ES2278519B1

ES2278519B1 - Dinucleotides for treatment of achondroplasia, reduce presence of mutated receptor in chondrocytes that shows decrease of pathological effects

Info

Publication number: ES2278519B1
Application number: ES200502229A
Authority: ES
Inventors: Jesus Pintor Just; Ana Isabel Guzman Aranguez; Marta Irazu Martinez; Asuncion Peral Cerda
Original assignee: FUNDACION MAGAR
Current assignee: FUNDACION MAGAR
Priority date: 2005-09-02
Filing date: 2005-09-02
Publication date: 2008-09-16
Anticipated expiration: 2025-09-02
Also published as: ES2278519A1

Abstract

The dinucleotides reduce the presence of a mutated receptor in chondrocytes that shows a decrease of the pathological effects associated with the disease. The dinucleotides of formula (I) reduce the presence of a mutated receptor in chondrocytes that shows a decrease of the pathological effects associated with the disease. [variables not defined] [Image].

Description

Tratamiento de la acondroplasia por la administración de dinucleótidos.Treatment of achondroplasia due to administration of dinucleotides.

Object of the invention

La presente invención describe el empleo de determinados dinucleótidos para el tratamiento de la patología denominada acondroplasia o enanismo.The present invention describes the use of certain dinucleotides for the treatment of pathology called achondroplasia or dwarfism.

State of the art

El crecimiento de los huesos largos depende de la proliferación y maduración de las células que constituyen el cartílago, los condrocitos. Estas células van a ser reemplazadas de modo gradual por el tejido óseo durante el crecimiento del individuo. Los condrocitos en su proceso de maduración pasan por una primera etapa en la que se hipertrofian para posteriormente morir en un proceso de tipo apoptótico (Roach et al., 1995; Gibson et al., 1997).The growth of long bones depends on the proliferation and maturation of the cells that make up the cartilage, the chondrocytes. These cells will be gradually replaced by bone tissue during the growth of the individual. Chondrocytes in their maturation process go through a first stage in which they hypertrophy to later die in an apoptotic type process (Roach et al ., 1995; Gibson et al ., 1997).

El crecimiento y desarrollo de los condrocitos en los cartílagos que flanquean los huesos está controlado por los factores de crecimiento de fibroblastos. Estos factores activan cuatro tipos diferentes de receptores denominados FGFR1, FGFR2, FGFR3 y FGFR4, estando los tres primeros implicados en enfermedades congénitas esqueléticas y craneales. Dentro de estas alteraciones destaca la acondroplasia, la forma más común de enanismo congénito. Esta patología se caracteriza por una talla baja desproporcionada y otras anormalidades del esqueleto (Anon,
1988).The growth and development of chondrocytes in the cartilage that flank the bones is controlled by fibroblast growth factors. These factors activate four different types of receptors called FGFR1, FGFR2, FGFR3 and FGFR4, the first three being involved in congenital skeletal and cranial diseases. Among these alterations, achondroplasia , the most common form of congenital dwarfism, stands out . This pathology is characterized by a disproportionate short stature and other abnormalities of the skeleton (Anon,
1988).

El receptor alterado en la acondroplasia es el FGFR3, cuyo gen se localiza en el extremo distal del brazo corto del cromosoma 4 (4p16.3). Los estudios realizados en distintas poblaciones, han establecido la existencia de una mutación frecuente (Gly380Arg) en el dominio transmembrana del receptor (Shiang et al., 1994; Rousseau et al., 1994). Junto con esta modificación, observada en el 97% de los casos, también se han identificado otras mutaciones menos frecuentes como el cambio de una Gly 375 por Cys fuera de la región transmembrana (Superti-Furga et al., 1995; Chen et al., 1999), la sustitución de la Gly 346 por Glu (Prinos et al., 1995), así como el reemplazamiento de la Lys 650 por Met en el caso de la forma más severa de acondroplasia (Iwata et al.,
2000).The altered receptor in achondroplasia is FGFR3, whose gene is located at the distal end of the short arm of chromosome 4 (4p16.3). Studies in different populations have established the existence of a frequent mutation (Gly380Arg) in the transmembrane domain of the recipient (Shiang et al ., 1994; Rousseau et al ., 1994). Along with this modification, observed in 97% of cases, other less frequent mutations have also been identified, such as the exchange of a Gly 375 for Cys outside the transmembrane region (Superti-Furga et al ., 1995; Chen et al . , 1999), the replacement of Gly 346 with Glu (Prinos et al ., 1995), as well as the replacement of Lys 650 with Met in the case of the most severe form of achondroplasia (Iwata et al .,
2000).

Ratones knock-out para el gen del receptor FGFR3 presentan un aumento del crecimiento endocondrial, expansión de la placa de crecimiento ósea y aumento de la proliferación de condrocitos, lo que ha permitido sugerir el papel del receptor FGFR3 como regulador negativo del crecimiento del hueso (Colvin et al., 1996; Goldfarb 1996; Burke et al., 1998), si bien las rutas a través de las cuales influye en los procesos de proliferación y diferenciación de los condrocitos no han sido totalmente esclarecidas.Knock-out mice for the FGFR3 receptor gene show an increase in endocondrial growth, expansion of bone growth plate and increased proliferation of chondrocytes, which has allowed us to suggest the role of the FGFR3 receptor as a negative regulator of bone growth ( Colvin et al ., 1996; Goldfarb 1996; Burke et al ., 1998), although the routes through which it influences the processes of proliferation and differentiation of chondrocytes have not been fully clarified.

Como miembro de la familia de receptores con actividad tirosina quinasa, este receptor se activa en presencia del FGF y de proteoglicanos del grupo heparán sulfato, para producir la dimerización del receptor y la posterior autofosforilación de los residuos de tirosina. Estos residuos fosforilados de tirosina sirven como sitios de unión de proteínas y efectores que propagan las señales de FGFR3 (Schlessinger et al., 2000; Hart et al., 2001). Los desórdenes en el crecimiento del hueso son resultado del aumento de la transmisión de señales por parte del receptor mutado como consecuencia de la activación sostenida del mismo. Varios mecanismos han sido propuestos para explicar cómo se produce esta activación continuada de FGFR3. Por ejemplo, en el caso de la mutación G380R, la más típica de la acondroplasia y que afecta a la región transmembrana, se origina una estabilización del dímero, prerrequisito para la activación del receptor y que le conduce a activarse incluso en ausencia del agonista (Naski et al., 1996; Webster and Donahue, 1996).As a member of the family of receptors with tyrosine kinase activity, this receptor is activated in the presence of FGF and heparan sulfate group proteoglycans, to produce dimerization of the receptor and subsequent autophosphorylation of tyrosine residues. These phosphorylated tyrosine residues serve as protein binding sites and effectors that propagate the FGFR3 signals (Schlessinger et al ., 2000; Hart et al ., 2001). Disorders in bone growth are the result of increased signal transmission by the mutated receptor as a result of its sustained activation. Several mechanisms have been proposed to explain how this continuous activation of FGFR3 occurs. For example, in the case of the G380R mutation, the most typical of achondroplasia and which affects the transmembrane region, a dimer stabilization occurs, a prerequisite for receptor activation and which leads to activation even in the absence of the agonist ( Naski et al ., 1996; Webster and Donahue, 1996).

