ES2283220B1 - USE OF DELTA OPIOID RECEIVER AGONISTS IN THE PREPARATION OF PHARMACEUTICAL COMPOSITIONS, PHARMACEUTICAL COMPOSITIONS CONTAINING THEM AND THEIR APPLICATIONS IN TUMOR TREATMENT. - Google Patents
USE OF DELTA OPIOID RECEIVER AGONISTS IN THE PREPARATION OF PHARMACEUTICAL COMPOSITIONS, PHARMACEUTICAL COMPOSITIONS CONTAINING THEM AND THEIR APPLICATIONS IN TUMOR TREATMENT. Download PDFInfo
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- ES2283220B1 ES2283220B1 ES200600960A ES200600960A ES2283220B1 ES 2283220 B1 ES2283220 B1 ES 2283220B1 ES 200600960 A ES200600960 A ES 200600960A ES 200600960 A ES200600960 A ES 200600960A ES 2283220 B1 ES2283220 B1 ES 2283220B1
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Abstract
Uso de agonistas del receptor opioide delta en la elaboración de composiciones farmacéuticas, composiciones farmacéuticas que las contienen y sus aplicaciones en el tratamiento de tumores. Esta invención se fundamenta en el efecto bloqueante del agonista DPDPE sobre la cascada de señalización intramolecular, iniciada por el par CXCL12-CXCR4, que induce la progresión tumoral y la formación de metástasis. Así, la invención describe que el tratamiento con DPDPE in vivo reduce las metástasis en modelos animales, mediante un mecanismo que no implica la respuesta inmune.Use of opioid delta receptor agonists in the preparation of pharmaceutical compositions, pharmaceutical compositions containing them and their applications in the treatment of tumors. This invention is based on the blocking effect of the DPDPE agonist on the intramolecular signaling cascade, initiated by the CXCL12-CXCR4 pair, which induces tumor progression and metastasis formation. Thus, the invention describes that treatment with DPDPE in vivo reduces metastases in animal models, by means of a mechanism that does not involve the immune response.
Description
Uso de agonistas del receptor opioide delta en la elaboración de composiciones farmacéuticas, composiciones farmacéuticas que las contienen y sus aplicaciones en el tratamiento de tumores.Use of delta opioid receptor agonists in the preparation of pharmaceutical compositions, compositions pharmaceuticals that contain them and their applications in treatment of tumors
La invención se encuadra en el sector biomédico, concretamente en el campo del desarrollo de composiciones farmacéuticas para el tratamiento de enfermedades tumorales, y más específicamente, en la utilización de agonistas del receptor opioide delta (por ejemplo, el DPDPE, D-Pen-D-Pen-encefalina), como nuevos agentes antitumorales que reducen específicamente la progresión tumoral y la aparición de metástasis por diseminación sanguínea.The invention falls within the biomedical sector, specifically in the field of composition development pharmaceuticals for the treatment of tumor diseases, and more specifically, in the use of receptor agonists Delta opioid (for example, the DPDPE, D-Pen-D-Pen-enkephalin), as new antitumor agents that specifically reduce the tumor progression and the appearance of metastasis by dissemination blood
El receptor de quimioquina CXCR4 interviene en el establecimiento de metástasis al promover la migración y la adhesión de las células tumorales a los nódulos linfáticos, los pulmones y el hígado, cuando su ligando, CXCL12, es sobreexpresado en esas localizaciones (Benovic, J.L. and Marchese, A. 2004.; Muller, A. et al, 2001; Balkwill, F., 2004). CXCR4 está sobreexpresado en numerosos tipos de células tumorales y su presencia anticipa resultados clínicos poco satisfactorios (Kang H et al. 2005; Salvucci o et al. 2005; Wang N et al. 2005). Además, se ha constatado que el uso de antagonistas de CXCR4, como el AMD 3100, anticuerpos bloqueantes o estrategias de ARN interferente reducen las metástasis en varios modelos murinos (Liang, Z. et al, 2004; Lapteva, N. et al, 2005; Takenaga, M. et al, 2004). CXCL12 es liberado en grandes cantidades a nivel de nódulos linfáticos, pulmones e hígados, órganos donde las metástasis tienden a aparecer. En resumen, hay evidencias suficientes para relacionar el par CXCR4-CLCX12 con el desarrollo de metástasis, y por tanto, para considerarlo una diana en el diseño de potenciales fármacos contra el cáncer.The CXCR4 chemokine receptor is involved in the establishment of metastases by promoting the migration and adhesion of tumor cells to lymph nodes, lungs and liver, when its ligand, CXCL12, is overexpressed in these locations (Benovic, JL and Marchese , A. 2004 .; Muller, A. et al , 2001; Balkwill, F., 2004). CXCR4 is overexpressed in numerous types of tumor cells and its presence anticipates unsatisfactory clinical results (Kang H et al . 2005; Salvucci o et al . 2005; Wang N et al . 2005). In addition, it has been found that the use of CXCR4 antagonists, such as AMD 3100, blocking antibodies or interfering RNA strategies reduce metastases in several murine models (Liang, Z. et al , 2004; Lapteva, N. et al , 2005 ; Takenaga, M. et al , 2004). CXCL12 is released in large quantities at the level of lymph nodes, lungs and livers, organs where metastases tend to appear. In summary, there is sufficient evidence to relate the CXCR4-CLCX12 pair with the development of metastases, and therefore, to consider it a target in the design of potential cancer drugs.
