medio condicionado (Análisis ANOVA, 3 columnas), ** p=0,005 cuando se compara el efecto con el del medio condicionado (Análisis ANOVA, 4 columnas). ***p=0,0107 cuando se compara el efecto con el de los agonistas por separado (ANOVA, 3 columnas). Crtl indica medio control; MC indica medio condicionado de calcificación. (A) The figure reflects the enzymatic activity of alkaline phosphatase expressed as product picomoles generated by μgrams of protein and hour, and is representative of 4 independent duplicate experiments. (B) In order to compare independent experiments, the figure shows the increase in enzyme activity induced with respect to enzyme activity in the presence of a control medium. The bars correspond to the standard deviation. * Asterisk indicates p = 0.0160 when compared with the effect of conditioned medium (ANOVA Analysis, 3 columns), ** p = 0.005 when the effect is compared with that of conditioned medium (ANOVA Analysis, 4 columns). *** p = 0.0107 when comparing the effect with that of the agonists separately (ANOVA, 3 columns). Crtl indicates medium control; MC indicates conditioned medium of calcification.
FIG. 5. Muestra el efecto de inhibición de la sinergia con algunos antagonistas de receptores de S1 P. FIG. 5. It shows the effect of inhibition of synergy with some S1 P receptor antagonists.
(A-D) Las células se pre-trataron con antagonistas farmacológicos de receptores de S1 P, posteriormente se activaron con S1 P y LPS, y se realizó el análisis como en la figura 3. El pre-tratamiento con suramina, antagonista de SI P3, bloquea el efecto sinérgico del tratamiento combinado con S1 P y LPS en la inducción de la expresión de COX-2 (5A), de ICAM-1 (5A); el tratamiento con PTX inhibe en menor medida que suramina (5A). Nuevos experimentos revelan que el pre-tratamiento con FTY720, antagonista funcional de S1 Pi , bloquea el efecto sinérgico del tratamiento combinado con S1 P y LPS en la inducción de la expresión de ICAM-1 (5B). En cuanto al efecto sinérgico del tratamiento combinado con S1 P y LPS en la inducción de la presencia de la forma soluble de ICAM en el medio extracelular, dicho efecto se inhibe con el pre-tratamiento con suramina, inhibidor de SI P3 (5C) y en menor medida con PTX (5C). Nuevos experimentos demuestran que el pre-tratamiento con W146, inhibidor de S1 Pi , bloquea el efecto sinérgico del tratamiento con S1 P y LPS en la inducción de la presencia de la forma soluble de ICAM-1 en el medio extracelular (5D). En nuevos experimentos se ha probado el efecto de otros antagonistas de receptores de S1 P como VPC 23019, inhibidor de S1 Pi y SI P3; pero el disolvente en que se diluye dicho producto interfiere con la medida de slCAM-1 y enmascara el posible efecto inhibidor de los antagonistas de receptores de S1 P ya que el disolvente per sé produce un aumento de slCAM-1 (5E). En los paneles A-B un anticuerpo anti-p-tubulina se usó para comprobar la uniformidad de carga. Los resultados del panel A son representativos de más de 5 experimentos de western blot independientes. El panel B es representativo de 2 experimentos independientes. Los paneles C y D son representativos de 3 experimentos de ELISA independientes. El panel E es representativo de 2 experimentos independientes. FTY indica FTY720; L, LPS; PTX, toxina pertussis (inhibidor de Gi, inhibe la señalización de varios receptores de S1 P); S, S1 P; Sura, Suramina. Disolvente de VPC se refiere al disolvente usado para reconstituir VPC 23019, y es dimetil sulfóxido: ácido clorhídrico 1 N: albúmina de suero bovino deslipidada (en proporción 0,95:0,05:20, v/v/v). (F) El ensayo de ARN de interferencia se realizó en células transfectadas con dúplex de ARN de interferencia específico para receptores S1 Pi, SI P2, SI P3 y SI P4 con objeto de bloquear su expresión. Como se observa, la inhibición de algunos receptores de S1 P bloquea el efecto de sinergia en la inducción de moléculas pro-inflamatorias. Los receptores de S1 P se pueden nombrar con la nomenclatura antigua en la que S1 PX equivale a EDG de la siguiente manera: S1 Pi/EDG1 , S1 P2/EDG5, S1 P3/EDG3 y SI P4/EDG6. (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). As for the synergistic effect of the combined treatment with S1 P and LPS in the induction of the presence of the soluble form of ICAM in the extracellular medium, said effect is inhibited with pre-treatment. with suramin, SI P3 inhibitor (5C) and to a lesser extent with PTX (5C). New experiments show that pre-treatment with W146, S1 Pi inhibitor, blocks the synergistic effect of treatment with S1 P and LPS in inducing the presence of the soluble form of ICAM-1 in the extracellular medium (5D). In new experiments, the effect of other S1 P receptor antagonists such as VPC 23019, S1 Pi inhibitor and SI P3 has been tested; but the solvent in which said product is diluted interferes with the measurement of slCAM-1 and masks the possible inhibitory effect of S1 P receptor antagonists since the solvent per se produces an increase in slCAM-1 (5E). In AB panels an anti-p-tubulin antibody was used to check load uniformity. The results of panel A are representative of more than 5 independent western blot experiments. Panel B is representative of 2 independent experiments. Panels C and D are representative of 3 independent ELISA experiments. Panel E is representative of 2 independent experiments. 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). (F) 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. As noted, the inhibition of some S1 P receptors blocks the effect of synergy in the induction of pro-inflammatory molecules. 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. Muestra el efecto de inhibición del efecto aditivo en la inducción de la actividad enzimática de fosfatasa alcalina con algunos antagonistas de receptores de S1 P. 