Independientemente de cómo se origine la activación sostenida del receptor, una vez que ésta se produce se altera el equilibrio normal entre los procesos de proliferación y maduración inhibiéndose el correcto crecimiento de la placa ósea.Regardless of how the Sustained activation of the receptor, once this occurs disrupts the normal balance between proliferation processes and maturation inhibiting the correct growth of plaque that is.

Gran parte de los estudios que se han realizado hasta el momento están dirigidos a determinar las vías de señalización celular empleadas por el receptor FGFR3 activado para mediar sus efectos sobre la proliferación y diferenciación de condrocitos. Así distintas investigaciones han identificado a la ruta de señalización de STAT1 como la vía a través de la cual el receptor FGFR3 inhibe la proliferación de los condrocitos (Li et al., 1999; Sahni et al., 1999, 2001). La activación de STAT1 provoca el aumento de expresión del inhibidor de ciclo celular p21, bloqueándose de esta forma el crecimiento celular.Much of the studies that have been conducted so far are aimed at determining the cell signaling pathways used by the activated FGFR3 receptor to mediate their effects on chondrocyte proliferation and differentiation. Thus, several investigations have identified the STAT1 signaling pathway as the pathway through which the FGFR3 receptor inhibits chondrocyte proliferation (Li et al ., 1999; Sahni et al ., 1999, 2001). The activation of STAT1 causes the expression of the cell cycle inhibitor p21 to increase, thus blocking cell growth.

Con respecto a la influencia del receptor FGFR3 en la diferenciación de los condrocitos existe aún una cierta controversia. Mientras algunos autores sostienen que la activación de FGFR3 acelera el proceso de diferenciación promoviendo el incremento de condrocitos hipertróficos (Minina et al., 2002; Dailey et al., 2003), otros plantean que la activación del receptor inhibe la diferenciación de condrocitos a través de la ruta de señalización celular de las MAP quinasas (Murakami et al., 2004). Además, también mediante esta cascada de señalización celular de las MAP quinasas, la activación constitutiva de FGFR3 provoca una menor síntesis de matriz extracelular, hecho que también influiría en la inhibición del crecimiento óseo (Yasoda et al., 2003).With regard to the influence of the FGFR3 receptor on the differentiation of chondrocytes there is still some controversy. While some authors argue that the activation of FGFR3 accelerates the differentiation process by promoting the increase of hypertrophic chondrocytes (Minina et al ., 2002; Dailey et al ., 2003), others suggest that receptor activation inhibits chondrocyte differentiation through of the cellular signaling pathway of MAP kinases (Murakami et al ., 2004). In addition, also by means of this cellular signaling cascade of MAP kinases, the constitutive activation of FGFR3 causes a lower synthesis of extracellular matrix, a fact that would also influence the inhibition of bone growth (Yasoda et al ., 2003).

Para abordar todos estos estudios están siendo empleados diversos sistemas modelo. Así, se ha diseñado un ratón modificado genéticamente al que se le ha incorporado el gen mutado del receptor FGFR3 (Wang et al., 1999; Segev et al., 2000).To address all these studies, various model systems are being used. Thus, a genetically modified mouse has been designed to which the mutated FGFR3 receptor gene has been incorporated (Wang et al ., 1999; Segev et al ., 2000).

Junto con este modelo animal también se utilizan cultivos primarios de condrocitos o células como las de condrosarcoma de rata (RCS) que presentan marcadores característicos de condrocitos (Rozenblatt-Rosen et al., 2001). Adicionalmente se emplean condrocitos de rata (RCJ) transfectados de manera estable tanto con el receptor FGFR3 humano intacto como con la forma mutada G380R (Monsonego-Ornan et al., 2000).Together with this animal model, primary cultures of chondrocytes or cells such as rat chondrosarcoma (RCS) that have characteristic markers of chondrocytes are also used (Rozenblatt-Rosen et al ., 2001). Additionally, rat chondrocytes (RCJ) stably transfected with both the intact human FGFR3 receptor and the mutated form G380R (Monsonego-Ornan et al ., 2000) are employed.

En cuanto a las estrategias para el tratamiento farmacológico de la acondroplasia, no existe en la actualidad ningún tratamiento comercializado. Este hecho no impide que existan un número relativamente discreto de patentes sobre el tema. Así por ejemplo, existen patentes basadas en formulaciones cuyo fin principal es la intervención de la guanilato ciclasa b de los condrocitos acondroplásicos (US 2003068313, US 2004198665). En otros casos el aumento del crecimiento y de la reabsorción ósea se lleva a cabo por medio de compuestos con un núcleo de pirazilo (EP 1512401). Por último, también se ha encontrado que los péptidos natriuréticos son capaces de modificar la condición característica de la acondroplasia y otras patologías óseas (US 2004138134, WO02074234).As for the strategies for treatment achondroplasia pharmacological, currently does not exist No commercialized treatment. This fact does not prevent them from existing a relatively discreet number of patents on the subject. So by For example, there are patents based on formulations whose purpose main intervention is the guanylate cyclase b of the achondroplastic chondrocytes (US 2003068313, US 2004198665). In other cases the increase in growth and bone resorption is carried out by means of compounds with a pyrazil core (EP 1512401). Finally, it has also been found that peptides Natriuretics are able to modify the characteristic condition of achondroplasia and other bone pathologies (US 2004138134, WO02074234).

En todos estos ejemplos se barajan tratamientos para la acondroplasia por medio de moléculas que nada tienen que ver con las descritas en la presente invención. Por otro lado, los mecanismos moleculares involucrados en las patentes descritas en este párrafo no afectan directamente al receptor de FGFR3 acondroplásico, sin embargo, en la presente patente si que se describe una disminución del mencionado receptor.In all these examples treatments are considered for achondroplasia by means of molecules that have nothing to see with those described in the present invention. On the other hand, the molecular mechanisms involved in the patents described in This paragraph does not directly affect the FGFR3 receptor achondroplastic, however, in the present patent if describes a decrease in said receiver.

Un grupo interesante de moléculas de naturaleza nucleotídica son los dinucleósidos polifosfatos. Esta familia de moléculas intervienen en un número importante de procesos biológicos (Baxi and Vishwanatha, 1995; Flores et al., 1999; McLennan,; Szalata, 2001; van der Giet et al., 2002). Un grupo de estos dinucleótidos, los diadenosin polifosfatos presentan un gran interés por su papel como neurotransmisores (Hoyle, 1990; Miras-Portugal et al., 1998; Pintor and Miras-Portugal, 1995). Desde el punto de vista molecular, los diadenosin polifosfatos son compuestos que están formados por dos adenosinas unidas entre si por una cadena de fosfatos. A estas biomoléculas se les suele denominar de forma abreviada como ApnA donde "n" es el numero de fosfatos que puentea ambas adenosinas.An interesting group of nucleotide molecules are dinucleoside polyphosphates. This family of molecules is involved in a significant number of biological processes (Baxi and Vishwanatha, 1995; Flores et al ., 1999; McLennan ;; Szalata, 2001; van der Giet et al ., 2002). A group of these dinucleotides, diadenosine polyphosphates have a great interest in their role as neurotransmitters (Hoyle, 1990; Miras-Portugal et al ., 1998; Pintor and Miras-Portugal, 1995). From the molecular point of view, diadenosine polyphosphates are compounds that are formed by two adenosines linked together by a phosphate chain. These biomolecules are usually referred to in an abbreviated form as ApnA where "n" is the number of phosphates that bridges both adenosines.