CXCR4 pertenece a la superfamilia de receptores acoplados a la proteína G (GPCR). Los receptores opioides también forman parte de esta familia. Los opiáceos endógenos (endorfinas) son pequeñas moléculas peptídicas, descubiertas inicialmente en tejidos cerebrales, que reducen la sensación de dolor. Los opiáceos se unen a los GPCRs mu, delta y kappa (Kirk-Othmer, 1996; Kieffer BL, 2000). Si bien se expresan a altos niveles en el cerebro, los opiáceos también han sido detectadas en células del sistema inmune y en algunos tipos de células malignas, como las de los melanoma, tumores de próstata y pulmón (Fichna, J. and Janecka, A. 2004), donde su papel es todavía desconocido. Distintos estudios con leucocitos in vitro han mostrado que los opiáceos pueden bloquear los receptores de quimioquinas mediante desensibilización heteróloga (Rogers, T.J. et al, 2000). La desensibilización heteróloga es un mecanismo bien estudiado en esta superfamilia de receptores: un GPCR resulta activado por un agonista, iniciándose un proceso de señalización que conduce a la inactivación de otro GPCR, produciéndose así una modulación cruzada.CXCR4 belongs to the superfamily of G protein-coupled receptors (GPCR). Opioid receptors are also part of this family. Endogenous opioids (endorphins) are small peptide molecules, initially discovered in brain tissues, which reduce the sensation of pain. Opioids bind to mu , delta and kappa GPCRs (Kirk-Othmer, 1996; Kieffer BL, 2000). Although they are expressed at high levels in the brain, opiates have also been detected in cells of the immune system and in some types of malignant cells, such as melanoma, prostate and lung tumors (Fichna, J. and Janecka, A 2004), where his role is still unknown. Different studies with leukocytes in vitro have shown that opioids can block chemokine receptors by heterologous desensitization (Rogers, TJ et al , 2000). Heterologous desensitization is a well-studied mechanism in this superfamily of receptors: a GPCR is activated by an agonist, initiating a signaling process that leads to the inactivation of another GPCR, thus producing a cross modulation.
Las metástasis tumorales constituyen la causa de mortalidad determinante en la mayoría de los cánceres malignos. Teniendo en cuenta la interacción cruzada que se puede establecer entre los opiáceos y receptores relacionados con la migración de células tumorales, esta invención descubre que a los opiáceos agonistas del receptor delta pueden actuar como agentes antitumorales eficaces en la reducción de metástasis.Tumor metastases are the cause of Determinant mortality in most malignant cancers. Taking into account the cross interaction that can be established between opioids and receptors related to the migration of tumor cells, this invention discovers that opiates Delta receptor agonists can act as agents effective antitumor agents in reducing metastases.
Un objeto de la presente invención lo constituye la utilización de agonistas del receptor delta opiáceo, en adelante utilización de un compuesto de la presente invención, en la elaboración de medicamentos o composiciones farmacéuticas para la prevención y/o tratamiento del cáncer.An object of the present invention constitutes it the use of opioid delta receptor agonists, in hereinafter use of a compound of the present invention, in the preparation of drugs or pharmaceutical compositions for Cancer prevention and / or treatment.