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) La figura refleja la actividad enzimática de fosfatasa alcalina expresada como picomoles de producto generados por μgramos de proteína y hora, y es representativa de 3 experimentos independientes en duplicado. El pretratamiento de células intersticiales de válvulas aórticas control con VPC23019 inhibe el efecto aditivo en la inducción de la actividad enzimática de fosfatasa alcalina inducido por S1 P y LPS observado en la Figura 4A. El pretratamiento con JTE013, inhibidor de SI P2, inhibe el efecto aditivo, aunque en menor medida que VPC23019. (B) La figura incluye los datos de nuevos experimentos realizados. En la figura se representa el % de inhibición (valor medio ± error estándar) de la actividad enzimática de la fosfatasa alcalina inducida por el tratamiento combinado con S1 P y LPS (valor 100), y es representativa de 5-7 experimentos independientes. Los nuevos experimentos demuestran que el pretratamiento de células intersticiales de válvulas aórticas control con suramina, inhibidor de SI P3, y con toxina pertussis, bloquea de modo significativo el efecto aditivo en la inducción de la actividad enzimática de fosfatasa alcalina inducido por S1 P y LPS observado en la Figura 4A. Además, los nuevos experimentos confirman que el pretratamiento con JTE013 y con VPC23019 inhibe de modo significativo el efecto aditivo en la inducción de la actividad enzimática de fosfatasa alcalina inducido por S1 P y LPS (5B), y revelan que JTE013 es el inhibidor más potente. ALP indica fosfatasa alcalina, JTE, JTE013; MC medio condicionado de calcificación; LPS, lipopolisacárido; PTX, toxina pertussis; Sur, suramina; S1 P, esfingosina 1 -fosfato; VPC, VPC23019. * Asterisco indica que P < 0,05 con respecto al valor de referencia LPS + S1 P (Análisis de "t de student"). (A) The figure reflects the enzymatic activity of alkaline phosphatase expressed as product picomoles generated by μgrams 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. In addition, the new experiments confirm that pretreatment with JTE013 and with VPC23019 significantly inhibits the additive effect in the induction of the enzymatic activity of alkaline phosphatase induced by S1 P and LPS (5B), and reveals that JTE013 is the most potent inhibitor . ALP indicates alkaline phosphatase, JTE, JTE013; MC conditioned medium calcification; LPS, lipopolysaccharide; PTX, pertussis toxin; South, suramina; S1 P, sphingosine 1-phosphate; VPC, VPC23019. * Asterisk indicates that P <0.05 with respect to the reference value LPS + S1 P (Analysis of "student t").
EJEMPLOS EXAMPLES
Los siguientes ejemplos específicos que se proporcionan en este documento de patente sirven para ilustrar la naturaleza de la presente invención. Estos ejemplos se incluyen solamente con fines ilustrativos y no han de ser interpretados como limitaciones a la invención que aquí se reivindica. Por tanto, los ejemplos descritos más adelante ilustran la invención sin limitar el campo de aplicación de la misma. The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.
En los ejemplos y/o figuras de la presente invención se puede emplear la nomenclatura antigua de los receptores de S1 P. Los receptores de S1 P se pueden denominar como receptores EDG con la siguiente equivalencia: S1 Pi/EDG1 , S1 P2/EDG5, S1 P3/EDG3, SI P4/EDG6, SI P5/EDG8. In the examples and / or figures of the present invention, the old nomenclature of the S1 P receptors can be used. 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.
EJEMPLO 1. EXPRESIÓN DE RECEPTORES DE S1 P EN CÉLULAS INTERSTICIALES DE VÁLVULAS CARDÍACAS EXAMPLE 1. EXPRESSION OF S1 P RECEPTORS IN HEART VALVES INTERSTICIAL CELLS
Técnica 1a: Aislamiento y cultivo primario de células intersticiales procedentes de válvulas aórticas y pulmonares humanas. Se aislaron células intersticiales de válvulas aórticas de pacientes con estenosis aórtica calcificada a los que se reemplazó quirúrgicamente la válvula aórtica (estenóticas), y de válvulas aórticas y pulmonares procedentes de pacientes sometidos a trasplante cardiaco sin enfermedad valvular conocida (control no estenótica). Las muestras empleadas en la presente invención proceden de pacientes que están en el estadio avanzado de la estenosis aórtica y es calcificada; en estadios iniciales la estenosis aórtica es asintomática. El aislamiento celular se realizó según un método recientemente descrito basado en el tratamiento secuencial con colagenasa tipo I I (Meng et al. 2008, Am J Physiol Cell Physiol, 294: 29-35). La suspensión celular se centrífugo, se añadió medio M199 suplementado con penicilina G, estreptomicina, anfotericina B y 10% de suero de ternera fetal, y las células intersticiales se cultivaron en un incubador con atmósfera humidificada y un 5% de CO2. El cultivo primario se dejó crecer durante 7-10 días en monocapas hasta confluencia. La expresión de a2-actina se utilizó como marcador de células de estirpe de miofibroblasto. Las células intersticiales en cultivo son fundamentalmente miofibroblastos como se había descrito anteriormente (Meng et al. 2008, Am J Physiol Cell Physiol, 294:29- 35). Technical 1a: Isolation and primary culture of interstitial cells from human aortic and pulmonary valves. 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. 2008, Am J Physiol Cell Physiol, 294: 29-35). The cell suspension was centrifuged, M199 medium supplemented with penicillin G, streptomycin, amphotericin B and 10% fetal calf serum was added, and the interstitial cells were grown in a humidified atmosphere incubator and 5% CO2. The primary culture was allowed to grow for 7-10 days in monolayers until confluence. Expression a2-actin was used as a marker for myofibroblast lineage cells. Interstitial cells in culture are primarily myofibroblasts as previously described (Meng et al. 2008, Am J Physiol Cell Physiol, 294: 29-35).
Resultados: No se observaron diferencias morfológicas entre células procedentes de válvula aórtica y de válvula pulmonar, ni entre células aisladas de válvulas control y estenóticas. Results: No morphological differences were observed between cells from aortic valve and pulmonary valve, nor between cells isolated from control and stenotic valves.