La gran variabilidad de compuestos pertenecientes a esta familia de dinúcleotidos se establece en base a dos aspectos fundamentales, por una parte a la longitud de la cadena de fosfatos y por otro lado las bases nitrogenadas que posea el dinucleótido. Es precisamente la variabilidad en las bases nitrogenadas uno de los aspectos más atractivos de estas moléculas. La presencia de dinucleótidos con bases clásicas como la adenina, la guanina, el uracilo o la citidina, no son impedimento para que existan otras bases denominadas raras, tales como la 5-metilcitosina, 6-metiladenina, 2-metilguanina, 5-hidroximetiluracilo o el 2-tiouracilo que puedan dar lugar de manera natural o a través de procesos de síntesis a nuevos compuestos dinucleotídicos farmacológicamente activos (Nahum et al., 2006; Yerxa et al., 2002).The great variability of compounds belonging to this family of dinucleotides is established based on two fundamental aspects, on the one hand the length of the phosphate chain and on the other hand the nitrogenous bases that the dinucleotide possesses. It is precisely the variability in nitrogen bases one of the most attractive aspects of these molecules. The presence of dinucleotides with classical bases such as adenine, guanine, uracil or citidine, are not an impediment to the existence of other so-called rare bases, such as 5-methylcytosine, 6-methyladenine, 2-methylguanine, 5-hydroxymethyluracil or 2-thiouracil that can give rise naturally or through synthesis processes to new pharmacologically active dinucleotide compounds (Nahum et al ., 2006; Yerxa et al ., 2002).

Dadas las propiedades que presentan los dinucleótidos estas moléculas han sido estudiadas con fines terapéuticos de manera que en la actualidad existen también patentes que describen el empleo de dinucleótidos para el tratamiento de diversas patologías. Así por ejemplo las patentes US 2003207825 y CN 1575181 tratan del empleo de los dinucleósidos polifosfatos para el tratamiento de procesos edematosos oculares especialmente enfocados a la retina. Por otro lado la patente US 2003207825 describe el uso de estos mismos compuestos para el tratamiento de afecciones relacionadas con las secreciones mucosas, de fluidos en general así como para procesos de agregación plaquetaria. En este mismo orden de cosas la patente US 2003036527 describe el empleo de dinucleósidos polifosfatos para la limpieza de mucosidades en el ojo, pulmones, oídos etc. Por último, la patente WO0112644 describe el empleo de dinucleósidos polifosfatos como agentes terapéuticos para el tratamiento de las infecciones con el virus VIH. En estos ejemplos aunque se emplean dinucleósidos polifosfatos como los que se describen en esta patente, en ningún caso creemos que las aplicaciones tengan algo que ver con lo descrito en la presente memoria.Given the properties that the dinucleotides these molecules have been studied for purposes therapeutic so that currently there are also patents describing the use of dinucleotides for the Treatment of various pathologies. So for example US patents 2003207825 and CN 1575181 deal with the use of dinucleosides polyphosphates for the treatment of ocular edematous processes especially focused on the retina. On the other hand the US patent 2003207825 describes the use of these same compounds for the treatment of conditions related to mucous secretions, of fluids in general as well as for aggregation processes platelet In this same order of things the patent US 2003036527 describes the use of dinucleoside polyphosphates for cleaning of mucus in the eye, lungs, ears etc. Finally, the WO0112644 describes the use of dinucleoside polyphosphates as therapeutic agents for the treatment of infections with the HIV virus. In these examples although dinucleosides are used polyphosphates such as those described in this patent, in any case we believe that the applications have something to do with what described herein.

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Sahni M, Ambrosetti DC, Mansukhani A, Gertner R, Levy D, Basilico C. FGF signaling inhibits chondrocyte proliferation and regulates bone development through the STAT-1 pathway. Genes Dev. 1999; 13(11):1361-6. Sahni M, Ambrosetti DC, Mansukhani A, Gertner R, Levy D, Basilico C. FGF signaling inhibits chondrocyte proliferation and regulates bone development through the STAT-1 pathway. Dev Genes 1999 ; 13 (11): 1361-6.

Schlessinger J, Plotnikov AN, Ibrahimi OA, Eliseenkova AV, Yeh BK, Yayon A, Linhardt RJ, Mohammadi M. Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization. Mol Cell. 2000; 6(3):743-50. Schlessinger J, Plotnikov AN, Ibrahimi OA, Eliseenkova AV, Yeh BK, Yayon A, Linhardt RJ, Mohammadi M. Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization. Mol Cell 2000 ; 6 (3): 743-50.

Segev O, Chumakov I, Nevo Z, Givol D, Madar-Shapiro L, Sheinin Y, Weinreb M, Yayon A. Restrained chondrocyte proliferation and maturation with abnormal growth plate vascularization and ossification in human FGFR-3(G380R) transgenic mice. Hum Mol Genet. 2000; 9(2):249-58. Segev O, Chumakov I, Nevo Z, Givol D, Madar-Shapiro L, Sheinin Y, Weinreb M, Yayon A. Restrained chondrocyte proliferation and maturation with abnormal growth plate vascularization and ossification in human FGFR-3 (G380R) transgenic mice. Hum Mol Genet . 2000 ; 9 (2): 249-58.

Shiang R, Thompson LM, Zhu YZ, Church DM, Fielder TJ, Bocian M, Winokur ST, Wasmuth JJ. Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia. Cell. 1994; 78(2):335-42. Shiang R, Thompson LM, Zhu YZ, Church DM, Fielder TJ, Bocian M, Winokur ST, Wasmuth JJ. Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia. Cell 1994 ; 78 (2): 335-42.

Superti-Furga A, Eich G, Bucher HU, Wisser J, Giedion A, Gitzelmann R, Steinmann B. A glycine 375-to-cysteine substitution in the transmembrane domain of the fibroblast growth factor receptor-3 in a newborn with achondroplasia. Eur J Pediatr. 1995; 154(3):215-9. Superti-Furga A, Eich G, Bucher HU, Wisser J, Giedion A, Gitzelmann R, Steinmann B. A glycine 375-to-cysteine substitution in the transmembrane domain of the fibroblast growth factor receptor-3 in a newborn with achondroplasia. Eur J Pediatr . 1995 ; 154 (3): 215-9.

Szalata, M., 2001, Dinucleoside polyphosphates: occurrence, metabolism and function, Postepy Biochemii 47, 105-113. Szalata , M., 2001 , Dinucleoside polyphosphates: occurrence, metabolism and function, Postepy Biochemii 47, 105-113.

Szweykowska-Kuliñska Z, Jarmolowski A, Figlerowicz M. RNA interference and its role in the regulation of eucaryotic gene expression Acta Biochem Pol 2003; 50(1):217-229. Szweykowska-Kuliñska Z, Jarmolowski A, Figlerowicz M. RNA interference and its role in the regulation of eucaryotic gene expression Acta Biochem Pol 2003 ; 50 (1): 217-229.

van der Giet, M., S. Schmidt, M. Tolle, J. Jankowski, H. Schluter, W. Zidek and M. Tepel, 2002, Effects of dinucleoside polyphosphates on regulation of coronary vascular tone, European Journal Of Pharmacology 448, 207-213.van der Giet , M., S. Schmidt , M. Tolle , J. Jankowski , H. Schluter , W. Zidek and M. Tepel , 2002 , Effects of dinucleoside polyphosphates on regulation of coronary vascular tone, European Journal Of Pharmacology 448, 207-213.