Un objeto particular de la presente invención lo constituye la utilización de un compuesto de la invención en el que el compuesto agonista pertenece al siguiente grupo, ya sean de estructura peptídica o no: compuestos de diarilmetilpiperazina (U.S. Pat 20060004016, Chang; Kwen-Jen 2006, Enantiomerically pure opioid diarylmethylpiperazine as a cardioprotection agent), TAN-67 (Fryer et al. in 274 J. Biol. Chem. 451-457, 2000), derivados tetracíclicos de piridina y pirazina (WO 99/04795, Toray Industries Inc.), pirrolooctahidroisoquinolonas (Dondio, G. et al. Discovery of a novel class of substituted pyrrolooctahydroisoquinolines as potent and selective.delta. opioid agonists, based on an extension of the message-address concept. J. Med. Chem. 1997, 40: 3192-3198), el compuesto BW373U86 (Chang, KJ. et al. A novel potent and selective nonpeptidic delta opioid receptor agonist, BW373U86. J. Pharm. Exp. Ther. 1993, 267, 852-857), el compuesto SNC 80 (Calderon, SN. et al. Probes for narcotic receptor mediated phenomena 19. Synthesis of (+)-4-[(.alpha.R)-.alpha.-(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC 80) A highly selective, nonpeptide.delta. opioid receptor agonist. J. Med. Chem. 1994, 37, 2125-2128), DPDPE y deltorfina I y II (U.S. Pat. 5,389,645 y 5,985,880, DPDPE [D-Pen.sup.2, D-Pen.sup.5]-(enkephalin) y heptapéptidos [deltorfina I y II] respectivamente), bifalina (Crystal structure of biphalin sulfate: a multireceptor opioid peptide. Flippen-Anderson JL et al. J Pept Res 2002, 59: 123-33), el péptido DADLE ([D-Ala2, D-Leu5] enkephalin (DADLE) protects liver against ischemia-reperfusion injury in the rat. Yamanouchi K, et al. J Surg Res. 2003, 114: 72-7) y la metadona.A particular object of the present invention is the use of a compound of the invention in which the agonist compound belongs to the following group, whether peptide or not: diarylmethylpiperazine compounds (US Pat 20060004016, Chang; Kwen-Jen 2006 , Enantiomerically pure opioid diarylmethylpiperazine as a cardioprotection agent), TAN-67 (Fryer et al . In 274 J. Biol. Chem. 451-457, 2000), tetracyclic derivatives of pyridine and pyrazine (WO 99/04795, Toray Industries Inc. ), pyrrolooctahydroisoquinolones (Dondio, G. et al . Discovery of a novel class of substituted pyrrolooctahydroisoquinolines as potent and selective.delta. opioid agonists, based on an extension of the message-address concept. J. Med. Chem. 1997, 40: 3192-3198), compound BW373U86 (Chang, KJ. Et al . A novel potent and selective nonpeptidic delta opioid receptor agonist, BW373U86. J. Pharm. Exp. Ther. 1993, 267, 852-857), compound SNC 80 (Calderon, SN. Et al . Probes for na rcotic receptor mediated phenomena 19. Synthesis of (+) - 4 - [(. alpha.R) -. alpha .- (2S, 5R) -4-allyl-2,5-dimethyl-1-piperazinyl) -3-methoxybenzyl ] -N, N-diethylbenzamide (SNC 80) A highly selective, nonpeptide.delta. agonist opioid receptor. J. Med. Chem. 1994, 37, 2125-2128), DPDPE and deltorfina I and II (US Pat. 5,389,645 and 5,985,880, DPDPE [D-Pen.sup.2, D-Pen.sup.5] - (enkephalin ) and heptapeptides [deltorphine I and II] respectively), bifalin (Crystal structure of biphalin sulfate: a multireceptor opioid peptide. Flippen-Anderson JL et al . J Pept Res 2002, 59: 123-33), the DADLE peptide ([D -Ala2, D-Leu5] enkephalin (DADLE) protects liver against ischemia-reperfusion injury in the rat. Yamanouchi K, et al. J Surg Res. 2003, 114: 72-7) and methadone.
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Una realización particular de la presente invención lo constituye el uso de un compuesto de la invención en el que el compuesto agonista es el agonista D-Pen-D-Pen-encefalina (DPDPE).A particular embodiment of the present invention constitutes the use of a compound of the invention in the one that the agonist compound is the agonist D-Pen-D-Pen-enkephalin (DPDPE).