Técnica 1b: Extracción de ARN y ensayos de PCR cuantitativa. El ARN mensajero se analizó mediante PCR cuantitativa (Real Time Quantitative Reverse Transcription). Para ello se aisló el ARN total mediante el método de Trizol, y mediante "retro-transcripción" usando una transcriptasa reversa se obtuvo cDNA, proporcionando así el molde o sustrato a la polimerasa estable para la reacción de PCR. La PCR se realizó utilizando primers específicos de receptores de S1 P y SYBR-Green siguiendo las instrucciones del fabricante (MJ Research). Los valores obtenidos se normalizaron respecto a un control interno para evitar la variabilidad en la concentración inicial de ARN. La especificidad de la reacción se determinó realizando la curva de desnaturalización de cada población de ADNs. La expresión relativa se calculó como 2 ACT donde A CT es la diferencia en los valores del ciclo umbral (CT) entre el gen dado y el gen de referencia interno, valor que se normaliza con respecto al valor control. Los cebadores usados son los siguientes: S1 Pi humano, cebador directo 5'-TATCAGCGCGGACAAGGAGAACAG-3' (SEQ ID NO: 1 ); cebador reverso ATAGGCAGGCCACCCAGGATGAG-3' (SEQ ID NO: 2). S1 P2 humano, cebador directo 5'-TCGGCCTTCATCGTCATCCTCT-3' (SEQ ID NO: 3); cebador reverso 5'-CCTCCCGGGCAAACCACTG-3' (SEQ ID NO: 4). S1 P3 humano, cebador directo 5'-CTTGGTCATCTGCAGCTTCAT-3' (SEQ ID NO: 5); cebador reverso 5'-TCATTGTCAAGTGCCGCTCGAT-3' (SEQ ID NO: 6). SI P4 humano, cebador directo 5'-GAGAGCGGGGCCACCAAGAC- 3' (SEQ ID NO: 7); cebador reverso 5'-GGTTGACCGCCGAGTTGAGGAC-3' (SEQ ID NO: 8). S1 P5 humano, cebador directo 5'- ACAACTACACCGGCAAGCTC-3' (SEQ ID NO: 9); cebador reverso 5'- GCCCCGACAGTAGGATGTT-3' (SEQ ID NO: 10). Technique 1b: 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. The relative expression was calculated as 2 ACT where A CT is the difference in the threshold cycle (CT) values between the given gene and the internal reference gene, a value that is normalized with respect to the control value. 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). S1 P 3 human, direct primer 5 ' -CTTGGTCATCTGCAGCTTCAT-3 ' (SEQ ID NO: 5); reverse primer 5'-TCATTGTCAAGTGCCGCTCGAT-3 '(SEQ ID NO: 6). 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).
Resultados: Los datos obtenidos, una vez normalizados con el gen de referencia, actina, indican que todos los receptores de S1 P se expresan, siendo el receptor SI P2 el más abundante, seguido de SI P3 y S1 P4. En las válvulas estenóticas se observa reducción de la expresión de receptores S1 P4 y SI P5 (Figura 1 ). EJEMPLO 2. FUNCIONALIDAD DE RECEPTORES DE S1 P Y TLR4: ACTIVACIÓN DE CASCADAS DE SEÑALIZACIÓN INTRACELULARES Results: The data obtained, once normalized with the reference gene, actin, indicate that all S1 P receptors are expressed, with the SI P 2 receptor being the most abundant, followed by SI P3 and S1 P 4 . In the stenotic valves, a reduction in the expression of S1 P 4 and SI P5 receptors is observed (Figure 1). EXAMPLE 2. FUNCTIONALITY OF S1 PY TLR4 RECEIVERS: ACTIVATION OF INTRACELLULAR SIGNALING WATERFALLS
Técnica: Extractos citoplasmáticos y análisis de Western blot. - Agonistas: Para los ensayos in vitro, la S1 P (a concentraciones de 0,01 -1 μΜ) se usó como agonista de los receptores de S1 P, y el lipopolisacárido (LPS, a concentraciones de 0.1 -^g/ml) se usó como ligando del receptor TLR4, que es el receptor tipo Toll mas expresado en células intersticiales de válvulas aórticas y cuya expresión aumenta en la estenosis aórtica calcificada (López J, et al. Int. J. Cardiol. DOI: 10.1016/j.ijcard.2010.12.089). Technique: Cytoplasmic extracts and Western blot analysis. - Agonists: For in vitro assays, S1 P (at concentrations of 0.01 -1 μΜ) was used as an agonist for S1 P receptors, and lipopolysaccharide (LPS, at concentrations of 0.1 - ^ g / ml) It was used as a ligand of the TLR4 receptor, which is the most expressed Toll-like receptor in interstitial cells of aortic valves and whose expression increases in calcified aortic stenosis (López J, et al. Int. J. Cardiol. DOI: 10.1016 / j. ijcard.2010.12.089).
Nota: En la presente invención se puede utilizar el sistema "Digital object identifier" (DOI) para poder localizar el artículo científico ya que no cambia con el paso del tiempo, aunque el artículo sea reubicado en una dirección web distinta puesto que lleva la información incorporada en forma de metadatos. Note: In the present invention, 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.