Wang Y, Spatz MK, Kannan K, Hayk H, Avivi A, Gorivodsky M, Pines M, Yayon A, Lonai P, Givol D. A mouse model for achondroplasia produced by targeting fibroblast growth factor receptor 3. Proc Natl Acad Sci USA. 1999; 96(8):4455-60. Wang Y, Spatz MK, Kannan K, Hayk H, Avivi A, Gorivodsky M, Pines M, Yayon A, Lonai P, Givol D. A mouse model for achondroplasia produced by targeting fibroblast growth factor receptor 3. Proc Natl Acad Sci USA . 1999 ; 96 (8): 4455-60.

Webster MK, Donoghue DJ. Constitutive activation of fibroblast growth factor receptor 3 by the transmembrane domain point mutation found in achondroplasia. EMBO J. 1996; 15(3):520-7. Webster MK, Donoghue DJ. Constitutive activation of fibroblast growth factor receptor 3 by the transmembrane domain point mutation found in achondroplasia. EMBO J. 1996 ; 15 (3): 520-7.

Yasoda A, Komatsu Y, Chusho H, Miyazawa T, Ozasa A, Miura M, Kurihara T, Rogi T, Tanaka S, Suda M, Tamura N, Ogawa Y, Nakao K. Overexpression of CNP in chondrocytes rescues achondroplasia through a MAPK-dependent pathway. Nat Med. 2004; 10(1):80-6. Yasoda A, Komatsu Y, Chusho H, Miyazawa T, Ozasa A, Miura M, Kurihara T, Rogi T, Tanaka S, Suda M, Tamura N, Ogawa Y, Nakao K. Overexpression of CNP in chondrocytes rescues achondroplasia through a MAPK- dependent pathway. Nat Med . 2004 ; 10 (1): 80-6.

Yerxa, B.R., J.R. Sabater, C.W. Davis, M.J. Stutts, M. Lang-Furr, M. Picher, A.C. Jones, M. Cowlen, R. Dougherty and a. Boyer et, 2002, Pharmacology of INS37217 [P(1)-(uridine 5')-P(4)-(2'-deoxycytidine 5')tetraphosphate, tetrasodium salt], a next-generation P2Y(2) receptor agonist for the treatment of cystic fibrosis, The Journal Of Pharmacology And Experimental Therapeutics 302, 871-880. Yerxa , BR, JR Sabater , CW Davis , MJ Stutts , M. Lang-Furr , M. Picher , AC Jones , M. Cowlen , R. Dougherty and a. Boyer et, 2002 , Pharmacology of INS37217 [P (1) - (uridine 5 ') - P (4) - (2'-deoxycytidine 5') tetraphosphate, tetrasodium salt], a next-generation P2Y (2) agonist receptor for the treatment of cystic fibrosis, The Journal Of Pharmacology And Experimental Therapeutics 302, 871-880.

Detailed description of the invention

La presente invención se basa en el comportamiento de que los dinucleótidos son capaces de disminuir la presencia de los receptores FGFR3 acondroplásicos de las membranas plasmáticas de las células que los poseen (principalmente condrocitos).The present invention is based on the behavior that dinucleotides are able to decrease the presence of achondroplastic FGFR3 membrane receptors plasma cells that own them (mainly chondrocytes)

El aspecto más sobresaliente de esta invención tiene que ver con el crecimiento de los huesos como consecuencia de la disminución de los receptores de FGFR3 acondroplásicos.The most outstanding aspect of this invention it has to do with bone growth as a result of the decrease in achondroplastic FGFR3 receptors.

La invención provee nuevas composiciones de ciertos dinucleósidos polifosfatos y sus análogos los cuales podrían ser usados terapéuticamente como agonistas de los receptores purinérgicos. La invención también aporta los métodos para emplear dichas composiciones para tratar y prevenir aquellas patologías relacionadas con la falta de estatura como consecuencia de la mutación en el receptor para el FGF denominado FGFR3.The invention provides new compositions of certain polyphosphate dinucleosides and their analogues which could be used therapeutically as agonists of purinergic receptors The invention also provides the methods to use said compositions to treat and prevent those pathologies related to lack of height as a consequence of the mutation in the receptor for the FGF called FGFR3.

El método comprende la administración a un mamífero de una composición farmacéutica terapéuticamente efectiva de un ligando del tipo P2 (P2X y P2Y). Por cantidad terapéuticamente efectiva se entiende una cantidad efectiva para tratar o prevenir la patología, condición o desorden, la cual es a su vez una cantidad efectiva para inhibir, prevenir, parar o retrasar la progresión del estado patológico y por ende de sus posibles consecuencias.The method comprises administration to a mammal of a therapeutically effective pharmaceutical composition of a ligand of the type P2 (P2X and P2Y). By quantity therapeutically effective means an effective amount to treat or prevent the pathology, condition or disorder, which is a in turn an effective amount to inhibit, prevent, stop or delay the progression of the pathological state and therefore of its possible consequences.

La presente invención demuestra que la existencia de receptores del tipo P2, presentes en muchas células se encuentran también en los condrocitos, células del cartílago responsables de la patología acondroplasia. Los solicitantes consideran que la activación de receptores P2 con los nuevos compuestos de la presente invención activarán efectos celulares específicos los cuales resulten en efectos terapéuticos mediados por los receptores P2.The present invention demonstrates that the existence of P2 type receptors, present in many cells They are also found in chondrocytes, cartilage cells responsible for achondroplasia pathology. The appliers consider that the activation of P2 receptors with the new Compounds of the present invention will activate cellular effects specific which result in mid therapeutic effects by the P2 receptors.

Los agonistas P2 incluyen dinucleósidos polifosfatos como se muestra en la fórmula I. Uno de los aspectos más destacado es la presencia de residuos azucarados de cualquiera de los siguientes tipos: 3'-desoxiribofuranosil, 2',3'-didesoxiribofuranosil, arabinofuranosil, 3'-desoxiarabinofuaranosil, xilofuranosil, 2'-desoxixilofuranosil y lixofuranosil.P2 agonists include dinucleosides polyphosphates as shown in formula I. One of the aspects most prominent is the presence of sugary residues of any of the following types: 3'-deoxyribofuranosil, 2 ', 3'-dideoxyyribofuranosyl, arabinofuranosil, 3'-desoxiarabinofuaranosyl, xylofuranosil, 2'-deoxyxylofuranosil and lixofuranosil.