Otro objeto de la presente invención lo constituye una composición farmacéutica, en adelante composición farmacéutica de la presente invención, que comprende una cantidad terapéuticamente efectiva de un compuesto o agente agonista del receptor opioide delta, junto con, opcionalmente, uno o más adyuvantes y/o vehículos farmacéuticamente aceptables.Another object of the present invention is constitutes a pharmaceutical composition, hereinafter composition pharmaceutical of the present invention, which comprises an amount therapeutically effective of a compound or agonist agent of the Delta opioid receptor, along with, optionally, one or more pharmaceutically acceptable adjuvants and / or vehicles.
Otro objeto particular de la presente invención lo constituye la composición farmacéutica de la invención en la que el compuesto agonista pertenece al siguiente grupo, ya sean de estructura peptídica o no: compuestos de diarilmetilpiperazina, TAN-67, derivados tetracíclicos de piridina y pirazina, pirrolooctahidroisoquinolonas, el compuesto BW373U86, el compuesto SNC 80, DPDPE, deltorfina I y II, bifalina, el péptido DADLE y la metadona.Another particular object of the present invention it is constituted by the pharmaceutical composition of the invention in the that the agonist compound belongs to the following group, whether peptide structure or not: diarylmethylpiperazine compounds, TAN-67, tetracyclic derivatives of pyridine and pyrazine, pyrrolooctahydroisoquinolones, compound BW373U86, the compound SNC 80, DPDPE, deltorfina I and II, bifalina, the peptide DADLE and methadone.
Otro objeto particular de la presente invención lo constituye la composición farmacéutica de la invención en la que el compuesto es el agonista D-Pen-D-Pen-encefalina (DPDPE).Another particular object of the present invention it is constituted by the pharmaceutical composition of the invention in the that the compound is the agonist D-Pen-D-Pen-enkephalin (DPDPE).
Otro objeto de la presente invención lo constituye el uso de la composición farmacéutica de la invención en el tratamiento de un mamífero, preferentemente un ser humano, afectado por cáncer, en adelante uso de la composición farmacéutica de la presente invención, consistente en la administración de dicha composición terapéutica que reduce la progresión tumoral, por ejemplo, la migración, extravasación y asentamiento de células tumorales en localizaciones distintas de la original (metástasis).Another object of the present invention is constitutes the use of the pharmaceutical composition of the invention in the treatment of a mammal, preferably a human being, affected by cancer, hereinafter use of the pharmaceutical composition of the present invention, consisting of the administration of said therapeutic composition that reduces tumor progression, by example, migration, extravasation and cell settlement tumor in locations other than the original (metastasis).
La presenta invención ofrece una nueva estrategia terapéutica para la profilaxis y/o tratamiento de enfermedades tumorales humanas.The present invention offers a new therapeutic strategy for the prophylaxis and / or treatment of human tumor diseases
La invención se basa en que los inventores han identificado el efecto inactivante del compuesto DPDPE, agonista del receptor opioide delta, sobre la cascada de eventos moleculares inducida por el par, ligando-receptor, CXCL12-CXR4 que induce a la progresión tumoral, por ejemplo al establecimiento de metástasis. Este efecto inactivante se produce tras su unión al receptor opiáceo delta y por un proceso de modulación cruzada entre receptores GPCR (Ejemplo 1).The invention is based on the fact that the inventors have identified the inactivating effect of the DPDPE compound, agonist delta opioid receptor, on the cascade of molecular events pair-induced, ligand-receptor, CXCL12-CXR4 that induces tumor progression, by example to the establishment of metastases. This inactivating effect occurs after its binding to the opioid delta receptor and by a process cross modulation between GPCR receptors (Example 1).
Los inventores han descubierto que el DPDPE podría ejercer una desensibilización heteróloga sobre CLCX12-CXR4, reduciendo la adhesión celular, la migración celular, la extravasación y el anclaje en órganos distantes de las células tumorales (Ejemplo 2). Así, el tratamiento con DPDPE en animales que habían sido inyectados con células de melanona redujo la aparición de focos de metástasis en el pulmón (Ejemplo 2).The inventors have discovered that DPDPE could exercise heterologous desensitization on CLCX12-CXR4, reducing cell adhesion, the cell migration, extravasation and anchoring in organs distant from the tumor cells (Example 2). So, the treatment with DPDPE in animals that had been injected with cells from melanone reduced the appearance of foci of metastases in the lung (Example 2).