- Análisis de las cascadas de activación: usamos el método descrito anteriormente (López J, et al. Int. J. Cardiol. DOI: 10.1016/j.ijcard.2010.12.089). Las células se trataron con S1 P en combinación o no con LPS durante 15, 30, y 60 min. A continuación las células se lisaron con tampón TNE (Tris-HCI 20mM pH 7,4, NaCI 150 mM, EDTA 5 mM, NP40 al 0,01 %) durante 10 min en hielo. Las proteínas procedentes de lisados celulares, 30-50 μg de proteína total, se separaron por métodos de electroforesis en geles de poliacrilamida (SDS-PAGE) y se transfirieron a una membrana de nitrocelulosa mediante sistemas de transferencia en líquido. La membrana se incubó primeramente en TBS+5% leche, a continuación con el anticuerpo primario correspondiente durante toda la noche a 4°C, y posteriormente con un anticuerpo secundario durante 45 min. Las proteínas se visualizaron mediante quimioluminiscencia usando un kit de revelado de ECL (Amersham/GE). Se usaron anticuerpos frente a MAP quinasas (Cell Signalling): anti-P-p38 y como control de la uniformidad de la carga se usó un anticuerpo frente a ERK, quinasa regulada por señales extracelulares. Resultados. El tratamiento con S1 P promueve la activación de MAPK p38 con lo que se puede concluir que los receptores de S1 P son funcionales en células intersticiales de válvulas aórticas control (Figura 2A) y estenótica (Figura 2B). Además la activación conjunta de receptores de S1 P y TLR4 tiene efecto aditivo en la inducción de la activación de MAPK p38. La activación de MAPK p38 por LPS indica que los receptores TLR4 son funcionales, como se ha demostrado en un estudio previo (López J, et al. Int. J. Cardiol. DOI: 10.1016/j.ijcard.2010.12.089). EJEMPLO 3. MEDIADORES PRO-INFLAMATORIOS: EFECTO DE SINERGIA DE RECEPTORES DE S1 P Y TLR4 - Analysis of the activation cascades: we use the method described above (López J, et al. Int. J. Cardiol. DOI: 10.1016 / j.ijcard.2010.12.089). The cells were treated with S1 P in combination or not with LPS for 15, 30, and 60 min. The cells were then lysed with TNE buffer (20mM Tris-HCI pH 7.4, 150 mM NaCI, 5 mM EDTA, 0.01% NP40) for 10 min on ice. Proteins from cell lysates, 30-50 μg of total protein, were separated by polyacrylamide gels (SDS-PAGE) electrophoresis methods and transferred to a nitrocellulose membrane by liquid transfer systems. The membrane was first incubated in TBS + 5% milk, then with the corresponding primary antibody overnight at 4 ° C, and then with a secondary antibody for 45 min. Proteins were visualized by chemiluminescence using an ECL development kit (Amersham / GE). Antibodies against MAP kinases (Cell Signaling): anti-P-p38 were used and as a control of the uniformity of the load an antibody against ERK, kinase regulated by extracellular signals was used. Results Treatment with S1 P promotes the activation of MAPK p38 with which it can be concluded that S1 P receptors are functional in interstitial cells of control aortic valves (Figure 2A) and stenotic (Figure 2B). In addition, the joint activation of S1 P and TLR4 receptors has an additive effect in the induction of MAPK p38 activation. MAPK activation p38 by LPS indicates that TLR4 receptors are functional, as demonstrated in a previous study (López J, et al. Int. J. Cardiol. DOI: 10.1016 / j.ijcard.2010.12.089). EXAMPLE 3. PRO-INFLAMMATORY MEDIATORS: S1 PY TLR4 RECEIVER SYNERGY EFFECT
Técnica: Extractos citoplasmáticos celulares y análisis de Western blot; método de ELISA para cuantificar los niveles de mediadores en el medio extracelular. Se realizó el mismo método que en el ejemplo 2 usando anticuerpos específicos para la enzima COX-2 y la molécula de adhesión ICAM-1 (Santa Cruz). Se realizaron estudios de cinética de inducción y de dosis-respuesta. Como control de la carga se usaron anti- β actina o anti-p-tubulina (Sigma). Resultados: El tratamiento celular con 1 μg/ml LPS induce la expresión de los mediadores pro-inflamatorios ciclooxigenasa (COX)-2 e ICAM-1 a tiempos largos 12-24h, y 1 μΜ S1 P induce su expresión, aunque en menor medida. Sorprendentemente, el tratamiento combinado con S1 P 1 μΜ y LPS de E. coli 1 μg/ml induce la expresión de COX-2 e ICAM-1 en mayor medida que el efecto de la suma de los agonistas, lo que indica un efecto de sinergia de acción entre receptores de S1 P y TLR4 en células intersticiales de válvula aórtica (Figura 3A, panel izquierdo). Technique: Cellular cytoplasmic extracts and Western blot analysis; ELISA method to quantify mediator levels in the extracellular environment. The same method as in example 2 was performed using antibodies specific for the COX-2 enzyme and the adhesion molecule ICAM-1 (Santa Cruz). Induction and dose-response kinetics studies were performed. Anti-β actin or anti-p-tubulin (Sigma) was used as load control. Results: Cell treatment with 1 ug / ml LPS induces the expression of proinflammatory mediators cyclooxygenase (COX) -2 and ICAM-1 to long 12-24h, and 1 μΜ S1P induces expression, albeit less . Surprisingly, combination treatment with S1 P1 μΜ and E. coli LPS 1 ug / ml induced the expression of COX-2 and ICAM-1 to a greater extent than the effect of the addition of agonists, indicating an effect of synergy of action between S1 P and TLR4 receptors in aortic valve interstitial cells (Figure 3A, left panel).
A continuación se estudió la cinética del efecto. La inducción de COX-2 se observa a 8, 12 y 24h después de la estimulación celular, incluso se observa en menor medida a tiempos tardíos como 48h y 72h (Figura 3B). En cuanto a la dosis respuesta, manteniendo constante la concentración de LPS a 1 μg/ml y el tiempo de incubación de 12 h, se varió la dosis de S1 P. El efecto sinérgico se observa a dosis fisiológicas de S1 P, entre 0,01 -1 μΜ, y en combinación con un ligando de TLR4 (LPS) pero no de TLR2 (Pam3CSK4). Next, the kinetics of the effect were studied. The induction of COX-2 is observed at 8, 12 and 24h after cell stimulation, it is even observed to a lesser extent at late times such as 48h and 72h (Figure 3B). Regarding the dose response, keeping the concentration of LPS constant at 1 μg / ml and the incubation time of 12 h, the dose of S1 P was varied. The synergistic effect is observed at physiological doses of S1 P, between 0, 01 -1 μΜ, and in combination with a ligand of TLR4 (LPS) but not of TLR2 (Pam3CSK4).
Seguidamente se cuantificaron los niveles de la forma soluble de la proteína ICAM-1 en el medio extracelular de células intersticiales. Se observó que el tratamiento de las células con LPS 1 μ9/ηπΙ induce la secreción al medio extracelular de slCAM-1 , y sorprendentemente el tratamiento combinado con S1 P 1 μΜ y LPS 1 μ9/ηιΙ induce un aumento de los niveles de slCAM-1 superior a la suma simple del efecto de los agonistas, lo que indica un efecto de sinergia de acción entre receptores de S1 P y TLR4 (Figura 3C). The levels of the soluble form of the ICAM-1 protein in the extracellular medium of interstitial cells were then quantified. It was observed that the Treatment of cells with LPS 1 μ9 / ηπΙ induces secretion to the extracellular medium of slCAM-1, and surprisingly the combined treatment with S1 P 1 μΜ and LPS 1 μ9 / ηιΙ induces an increase in slCAM-1 levels greater than simple sum of the effect of the agonists, which indicates an effect of synergy of action between S1 P and TLR4 receptors (Figure 3C).