1one

En donde:In where:

X es oxígeno, metileno, dihalometileno o iminoX is oxygen, methylene, dihalomethylene or imino

n = 0, 1 ó 2n = 0, 1 or 2

m = 0, 1 ó 2m = 0, 1 or 2

n + m = 0, 1, 2, 3 ó 4n + m = 0, 1, 2, 3 or 4

Z = OH ó HZ = OH or H

Z' = OH ó HZ '= OH or H

Y = OH ó HY = OH or H

Y' = OH ó H y ademásY '= OH or H and also

B y B' son ambos e independientemente un anillo de purina o un anillo de pirimidina como se definen en las fórmulas II y III, unidas por las posiciones 9- ó 1- respectivamenteB and B 'are both and independently a ring of purine or a pyrimidine ring as defined in the formulas II and III, joined by positions 9- or 1- respectively

22

R_{4} es oxo, hidroxi, mercapto, tiona, amino, ciano, C_{7-12} arilaloxi, C_{1-6} alquiltio, C_{1-6} alquiloxi, C_{1-6} alquilamino o diC_{1-4} alquilamino, donde los grupos alquilo están unidos opcionalmente para formar un heterociclo.R 4 is oxo, hydroxy, mercapto, thione, amino, cyano, C 7-12 aryloxy, C 1-6 alkylthio, C 1-6 alkyloxy, C 1-6 alkylamino or diC 1-4 alkylamino, where the alkyl groups they are optionally linked to form a heterocycle.

R_{5} es hidrógeno, acetil, benzoil, C_{1-6} alquil, C_{1-8} alquinoil, arolil o puede estar ausente.R 5 is hydrogen, acetyl, benzoyl, C 1-6 alkyl, C 1-8 alkynyl, arolyl or may be absent.

R_{6} es hidroxi, oxo, mercapto, tiona, C_{1-4} alcoxi, C_{7-12} arilalcoxi, C_{1-6} alquiltio, S-fenil, ariltio, arilalquiltio, triazolil, amino, C_{2-6} alquilamino, C_{2-5} amino disustituido o di C_{2-4} alquilamino, donde los grupos dialquil pueden ser opcionales ligados para formar un heterociclo o unidos para formar un anillo sustituido como morfolino, pirrolo etc.R 6 is hydroxy, oxo, mercapto, thione, C 1-4 alkoxy, C 7-12 arylalkoxy, C 1-6 alkylthio, S-phenyl, arylthio, arylalkylthio, triazolyl, amino, C 2-6 alkylamino, C 2-5 disubstituted amino or di C 2-4 alkylamino, where dialkyl groups can be optional linked to form a heterocycle or attached to form a substituted ring as morpholino, pirrolo etc.

R_{5} y R_{6} conjuntamente pueden formar un anillo imidazol de 5 miembros entre las posiciones 3 y 4 del anillo de pirimidina y formar un derivado 3,N^{4}-etenocitosina donde el grupo eteno es sustituido opcionalmente en las posiciones 4 ó 5 donde al menos un hidrógeno de C_{1-4} alquil, fenil, feniloxi es sustituido opcionalmente por un grupo de entre halógenos, hidroxi, C_{1-4} alcoxi, C_{1-4} alquil, C_{6-10} aril, C_{7-12} arilalquil, carboxi, ciano, nitro, sulfonamido, sulfonato, fosfato, ácido sulfónico, amino, C_{1-4} alquilamino y di C_{3-4} alquilamino, donde los grupos dialquil pueden estar opcionalmente unidos para formar un heterociclo.R 5 and R 6 together can form a 5-member imidazole ring between positions 3 and 4 of the ring of pyrimidine and form a derivative 3, N 4 -ethenocytosine where the ethene group is optionally substituted in positions 4 or 5 where at least one C 1-4 alkyl, phenyl, phenyloxy hydrogen is optionally substituted by a group of halogens, hydroxy, C 1-4 alkoxy, C 1-4 alkyl, C 6-10 aryl, C 7-12 arylalkyl, carboxy, cyano, nitro, sulfonamido, sulfonate, phosphate, sulfonic acid, amino, C 1-4 alkylamino and di C 3-4 alkylamino, where dialkyl groups they can be optionally linked to form a heterocycle.

R_{7} puede estar formado por hidrógeno, hidroxilo, ciano, nitro, C_{2-8} alquinil, halógeno, C_{1-6} alquil, CF_{3}, C_{2-3} alquenil, C_{2-3} alquinil, alilamino, bromovinil, etil propenoato, ácido propenoico y C_{2-8} alquenil.R 7 may be formed by hydrogen, hydroxyl, cyano, nitro, C 2-8 alkynyl, halogen, C 1-6 alkyl, CF 3, C_ {2-3} alkenyl, C_ {2-3} alkynyl, allylamino, bromovinyl, ethyl propenoate, propenoic acid and C 2-8 alkenyl.

R_{6} y R_{7} conjuntamente pueden formar un anillo saturado o insaturado de 5 ó 6 miembros unidos por medio de N, O u S a R_{6}, ese anillo podría contener sustituyentes.R 6 and R 7 together can form a saturated or unsaturated ring of 5 or 6 members joined by means of N, O or S at R 6, that ring could contain substituents.

R_{8} puede ser hidrógeno, amino, C_{1-4} alquilamino, C_{1-4} alcoxi, C_{7-12} arilalcoxi, C_{1-6} alquiltio, C_{7-12} arilalquiltio, carboxamidometil, carboximetil, metoxi, metiltio, fenoxi y feniltio de modo que cuando R_{8} es amino o amino sustituido, R_{7} es hidrógeno.R 8 can be hydrogen, amino, C 1-4 alkylamino, C 1-4 alkoxy, C 7-12 arylalkoxy, C 1-6 alkylthio, C 7-12 arylalkylthio, carboxyamidomethyl, carboxymethyl, methoxy, methylthio, phenoxy and phenylthio so that when R 8 is amino or amino substituted, R 7 is hydrogen.

Los diferentes agentes serán administrados en cantidades farmacológicamente suficientes para lograr concentraciones en el cartílago que varíen entre 10^{-7} a 100 g/litro.The different agents will be administered in pharmacologically sufficient amounts to achieve cartilage concentrations ranging from 10-7 to 100 g / liter

Su administración se podrá realizar de maneras diversas, por medio de vehículos portadores como gotas de líquido, lavados de líquido, geles ungüentos sprays o liposomas.Your administration can be done in ways various, by means of carrier vehicles such as drops of liquid, liquid washes, spray ointment gels or liposomes.

La administración de estas sustancias se realizará empleando dispositivos catéter-bomba, de liberación En donde R_{1} es cloro, amino, hidrógeno, ceto, amino monosustituido, amino disustituido, alquiltio, ariltio, aralquiltio, y donde en la sustitución del sulfuro contenga hasta un máximo de 20 átomos de carbono, con o sin insaturaciones.The administration of these substances is will perform using catheter-pump devices, of release Where R 1 is chlorine, amino, hydrogen, keto, amino monosubstituted, disubstituted amino, alkylthio, arylthio, aralkylthio, and where in the sulfide substitution it contains up to a maximum of 20 carbon atoms, with or without unsaturations.

R_{2}, es alquenil, hidroxi, oxo, amino, mercapto, tiona, alquiltio, ariltio, aralquiltio, aciltio, alquiloxi, ariloxi, aralquiloxi, alquilaminos monosustituidos, cicloalquilamino monosustituidos heterociclicos, arilamino monosustituidos, diaralquilamino, diarilamino, dialquilamino, acilamino o diacilamino.R2 is alkenyl, hydroxy, oxo, amino, mercapto, tiona, alkylthio, arylthio, aralkylthio, acylthio, alkyloxy, aryloxy, aralkyloxy, monosubstituted alkylaminos, heterocyclic monosubstituted cycloalkylamino, arylamino monosubstituted, diaralkylamino, diarylamino, dialkylamino, acylamino or diacylamino.