Actualmente se están realizando ensayos clínicos de las propiedades analgésicas de DPDPE (Quock RM et al. 1999). Esta invención muestra que la aplicación del DPDPE podría también extenderse a la eliminación de metástasis.Clinical trials of the analgesic properties of DPDPE are currently being conducted (Quock RM et al . 1999). This invention shows that the application of DPDPE could also extend to the elimination of metastases.
La utilización del DPDPE en el tratamiento de tumores ofrece las siguientes ventajas: 1) podría ayudar a remitir metástasis de numerosos tumores (de pulmón, hígado, nódulos, mama, próstata) en cuyas células aparecen receptores del agonista DPDPE y CXCR4; además, 2) podría representar una alternativa menos agresiva a los tratamientos de quimioterapia y radioterapia actuales; 3) considerando el efecto analgésico, se ha observado que en ratas el DPDPE causa menos efectos indeseados que la morfina, un opiáceo se administra a enfermos de cáncer en estado crítico (Cheng, et al, 1993); 3) por último, actualmente se buscan opiáceos que no atraviesen la barrera hematoencefálica y permanezcan en periferia.The use of DPDPE in the treatment of tumors offers the following advantages: 1) it could help to remit metastases from numerous tumors (lung, liver, nodules, breast, prostate) in whose cells appear DPDPE and CXCR4 agonist receptors; in addition, 2) it could represent a less aggressive alternative to current chemotherapy and radiotherapy treatments; 3) considering the analgesic effect, it has been observed that in rats DPDPE causes less unwanted effects than morphine, an opioid is administered to critically ill cancer patients (Cheng, et al , 1993); 3) Finally, opiates are currently being sought that do not cross the blood brain barrier and remain in the periphery.
Así, un objeto de la presente invención lo constituye la utilización de agonistas del receptor delta opiáceo, en adelante utilización de un compuesto de la presente invención, en la elaboración de medicamentos o composiciones farmacéuticas para la prevención y/o tratamiento de enfermedades tumorales humanas y animales.Thus, an object of the present invention is constitutes the use of opioid delta receptor agonists, hereinafter use of a compound of the present invention, in the preparation of drugs or pharmaceutical compositions for the prevention and / or treatment of human tumor diseases and animals.
Tal como se utiliza en la presente invención el término agonista del receptor opiáceo delta es un compuesto que se une o interactúa con el receptor opiáceo delta y cuya interacción induce sus efectos terapéuticos, es decir, analgésicos y/o antitumorales.As used in the present invention the term opioid delta receptor agonist is a compound that binds or interacts with the opioid delta receptor and whose interaction induces its therapeutic effects, that is, analgesics and / or anti-tumor
Un objeto particular de la presente invención lo constituye la utilización de un compuesto de la invención en el que el compuesto agonista pertenece al siguiente grupo, ya sean de estructura peptídica o no: compuestos de diarilmetilpiperazina (U.S. Pat 20060004016, Chang; Kwen-Jen 2006, Enantiomerically pure opioid diarylmethylpiperazine as a cardioprotection agent), TAN-67 (Fryer et al. in 274 J. Biol. Chem. 451-457, 2000), derivados tetracíclicos de piridina y pirazina (WO 99/04795, Toray Industries Inc.), pirrolooctahidroisoquinolonas (Dondio, G. et al. Discovery of a novel class of substituted pyrrolooctahydroisoquinolines as potent and selective.delta. opioid agonists, based on an extension of the message-address concept. J. Med. Chem. 1997, 40: 3192-3198), el compuesto BW373U86 (Chang, KJ. et al. A novel potent and selective nonpeptidic delta opioid receptor agonist, BW373U86. J. Pharm. Exp. Ther. 1993, 267, 852-857), el compuesto SNC 80 (Calderon, SN. et al. Probes for narcotic receptor mediated phenomena 19. Synthesis of (+)-4-[(.alpha.R)-.alpha.