Tal como se ha indicado, se observó que la activación simultánea de los receptores de S1 P y TLR4 en las células intersticiales de válvula aórtica promueve un efecto de sinergia en la inducción mantenida y exacerbada de mediadores pro-inflamatorios, lo que podría contribuir al proceso de inflamación crónica que se asocia a la enfermedad de la estenosis aórtica calcificada. En concreto el tratamiento de células intersticiales con los agonistas correspondientes, S1 P y LPS respectivamente, induce un efecto sinérgico en la expresión de (i) COX-2, enzima inducible y precursora de mediadores inflamatorios lipidíeos como prostaglandinas y prostaciclinas, (ii) ICAM-1 , molécula de adhesión asociada a membranas, iii) la forma soluble de ICAM-1 , que se ha asociado al aumento de la prevalencia y la severidad de la calcificación de la válvula aórtica (Shavelle et al. 2008. J.Heart Valve Dis., 17: 388-95) (Figura 3A). El efecto sinérgico se observa a dosis fisiológicamente relevantes de S1 P (Figura 3A), y se mantiene durante muchas horas (Figura 3B). Inesperadamente, el efecto de inducción es mucho menos marcado en células de origen pulmonar (Figura 3A, panel derecho) que en las de origen aórtico (Figura 3A, panel izquierdo), lo que estaría de acuerdo con el hecho de que las válvulas pulmonares raramente sufren procesos patológicos de estenosis. As indicated, it was observed that the simultaneous activation of S1 P and TLR4 receptors in aortic valve interstitial cells promotes a synergy effect in the sustained and exacerbated induction of pro-inflammatory mediators, which could contribute to the process of chronic inflammation that is associated with the disease of calcific aortic stenosis. Specifically, the treatment of 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). Unexpectedly, the effect of induction is much less marked pulmonary origin cells (Figure 3A, right panel) in the aortic origin (Figure 3A, left panel), which would agree with the fact that the pulmonary valves rarely They suffer pathological processes of stenosis.
EJEMPLO 4. CALCIFICACIÓN IN VITRO: EFECTO ADITIVO DE RECEPTORES DE S1 P Y TLR4 Técnica: Medida de la expresión y de la actividad de un marcador temprano de calcificación, fosfatasa alcalina (ALP). Se usó el método descrito anteriormente (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). Brevemente, se sembraron 20.000 células intersticiales de válvulas control por pocilio en una placa de 24 pocilios, y se cultivaron en medio condicionado (Medio M199 suplementado con glicerofosfato 10 mM, vitamina D3 10 nM, dexametasona 10 nM) solo o en presencia de 0, 1 μΜ S1 P, ^g/ml LPS, o de una combinación de ambos. En paralelo las células se cultivaron en el medio M199 usado habitualmente para su cultivo (medio control, Ctrl). Se añadió medio fresco 2 veces a la semana. A los 15-21 días de cultivo se midió la actividad de ALP. 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) . Briefly, 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 µΜ S1 P, ^ g / ml LPS, or a combination of both. In parallel, 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.
Actividad enzimática de ALP (método cuantitativo). Las células se lisaron en un tampón compuesto por Tris-HCI 20 mM, NaCI 150 mM, NP40 al 0.2%, y glicerol al 10%, pH 8). La actividad enzimática se midió en los lisados celulares (<5μgr de proteína) usando un kit fluorométrico muy sensible, en el que el sustrato está acoplado a un marcador fluorescente, metilumbiliferona fosfato (MUP). En un tampón de glicina se incubaron el sustrato MUP y las muestras durante 30 min a 25 °C y protegido de la luz. A continuación se paró la reacción, y se midió la fluorescencia a la longitud de onda de 360 excitación/440 nm emisión. La actividad de fosfatasa alcalina se calculó usando una curva estándar y normalizando frente a la concentración de proteína. Los resultados se expresaron como pmol^g de proteína por hora de reacción. Resultados: Se estudió la inducción de la actividad enzimática de la fosfatasa alcalina (ALP), marcador temprano de calcificación (Figura 4A-B). Los resultados indican que S1 P tiene un efecto pro-osteogénico, pues induce la actividad de ALP con respecto a los valores obtenidos con el medio condicionado y el medio control (Figura 4B). El efecto de S1 P es similar al observado con LPS, que recientemente se ha descrito como un activador de la expresión y actividad de ALP (Yang X, et al. 2009. J Am Coll Cardiol, 53: 491 - 500; López J, et al. Int. J. Cardiol. DOI: 10.1016/j.ijcard.2010.12.089). Más aún, el tratamiento combinado de S1 P con LPS tiene un efecto aditivo en el aumento de la actividad enzimática del marcador temprano de calcificación ALP. Por tanto, se puede concluir que los receptores S1 P/EDG y TLR4 cooperan en la inducción de calcificación in vitro, lo que podría contribuir al proceso de osteogénesis característico de la estenosis aórtica calcificada. EJEMPLO 5. BLOQUEO DE LA SINERGIA EN LA INDUCCIÓN DE MOLÉCULAS PRO-INFLAMATORIAS MEDIANTE ANTAGONISTAS DE ALGUNOS RECEPTORES DE S1 P Técnica: Ensayos de inhibición farmacológica con antagonistas comerciales y análisis por Western blot y ELISA (tal como se ha descrito en el ejemplo 3). Se estudió el efecto de antagonistas comerciales específicos del receptor S1 Pi (10 μΜ VPC23019, Avanti Polar, 10 μΜ W146, Cayman Chem.; 1 μΜ FTY720, Cayman Chem.), de S1 P3 (10 μΜ de suramina, Biomol, 10 μΜ VPC23019, Avanti Polar), de S1 P2 (10 μΜ de JTE013, Tocris), y el efecto de la 100 ng/ml de toxina pertussis (Sigma Aldrich). La toxina y los antagonistas se preincubaron durante 1 h y después se incubaron con S1 P 0, 1 μΜ + LPS ^g/ml durante 12 horas y a continuación los lisados celulares se analizaron como se ha descrito en el ejemplo 3 (expresión de COX-2 e ICAM-1 , y la secreción al medio extracelular de slCAM-1 ) y como en el ejemplo 4 (actividad enzimática de ALP). 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 (<5μgr protein) using a very sensitive fluorometric kit, in which the substrate is coupled to a fluorescent marker, methylumbiliferone phosphate (MUP). In a glycine buffer, the MUP substrate and samples were incubated for 30 min at 25 ° C and protected from light. The reaction was then stopped, and the fluorescence was measured at the wavelength of 360 excitation / 440 nm emission. 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. J Am Coll Cardiol, 53: 491-500; López J, et al. Int. J. Cardiol. DOI: 10.1016 / j.ijcard.2010.12.089). Moreover, the combined treatment of S1 P with LPS has an additive effect in increasing the enzymatic activity of the early ALP calcification marker. Therefore, it can be concluded that the S1 P / EDG and TLR4 receptors cooperate in the induction of in vitro calcification, which could contribute to the characteristic osteogenesis process of calcified aortic stenosis. EXAMPLE 5. BLOCK OF SYNERGY IN THE INDUCTION OF PRO-INFLAMMATORY MOLECULES BY ANTAGONISTS OF SOME P RECEIVERS Technique: Pharmacological inhibition assays with commercial antagonists and Western blot and ELISA analysis (as described in example 3) . The effect of specific commercial antagonists of the S1 Pi receptor (10 μΜ VPC23019, Avanti Polar, 10 μΜ W146, Cayman Chem .; 1 μΜ FTY720, Cayman Chem.), Of S1 P 3 (10 μΜ of suramin, Biomol, 10 was studied μΜ VPC23019, Avanti Polar), of S1 P 2 (10 μΜ of JTE013, Tocris), and the effect of 100 ng / ml of pertussis toxin (Sigma Aldrich). The toxin and antagonists were pre-incubated for 1 h and then incubated with S1 P 0.1 µL + 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).