R_{x}, es H, O ó está ausente.R_ {x}, is H, O or is absent.

R_{2} y R_{x} son tomados adicionalmente de modo conjunto para formar anillos de cinco miembros fusionándose el anillo imidazol de 1, N^{6}-eteno adenina derivados, opcionalmente sustituidos en las posiciones 4- o 5- del anillo eteno con alquil, aralquil.R2 and R2 are additionally taken from joint mode to form five-member rings merging the 1, N 6 -etene adenine imidazole ring derivatives, optionally substituted in positions 4- or 5- of ethene ring with alkyl, aralkyl.

R_{3} es hidrógeno, azido, alcoxi, ariloxi, aralquiloxi, alquiltio, ariltio o aralquiltio como se define más adelante.R 3 is hydrogen, azido, alkoxy, aryloxy, aralkyloxy, alkylthio, arylthio or aralkylthio as defined further ahead.

J es nitrógeno o carbono en previsión de que cuando J es nitrógeno, R_{3} está ausente.J is nitrogen or carbon in anticipation that when J is nitrogen, R 3 is absent.

Para todos los alquilos anteriormente definidos, estos pueden ser tanto lineales como ramificados.For all previously defined alkyls, These can be both linear and branched.

Los grupos arilo pueden ser opcionalmente mono, di o trisustituidos con alquil, aril amino, mono o dialquilamino, NO_{2}, N_{3}, ciano, carboxil, amido, sulfonamido, ácido sulfónico, fosfato o halógeno.The aryl groups may optionally be mono, di or trisubstituted with alkyl, aryl amino, mono or dialkylamino, NO 2, N 3, cyano, carboxy, amido, sulfonamido, acid sulfonic, phosphate or halogen.

33

continua o selectiva, o sistemas sencillos como gotas nasales, sprays nasales o nebulizados a las vías orales o nasofaríngeas, de forma oral, de forma inyectable o por medio de supositorios de manera que el principio activo contacte con los tejidos afectados en una cantidad terapéuticamente efectiva tras su absorción sistémica y circulación hasta dichas zonas.continuous or selective, or systems simple as nasal drops, nasal sprays or nebulized at oral or nasopharyngeal routes, orally, by injection or through suppositories so that the active substance contact the affected tissues in a therapeutically amount effective after its systemic absorption and circulation until these zones.

Brief description of the figures

Para facilitar la comprensión de la invención y formando parte de esta memoria descriptiva se acompañan una serie de figuras:To facilitate the understanding of the invention and being part of this descriptive report are accompanied by a series of figures:

Figura 1.- Marcaje del receptor FGFR3 acondroplásico en condrocitos. En los 2 paneles superiores, a la izquierda se ve el marcaje de color verde que corresponde a los receptores FGFR3 acondroplásicos. A su derecha las células vistas por microscopia de Nomarski. En los paneles inferiores se aprecia cómo la cantidad de receptor disminuye (menos verde) tras el tratamiento con el Ap_{4}A.Figure 1.- FGFR3 receptor labeling achondroplastic in chondrocytes. In the 2 upper panels, to the on the left you see the green marking that corresponds to the achondroplastic FGFR3 receptors. On your right the cells seen by microscopy of Nomarski. In the lower panels you can see how the amount of receptor decreases (less green) after treatment with Ap_ {A} A.

Figura 2.- Análisis de inmunotransferencia en el que en el recuadro superior se puede observar el efecto del Ap_{4}A (bandas de la derecha) en comparación con el control (bandas de la izquierda), donde se aprecia que las bandas de la derecha son menos intensas que las del control. Dichas bandas, tanto en el control como en el tratado representan el receptor de FGFR3. Debajo se representa en forma de diagrama de barras la cuantificación de dichas bandas, donde se evalúa que hay menos receptor FGFR3 en las células tratadas con Ap_{4}A que en las control.Figure 2.- Immunoblot analysis in the that in the upper box you can see the effect of Ap_ {4} A (bands on the right) compared to the control (bands on the left), where you can see that the bands of the right are less intense than the control. These bands, both the control and the treaty represent the receiver of FGFR3. Below is shown in the form of a bar chart the quantification of these bands, where it is evaluated that there is less FGFR3 receptor in cells treated with Ap4A than in the control.

Figura 3.- Análisis de inmunotransferencia en el que en el recuadro superior se puede observar el efecto del Up_{4}U (bandas de la derecha) en comparación con el control (bandas de la izquierda), donde se aprecia que las bandas de la derecha son menos intensas que las del control. Dichas bandas, tanto en el control como en el tratado representan el receptor de FGFR3. Debajo se representa en forma de diagrama de barras la cuantificación de dichas bandas, donde se evalúa que hay menos receptor FGFR3 en las células tratadas con Up_{4}U que en las control.Figure 3.- Immunoblot analysis in the that in the upper box you can see the effect of Up_ {4} U (bands on the right) compared to the control (bands on the left), where you can see that the bands of the right are less intense than the control. These bands, both the control and the treaty represent the receiver of FGFR3. Below is shown in the form of a bar chart the quantification of these bands, where it is evaluated that there is less FGFR3 receptor in cells treated with Up4U than in control.

Figura 4.- Análisis de inmunotransferencia en el que en el recuadro superior se puede observar el efecto del Ap_{3}A (bandas de la derecha) en comparación con el control (bandas de la izquierda), donde se aprecia que las bandas de la derecha son menos intensas que las del control. Dichas bandas, tanto en el control como en el tratado representan el receptor de FGFR3. Debajo se representa en forma de diagrama de barras la cuantificación de dichas bandas, donde se evalúa que hay menos receptor FGFR3 en las células tratadas con Ap_{3}A que en las control.Figure 4.- Immunoblot analysis in the that in the upper box you can see the effect of Ap_ {3} A (bands on the right) compared to the control (bands on the left), where you can see that the bands of the right are less intense than the control. These bands, both the control and the treaty represent the receiver of FGFR3. Below is shown in the form of a bar chart the quantification of these bands, where it is evaluated that there is less FGFR3 receptor in cells treated with Ap3A than in the control.

Figura 5.- Análisis de inmunotransferencia en el que en el recuadro superior se puede observar el efecto del Ap_{5}A (bandas de la derecha) en comparación con el control (bandas de la izquierda), donde se aprecia que las bandas de la derecha son menos intensas que las del control. Dichas bandas, tanto en el control como en el tratado representan el receptor de FGFR3. Debajo se representa en forma de diagrama de barras la cuantificación de dichas bandas, donde se evalúa que hay menos receptor FGFR3 en las células tratadas con Ap_{5}A que en las control.Figure 5.- Immunoblot analysis in the that in the upper box you can see the effect of Ap_ {5} A (bands on the right) compared to the control (bands on the left), where you can see that the bands of the right are less intense than the control. These bands, both the control and the treaty represent the receiver of FGFR3. Below is shown in the form of a bar chart the quantification of these bands, where it is evaluated that there is less FGFR3 receptor in cells treated with Ap5A than in the control.