Resultados: El estudio reveló que la suramina, inhibidor de S1 P3, bloquea el efecto sinérgico del tratamiento combinado con S1 P y LPS tanto a nivel de la inducción de la expresión de las moléculas pro-inflamatorias COX-2 (Figura 5A) como de ICAM-1 (Figura 5A). El tratamiento con PTX, que inhibe G¡/0 y por tanto la señalización intracelular de los receptores de S1 P especialmente S1 Pi,3, bloquea la respuesta sinérgica aunque en menor medida (Figuras 5A). Además, los nuevos experimentos demuestran que FTY720, inhibidor de S1 Pi inhibe la inducción de la expresión de la molécula pro-inflamatoria ICAM-1 (Figura 5B). En cuanto al efecto sinérgico del tratamiento combinado con S1 P y LPS en los niveles de slCAM-1 en el medio extracelular, este se inhibió con el pretratamiento con la suramina (Figura 5C), con PTX (Figura 5C). Además, los nuevos experimentos demuestran que dicho efecto se puede bloquear con W146 (Figura 5D), que es un inhibidor de S1 Pi . Hay que hacer notar que se han probado el efecto de más inhibidores, como VPC 23019, antagonista de S1 Pi y SI P3. Sin embargo, el disolvente de dicho compuesto [dimetil sulfóxido: ácido clorhídrico 1 N: albúmina de suero bovino deslipidada (en proporción 0,95:0,05:20, v/v/v)], interfiere con el ensayo de ELISA de slCAM-1 , ya que produce un potente efecto inductor de slCAM-1 (5E), que enmascararía un potencial efecto del antagonista, por lo que no se puede probar el posible efecto inhibitorio de VPC 23019 en la secreción de slCAM-1 . Por tanto, del estudio farmacológico se puede concluir que S1 P3 y S1 Pi y probablemente otros receptores de S1 P, están implicados en el efecto de sinergia observada, y que bloqueando dichos receptores se reduciría la inflamación asociada a COX- 2 e ICAM-1 , es más se inhibirían los niveles de un biomarcador asociado al aumento de prevalencia y severidad de la calcificación de la válvula aórtica, slCAM-1 . Results: The study revealed that suramin, S1 P 3 inhibitor, blocks the synergistic effect of the combined treatment with S1 P and LPS both at the level of induction of the expression of COX-2 pro-inflammatory molecules (Figure 5A) and of ICAM-1 (Figure 5A). PTX treatment, which inhibits G¡ / 0 and therefore the intracellular signaling of S1 P receptors, especially S1 Pi, 3, blocks the synergistic response although to a lesser extent (Figures 5A). In addition, the new experiments demonstrate that FTY720, S1 Pi inhibitor inhibits the induction of the expression of the pro-inflammatory molecule ICAM-1 (Figure 5B). Regarding the synergistic effect of the combined treatment with S1 P and LPS on the levels of slCAM-1 in the extracellular medium, this was inhibited by pretreatment with suramin (Figure 5C), with PTX (Figure 5C). In addition, the new experiments demonstrate that this effect can be blocked with W146 (Figure 5D), which is an inhibitor of S1 Pi. It should be noted that the effect of more inhibitors, such as VPC 23019, antagonist of S1 Pi and SI P3, have been tested. However, the solvent of said compound [dimethyl sulfoxide: 1 N hydrochloric acid: bovine serum albumin slipped (in proportion 0.95: 0.05: 20, v / v / v)], interferes with the ELISA test of slCAM-1, as it produces a potent inducing effect of slCAM-1 (5E), which would mask a potential effect of antagonist, so the possible inhibitory effect of VPC 23019 on the secretion of slCAM-1 cannot be tested. Therefore, from the pharmacological study it can be concluded that S1 P 3 and S1 Pi and probably other S1 P receptors are involved in the observed synergy effect, and that blocking said receptors would reduce the inflammation associated with COX-2 and ICAM- 1, moreover, the levels of a biomarker associated with the increased prevalence and severity of aortic valve calcification, slCAM-1, would be inhibited.
Técnica: Silenciamiento de la expresión génica de receptores de S1 P mediante oligonucleótidos de ARN (siRNA). Technique: Silence of gene expression of S1 P receptors by RNA oligonucleotides (siRNA).
Se usaron oligonucleótidos específicos de ARN de los receptores de S1 P/EDG validados en estudios previos del grupo (Dueñas A, et al. 2008. Cardiovasc. Res. 79: 537-544). Las células intersticiales se transfectaron usando el reactivo Dharmafect (Dharmacon) con 25 nM de siRNA de doble cadena diseñados para los receptores específicos (S1 Pi SI P2, SI P3 o S1 P4). Mediante PCR cuantitativa se comprobó la inhibición específica de la expresión de receptores S1 Pi SI P2, SI P3 o SI P4 con objeto de bloquear su expresión, y el resto de células se activaron con S1 P en presencia o no de LPS y se analizaron mediante ensayos de Western blot como en el ejemplo 3. RNA-specific oligonucleotides of the S1 P / EDG receptors validated in previous group studies were used (Dueñas A, et al. 2008. Cardiovasc. Res. 79: 537-544). Interstitial cells were transfected using the Dharmafect reagent (Dharmacon) with 25 nM double-stranded siRNA designed for specific receptors (S1 Pi SI P2, SI P3 or S1 P 4 ). The specific inhibition of the expression of S1 Pi receptors SI P 2 , SI P3 or SI P 4 was checked by quantitative PCR in order to block its expression, and the rest of the cells were activated with S1 P in the presence or absence of LPS and analyzed by Western blot assays as in example 3.
Resultados: Los ensayos de interferencia de ARN del receptor S1 P4 demuestran que el receptor S1 P4 (para el que no hay antagonista químico comercial específico, es por ello que se emplea el ARN de interferencia), está también implicado en el proceso de sinergia de receptores de S1 P y TLR4 en la inducción de moléculas pro-inflamatorias. Además, el silenciamiento del gen del receptor S1 Pi o del receptor SI P2, bloquea el efecto de sinergia (Figura 5F); y el silenciamiento de la expresión del receptor SI P3 también inhibe el efecto sinérgico, lo que está de acuerdo con los resultados observados en el estudio farmacológico. En conjunto, los resultados del ensayo de interferencia muestran que cuando se reducen los niveles de los receptores S1 Pi , S1 P2, SI P3 o SI P4, se bloquea el efecto de sinergia de receptores de S1 P y TLR4 en la inducción de moléculas pro-inflamatorias y como consecuencia dichos antagonistas de los receptores S1 P podrían tener aplicación terapéutica. Results: The 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. In addition, 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. Together, the results of the interference test show that when the levels of the S1 Pi, S1 P 2 , SI P3 or SI P 4 receptors are reduced, the synergy effect of S1 P and TLR4 receptors is blocked in the induction of pro-inflammatory molecules and as a consequence said antagonists of the S1 P receptors could have therapeutic application.
EJEMPLO 6. BLOQUEO DEL EFECTO ADITIVO DE S1 P Y TLR4 EN LA CALCIFICACION IN VITRO MEDIANTE ANTAGONISTAS DE ALGUNOS RECEPTORES DE S1 P EXAMPLE 6. BLOCK OF THE ADDITIVE EFFECT OF S1 P AND TLR4 IN THE IN VITRO CALCIFICATION BY ANTAGONISTS OF SOME P RECEIVERS
Técnica: Medida de la actividad de un marcador temprano de calcificación, ALP, tal como se ha descrito en el ejemplo 4. Pretratamiento celular con antagonistas de receptores de S1 P previa a la activación con S1 P y LPS como se ha descrito en el ejemplo 5. Technique: Measurement of activity of marker early calcification, ALP as described in Example 4. Pretreatment with cellular receptor antagonists S1 P prior to activation with LPS and S1 P as described in Example 5.
Resultados: El estudio reveló que VPC23019 (10 μΜ), antagonista de receptores S1 Pi y SI P3, inhibe el efecto aditivo de receptores S1 P/EDG y TLR4 en la actividad ALP. Además, JTE013 (10 μΜ), un inhibidor de S1 P2, y, inhibe dicha actividad, aunque en menor medida (Figura 6A). Se han realizado nuevos experimentos con estos y otros inhibidores que revelan que el pretratamiento con suramina (10 μΜ), un inhibidor de SI P3, y con toxina pertussis (100 ng/ml), que inhibe la señalización de varios receptores de S1 P, bloquea de modo estadísticamente significativo el efecto aditivo de S1 P y LPS en la actividad de ALP (Figura 6B). Además, los resultados confirman la inhibición de la actividad ALP con JTE013 y VPC23019 (Figura 6B) observada en la Figura 6A. Por tanto, los resultados muestran que cuando se reducen los niveles de S1 P3 o S1 P2, y probablemente S1 Pi , se bloquea el efecto aditivo de S1 P y TLR4 en la calcificación in vitro, y como consecuencia los antagonistas de dichos receptores podrían tener aplicación terapéutica. Results: The study revealed that VPC23019 (10 μΜ), antagonist of S1 Pi and SI P3 receptors, inhibits the additive effect of S1 P / EDG and TLR4 receptors on ALP activity. In addition, JTE013 (10 μΜ), an inhibitor of S1 P 2 , and , inhibits such activity, although to a lesser extent (Figure 6A). New experiments have been performed with these and other inhibitors that reveal that pretreatment with suramin (10 μΜ), an inhibitor of SI P3, and with pertussis toxin (100 ng / ml), which inhibits the signaling of several S1 P receptors, It statistically blocks the additive effect of S1 P and LPS on ALP activity (Figure 6B). In addition, the results confirm the inhibition of ALP activity with JTE013 and VPC23019 (Figure 6B) observed in Figure 6A. Therefore, the results show that when the levels of S1 P 3 or S1 P 2 are reduced, and probably S1 Pi , the additive effect of S1 P and TLR4 on in vitro calcification is blocked, and consequently the antagonists of said receptors They could have therapeutic application.

Claims

REIVINDICACIONES
Uso de al menos un inhibidor de un receptor de esfingosina-1 -fosfato (S1 P) para la elaboración de un medicamento para la prevención y/o el tratamiento de estenosis aórtica calcificada. Use of at least one inhibitor of a sphingosine-1-phosphate receptor (S1 P) for the preparation of a medicament for the prevention and / or treatment of calcified aortic stenosis.
Uso según la reivindicación 1 donde el receptor de S1 P es S1 Pi, SI P2,
Figure imgf000038_0001
Use according to claim 1 wherein the S1 P receiver is S1 Pi, SI P2,
Figure imgf000038_0001
Uso según cualquiera de las reivindicaciones 1 ó 2, donde el inhibidor es un antagonista, o cualquiera de sus sales, isómeros o solvatos farmacéuticamente aceptables. Use according to any one of claims 1 or 2, wherein the inhibitor is an antagonist, or any of its pharmaceutically acceptable salts, isomers or solvates.