Embodiment of the invention

Ejemplo 1Example one

Una muestra conteniendo a la adenosina 5' tetrafosfo 5' adenosina (Ap_{4}A, véase fórmula IV), un dinucleótido con la estructura general descrita en la fórmula I y con una base nitrogenada como la descrita en la fórmula II, fue incubado con condrocitos acondroplásicos (que presentan el receptor FGFR3 humano mutado).A sample containing 5 'adenosine tetraphosphorus 5 'adenosine (Ap 4 A, see formula IV), a dinucleotide with the general structure described in formula I and with a nitrogenous base as described in formula II, it was incubated with achondroplastic chondrocytes (presenting the receptor Mutated human FGFR3).

44

Una concentración de 100 \muM del dinucleótido Ap_{4}A fue incubada con las células durante 10 minutos. Este experimento se realizó en paralelo con otro idéntico en el que no se añadió el dinucleótido y ambos se compararon. Para medir y constatar las posibles modificaciones en la presencia del receptor FGFR3 (acondroplásico) se emplearon anticuerpos comerciales frente al mencionado receptor, y se verificaron las posibles modificaciones por técnicas inmunocitoquímicas y de inmunotransferencia (western-blot). La presencia del dinucleótido Ap_{4}A, cuando es comparado con el experimento control demuestra que los niveles del receptor FGFR3 disminuyen significativamente de la membrana de las células, tal y como se ve en la figura 1.A concentration of 100 µM of the dinucleotide Ap4A was incubated with the cells for 10 minutes. This experiment was performed in parallel with another identical one in which no Dinucleotide was added and both were compared. To measure and verify possible changes in the presence of the recipient FGFR3 (achondroplastic) commercial antibodies were used against to the mentioned receiver, and the possible ones were verified modifications by immunocytochemical techniques and immunoblot (western blot). The presence of Ap4 A dinucleotide, when compared to the experiment control demonstrates that FGFR3 receptor levels decrease significantly from the cell membrane, as seen in figure 1.

Los estudios realizados con la técnica de inmunotransferencia (western-blot) corroboran los resultados anteriores. Las células tratadas con Ap_{4}A presentan menos receptores FGFR3 mutados que las no tratadas, tal y como se aprecia en la figura 2.Studies conducted with the technique of Western blot corroborate the Previous results. Cells treated with Ap4A have fewer mutated FGFR3 receptors than untreated ones, as appreciate in figure 2.

Ejemplo 2Example 2

Una muestra conteniendo a la uridina 5' tetrafosfo 5' uridina (Up_{4}U, véase formula V), un dinucleótido con la estructura general descrita en la fórmula I y con una base nitrogenada como la descrita en la fórmula III, fue incubado con los condrocitos (que presentan el receptor FGFR3 mutado).A sample containing 5 'uridine 5 'uridine tetraphosphorus (Up 4 U, see formula V), a dinucleotide with the general structure described in formula I and with a base Nitrogenated as described in formula III, was incubated with chondrocytes (which have the mutated FGFR3 receptor).

55

Una concentración de 100 \muM del dinucleótido Up_{4}U fue incubada con las células durante 10 minutos. Este experimento se realizó en paralelo con otro idéntico en el que no se añadió el dinucleótido y ambos se compararon. Para medir y constatar las posibles modificaciones en la presencia del receptor FGFR3 (acondroplásico) se emplearon anticuerpos comerciales frente al mencionado receptor, y se verificaron las posibles modificaciones por técnicas de inmunotransferencia (western-blot). Como se observa en la figura 3, el dinucleótido Up_{4}U hizo desaparecer al receptor FGFR3 acondroplásico de las células.A concentration of 100 µM of the dinucleotide Up4U was incubated with the cells for 10 minutes. This experiment was performed in parallel with another identical one in which no Dinucleotide was added and both were compared. To measure and verify possible changes in the presence of the recipient FGFR3 (achondroplastic) commercial antibodies were used against to the mentioned receiver, and the possible ones were verified modifications by immunoblot techniques (western-blot). As seen in Figure 3, the dinucleotide Up_4U made the FGFR3 receptor disappear achondroplastic cells.

Ejemplo 3Example 3

Una muestra conteniendo a la adenosina 5' trifosfo 5' adenosina (Ap_{3}A, véase fórmula VI), un dinucleótido con la estructura general descrita en la fórmula I y con una base nitrogenada como la descrita en la fórmula II, fue incubado con condrocitos acondroplásicos (que presentan el receptor FGFR3 humano mutado).A sample containing 5 'adenosine 5 'adenosine triphosphate (Ap 3 A, see formula VI), a dinucleotide with the general structure described in formula I and with a nitrogenous base as described in formula II, it was incubated with achondroplastic chondrocytes (presenting the receptor Mutated human FGFR3).

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Una concentración de 100 \muM del dinucleótido Ap_{3}A fue incubada con las células durante 10 minutos. Este experimento se realizó en paralelo con otro idéntico en el que no se añadió el dinucleótido y ambos se compararon. Para medir y constatar las posibles modificaciones en la presencia del receptor FGFR3 (acondroplásico) se emplearon anticuerpos comerciales frente al mencionado receptor, y se verificaron las posibles modificaciones por técnicas de inmunotransferencia (western-blot).A concentration of 100 µM of the dinucleotide Ap3A was incubated with the cells for 10 minutes. This experiment was performed in parallel with another identical one in which no Dinucleotide was added and both were compared. To measure and verify possible changes in the presence of the recipient FGFR3 (achondroplastic) commercial antibodies were used against to the mentioned receiver, and the possible ones were verified modifications by immunoblot techniques (western-blot).

Los estudios realizados con la técnica de inmunotransferencia (western-blot) demuestran que las células tratadas con Ap_{3}A presentan menos receptores FGFR3 mutados que las no tratadas, tal y como se aprecia en la figura 4.Studies conducted with the technique of Western blot show that cells treated with Ap3A have fewer FGFR3 receptors mutated than untreated, as shown in the figure Four.

Ejemplo 4Example 4

Una muestra conteniendo a la adenosina 5' pentafosfo 5' adenosina (Ap_{5}A, véase fórmula VII), un dinucleótido con la estructura general descrita en la fórmula I y con una base nitrogenada como la descrita en la fórmula II, fue incubado con condrocitos acondroplásicos (que presentan el receptor FGFR3 humano mutado).A sample containing 5 'adenosine 5 'adenosine pentaphosphorus (Ap5 A, see formula VII), a dinucleotide with the general structure described in formula I and with a nitrogenous base as described in formula II, it was incubated with achondroplastic chondrocytes (presenting the receptor Mutated human FGFR3).

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Una concentración de 100 \muM del dinucleótido Ap_{5}A fue incubada con las células durante 10 minutos. Este experimento se realizó en paralelo con otro idéntico en el que no se añadió el dinucleótido y ambos se compararon. Para medir y constatar las posibles modificaciones en la presencia del receptor FGFR3 (acondroplásico) se emplearon anticuerpos comerciales frente al mencionado receptor, y se verificaron las posibles modificaciones por técnicas de inmunotransferencia (western-blot).A concentration of 100 µM of the dinucleotide Ap5A was incubated with the cells for 10 minutes. This experiment was performed in parallel with another identical one in which no Dinucleotide was added and both were compared. To measure and verify possible changes in the presence of the recipient FGFR3 (achondroplastic) commercial antibodies were used against to the mentioned receiver, and the possible ones were verified modifications by immunoblot techniques (western-blot).