Uso según la reivindicación 3 donde el antagonista se selecciona de la lista que además comprende FTY720, VPC23019, VPC25239, VPC01091 , Ascotricina A, Ascotricina B, W146, W123, BML-241 , JTE013, Suramina o toxina pertussis, o cualquiera de sus sales, isómeros o solvatos farmacéuticamente aceptables. Use according to claim 3 wherein the antagonist is selected from the list which further comprises FTY720, VPC23019, VPC25239, VPC01091, Ascotricin A, Ascotricin B, W146, W123, BML-241, JTE013, Suramine or pertussis toxin, or any of its salts , pharmaceutically acceptable isomers or solvates.
Uso según la reivindicación 4 donde el antagonista se selecciona de la lista que comprende VPC23019, JTE013, Suramina, W146, FTY720 o toxina pertussis, o cualquiera de sus sales, isómeros o solvatos farmacéuticamente aceptables. Use according to claim 4 wherein the antagonist is selected from the list comprising VPC23019, JTE013, Suramin, W146, FTY720 or pertussis toxin, or any of its salts, isomers or pharmaceutically acceptable solvates.
Uso según cualquiera de las reivindicaciones 1 ó 2 donde el inhibidor es un ARN de silenciamiento (siRNA). Use according to any one of claims 1 or 2 wherein the inhibitor is a silencing RNA (siRNA).
Uso según la reivindicación 6 donde si el receptor es S1 Pi , el siRNA es un ARN de doble cadena cuya secuencia sentido es SEQ ID NO: 1 1 y la cadena antisentido es SEQ ID NO: 12. Use according to claim 6 wherein if the receiver is S1 Pi, the siRNA is a double stranded RNA whose sense sequence is SEQ ID NO: 1 1 and the antisense chain is SEQ ID NO: 12.
8. Uso según la reivindicación 7, donde la secuencia de siRNA de doble cadena está codificada en una secuencia A-B de ADN, donde A es SEQ ID NO: 17 y B es SEQ ID NO: 18. 8. Use according to claim 7, wherein the double stranded siRNA sequence is encoded in a DNA A-B sequence, wherein A is SEQ ID NO: 17 and B is SEQ ID NO: 18.
9. Uso según la reivindicación 6 donde si el receptor es SI P3, el siRNA es un ARN de doble cadena cuya secuencia sentido es SEQ ID NO: 13 y la cadena antisentido es SEQ ID NO: 14. 9. Use according to claim 6 wherein if the receiver is SI P 3 , the siRNA is a double stranded RNA whose sense sequence is SEQ ID NO: 13 and the antisense chain is SEQ ID NO: 14.
10. Uso según la reivindicación 9, donde la secuencia de siRNA de doble cadena está codificada en una secuencia A-B de ADN, donde A es SEQ ID NO: 20 y B es SEQ ID NO: 21. 10. Use according to claim 9, wherein the double stranded siRNA sequence is encoded in a DNA A-B sequence, wherein A is SEQ ID NO: 20 and B is SEQ ID NO: 21.
1 1 . Uso según la reivindicación 6 donde si el receptor es S1 P4, el siRNA es un ARN de doble cadena cuya secuencia sentido es SEQ ID NO: 15 y la cadena antisentido es SEQ ID NO: 16. eleven . Use according to claim 6 wherein if the receiver is S1 P 4 , the siRNA is a double stranded RNA whose sense sequence is SEQ ID NO: 15 and the antisense chain is SEQ ID NO: 16.
12. Uso según la reivindicación 1 1 , donde la secuencia de siRNA de doble cadena está codificada en una secuencia A-B de ADN, donde A es SEQ ID NO: 23 y B es SEQ ID NO: 24. 12. Use according to claim 1, wherein the double-stranded siRNA sequence is encoded in a DNA A-B sequence, wherein A is SEQ ID NO: 23 and B is SEQ ID NO: 24.
13. Uso según la reivindicación 6, donde si el receptor es SI P2, el siRNA es un ARN de doble cadena cuya secuencia sentido es SEQ ID NO: 35 y la cadena antisentido es SEQ ID NO: 36. 13. Use according to claim 6, wherein if the receiver is SI P2, the siRNA is a double stranded RNA whose sense sequence is SEQ ID NO: 35 and the antisense chain is SEQ ID NO: 36.
14. Uso según la reivindicación 13, donde la secuencia de siRNA de doble cadena está codificada en una secuencia A-B de ADN, donde A es SEQ ID NO: 37 y B es SEQ ID NO: 38. 14. Use according to claim 13, wherein the double stranded siRNA sequence is encoded in a DNA A-B sequence, wherein A is SEQ ID NO: 37 and B is SEQ ID NO: 38.
15. Uso según cualquiera de las reivindicaciones 8, 10, 12 y 14, donde entre la secuencia A y B hay una secuencia nucleotídica espadadora de al menos un nucleótido de longitud. 15. Use according to any of claims 8, 10, 12 and 14, wherein between the sequence A and B there is a spacer nucleotide sequence of at least one nucleotide in length.
16. Uso según cualquiera de las reivindicaciones 1 a 15, donde el medicamento además comprende un vehículo y/o un excipiente farmacéuticamente aceptables. 16. Use according to any of claims 1 to 15, wherein the medicament further comprises a pharmaceutically acceptable carrier and / or excipient.
17. Uso según cualquiera de las reivindicaciones 1 a 16, donde el inhibidor del receptor de S1 P se usa en combinación con al menos un inhibidor del receptor de la inmunidad innata tipo Toll 4 (TLR-4). 17. Use according to any one of claims 1 to 16, wherein the S1 P receptor inhibitor is used in combination with at least one Toll 4 innate immunity receptor inhibitor (TLR-4).
18. Composición farmacéutica que comprende al menos un inhibidor de un receptor de S1 P y al menos un inhibidor del receptor TLR-4, o cualquiera de las sales, isómeros o solvatos farmacéuticamente aceptables de dichos inhibidores. 18. 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.
19. Composición farmacéutica según la reivindicación 18, donde dicha composición comprende, al menos, un vehículo y/o un excipiente farmacéuticamente aceptables. 19. Pharmaceutical composition according to claim 18, wherein said composition comprises at least one pharmaceutically acceptable carrier and / or excipient.
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