Los estudios realizados con la técnica de inmunotransferencia (western-blot) demuestran que las células tratadas con Ap_{5}A presentan menos receptores FGFR3 mutados que las no tratadas, tal y como se aprecia en la figura 5.Studies conducted with the technique of Western blot show that cells treated with Ap5A have fewer FGFR3 receptors mutated than untreated, as shown in the figure 5.

Claims

1. The use of a compound with the structure defined in formula I, for the preparation of a medicine intended for the treatment of achondroplasia where:

8

the sugars that form the structure of the 3'-deoxyribofuranosil type, 2 ', 3'-dideoxyyribofuranosyl, arabinofuranosil, 3'-desoxiarabinofuaranosyl, xylofuranosil, 2'-deoxyxylofuranosyl and lixofuranosil

Where also:

X is oxygen, methylene, dihalomethylene or imino

n = 0, 1 or 2

m = 0, 1 or 2

n + m = 0, 1, 2, 3 or 4

Z = OH or H

Z '= OH or H

Y = OH or H

Y '= OH or H and also

B and B 'are both and independently a purine or a pyrimidine as defined in formulas II and III, attached by positions 9- or 1- respectively

9

In where

R1 is chlorine, amino, hydrogen, amino monosubstituted, disubstituted amino, alkylthio, arylthio, aralkylthio, and where in the sulfide substitution it contains up to a maximum of 20 carbon atoms, with or without unsaturations.

R2 is alkenyl, hydroxy, oxo, amino, mercapto, tiona, alkylthio, arylthio, aralkylthio, acylthio, alkyloxy, aryloxy, aralkyloxy, monosubstituted alkylaminos, heterocyclic monosubstituted cycloalkylamino, arylamino monosubstituted, diaralkylamino, diarylamino, dialkylamino, acylamino or diacylamino.

R_ {x}, is H, O or is absent.

R2 and R2 are additionally taken from joint mode to form five-member rings merging the 1, N 6 -etene adenine imidazole ring derivatives, optionally substituted in positions 4- or 5- of ethene ring with alkyl, aralkyl.

R 3 is hydrogen, azido, alkoxy, aryloxy, aralkyloxy, alkylthio, arylthio or aralkylthio as defined further ahead.

J is nitrogen or carbon in anticipation that when J is nitrogen, R 3 is absent.

For all previously defined alkyls, These can be both linear and branched.

The aryl groups may optionally be mono, di or trisubstituted with lower alkyl, aryl amino, mono or dialkylamino, NO2, N3, cyano, carboxyl, amido, sulfonamido, sulfonic acid, phosphate or halogen.

10

R 4 is oxo, hydroxy, mercapto, thione, amino, cyano, C 7-12 aryloxy, C 1-6 alkylthio, C 1-6 alkyloxy, C 1-6 alkylamino or diC 1-4 alkylamino, where the alkyl groups they are optionally linked to form a heterocycle.

R 5 is hydrogen, acetyl, benzoyl, C 1-6 alkyl, C 1-8 alkynyl, arolyl or may be absent.

R 6 is hydroxy, oxo, mercapto, thione, C 1-4 alkoxy, C 7-12 arylalkoxy, C 1-6 alkylthio, S-phenyl, arylthio, arylalkylthio, triazolyl, amino, C 2-6 alkylamino, C 2-5 disubstituted amino or di C 2-4 alkylamino, where dialkyl groups can be optional linked to form a heterocycle or attached to form a substituted ring as morpholino, pirrolo etc.

R 5 and R 6 together can form a 5-member imidazole ring between positions 3 and 4 of the ring of pyrimidine and form a derivative 3, N 4 -ethenocytosine where the ethene group is optionally substituted in positions 4 or 5 where at least one C 1-4 alkyl, phenyl, phenyloxy hydrogen is optionally substituted by a group of halogens, hydroxy, C 1-4 alkoxy, C 1-4 alkyl, C 6-10 aryl, C 7-12 arylalkyl, carboxy, cyano, nitro, sulfonamido, sulfonate, phosphate, sulfonic acid, amino, C 1-4 alkylamino and di C 3-4 alkylamino, where dialkyl groups they can be optionally linked to form a heterocycle.

R 7 may be formed by hydrogen, hydroxyl, cyano, nitro, C 2-8 alkynyl, halogen, C 1-6 alkyl, CF 3, C_ {2-3} alkenyl, C_ {2-3} alkynyl, allylamino, bromovinyl, ethyl propenoate, propenoic acid and C 2-8 alkenyl.

R 6 and R 7 together can form a saturated or unsaturated ring of 5 or 6 members joined by means of N, O or S at R 6, that ring could contain substituents.

R 8 can be hydrogen, amino, C 1-4 alkylamino, C 1-4 alkoxy, C 7-12 arylalkoxy, C 1-6 alkylthio, C 7-12 arylalkylthio, carboxyamidomethyl, carboxymethyl, methoxy, methylthio, phenoxy and phenylthio so that when R 8 is amino or amino substituted, R 7 is hydrogen.

2. The use of a compound according to the claim 1, wherein the compound may be 5 'adenosine tetraphosphorus 5 'adenosine, Ap4A, as described in the formula IV, for the preparation of a medicine intended for treatment of achondroplasia.

3. The use of a compound according to the claim 1, wherein the compound may be uridine 5 ' tetraphosphorus 5 'uridine, Up4U, as described in formula V, for the preparation of a medicine intended for the treatment of achondroplasia

4. The use of a compound according to the claim 1, wherein the compound may be 5 'adenosine 5 'adenosine triphosphate, Ap 3 A, as described in the formula VI, for the preparation of a medicine intended for treatment of achondroplasia.

5. The use of a compound according to the claim 1, wherein the compound may be 5 'adenosine 5 'adenosine pentaphosphorus, Ap5 A, as described in the formula VII, for the preparation of a medicine intended for achondroplasia treatment.

6. The use according to the claims 1, 2, 3, 4 and 5 where said compounds are administered in a sufficient quantity to achieve concentrations in the cartilage that vary between 10-7 at 100 g / liter.

7. The use according to claims 1, 2, 3, 4, 5 and 6, where such administration implies the application of the compound described through a carrier vehicle selected from a group consisting of liquid drops, washed with liquid, gels, ointments, sprays and liposomes.

8. The use according to claim 7 where said application involves infusions of this compound to said cartilage through a device selected from a group consisting of catheter-pump systems, a continuous or selective release device.

9. The use according to claims 1, 2, 3, 4, 5 and 6, where such administration implies the systemic administration of this compound, applying a liquid / liquid suspension through nasal drops or nasal spray or liquid nebulized to the oral or nasopharyngeal pathways of said subject, such that a therapeutically effective amount of mentioned compound contact the affected tissues of said subject (cartilage) through systemic absorption and circulation.

10. The use according to the claims 1, 2, 3, 4, 5 and 6, wherein said systemic administration of said compound is carried out by administering an oral form of said compound, such that a therapeutically active amount of it contact cartilage through systemic absorption and circulation.

11. The use according to the claims 1, 2, 3, 4, 5 and 6, where said systemic administration of the mentioned compound be carried out, administering a form injectable thereof such that a therapeutically active amount from it contact cartilage through systemic absorption and circulation.

12. The use according to the claims 1, 2, 3, 4, 5 and 6, where said systemic administration of the said compound is carried out by applying a suppository of said compound such that a therapeutically amount active of it contact cartilage through absorption Systemic and circulation.