-(2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC 80) A highly selective, nonpeptide.delta. opioid receptor agonist. J. Med. Chem. 1994, 37, 2125-2128), DPDPE y deltorfina I y II (U.S. Pat. 5,389,645 y 5,985,880, DPDPE [D-Pen.sup.2, D-Pen.sup.5]-(enkephalin) y heptapéptidos [deltorpiin I and II] respectivamente), biphalin (Crystal structure of biphalin sulfate: a multireceptor opioid peptide. Flippen-Anderson JL et al. J Pept Res 2002, 59: 123-33), el péptido DADLE ([D-Ala2, D-Leu5] enkephalin (DADLE) protects liver against ischemia-reperfusion injury in the rat. Yamanouchi K, et al. J Surg Res. 2003, 114: 72- 7) y la metadona.A particular object of the present invention is the use of a compound of the invention in which the agonist compound belongs to the following group, whether peptide or not: diarylmethylpiperazine compounds (US Pat 20060004016, Chang; Kwen-Jen 2006 , Enantiomerically pure opioid diarylmethylpiperazine as a cardioprotection agent), TAN-67 (Fryer et al . In 274 J. Biol. Chem. 451-457, 2000), tetracyclic derivatives of pyridine and pyrazine (WO 99/04795, Toray Industries Inc. ), pyrrolooctahydroisoquinolones (Dondio, G. et al . Discovery of a novel class of substituted pyrrolooctahydroisoquinolines as potent and selective.delta. opioid agonists, based on an extension of the message-address concept. J. Med. Chem. 1997, 40: 3192-3198), compound BW373U86 (Chang, KJ. Et al . A novel potent and selective nonpeptidic delta opioid receptor agonist , BW373U86. J. Pharm. Exp. Ther. 1993, 267, 852-857), compound SNC 80 (Calderon, SN. Et al . Probes for n arcotic receptor mediated phenomena 19. Synthesis of (+) - 4 - [(. alpha.R) -. alpha .- (2S, 5R) -4-allyl-2,5-dimethyl-1-piperazinyl) -3-methoxybenzyl ] -N, N-diethylbenzamide (SNC 80) A highly selective, nonpeptide.delta. agonist opioid receptor. J. Med. Chem. 1994, 37, 2125-2128), DPDPE and deltorfina I and II (US Pat. 5,389,645 and 5,985,880, DPDPE [D-Pen.sup.2, D-Pen.sup.5] - (enkephalin ) and heptapeptides [deltorpiin I and II] respectively), biphalin (Crystal structure of biphalin sulfate: a multireceptor opioid peptide. Flippen-Anderson JL et al . J Pept Res 2002, 59: 123-33), the DADLE peptide ([D -Ala2, D-Leu5] enkephalin (DADLE) protects liver against ischemia-reperfusion injury in the rat. Yamanouchi K, et al. J Surg Res. 2003, 114: 72-7) and methadone.
Una realización particular de la presente invención lo constituye el uso de un compuesto de la invención en el que el compuesto agonista es el agonista D-Pen-D-Pen-encefalina (DPDPE).A particular embodiment of the present invention constitutes the use of a compound of the invention in the one that the agonist compound is the agonist D-Pen-D-Pen-enkephalin (DPDPE).
Tal como se utiliza en la presente invención el término "la utilización de un compuesto agonista" incluye además el uso de sus formas isoméricas, sales farmacéuticamente aceptables, derivados, solvatos, amidas, ésteres y éteres de los compuestos originales. Los compuestos agonistas de la presente invención utilizados pueden ser isómeros, incluyendo isómeros ópticos o enantiómeros. El uso de sus 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.As used in the present invention the term "the use of an agonist compound" includes In addition to the use of its isomeric forms, pharmaceutically salts acceptable, derivatives, solvates, amides, esters and ethers of the original compounds The agonist compounds of the present invention used may be isomers, including isomers optical or enantiomers. The use of its isomers, enantiomers or individual diastereoisomers and mixtures thereof fall within the scope of the present invention. The enantiomers or individual diastereoisomers, as well as mixtures thereof, can separated by conventional techniques.
Asimismo, dentro del alcance de esta invención se encuentran la utilización de los profármacos de los compuestos agonistas de la invención. El término "profármaco" tal como aquí se utiliza incluye a cualquier compuesto derivado de un compuesto agonista de la invención, por ejemplo, ésteres, incluyendo ésteres de ácidos carboxílicos, ésteres de aminoácidos, ésteres de fosfato, ésteres de sulfonato de sales metálicas, etc., carbamatos, amidas, etc., que, cuando se administra a un individuo es capaz de proporcionar, directa o indirectamente, dicho compuesto agonista de la invención en dicho individuo. Ventajosamente, dicho derivado es un compuesto que aumenta la biodisponibilidad del compuesto agonista cuando se administra a un individuo o que potencia la liberación del mismo en un compartimento biológico. La preparación de dicho profármaco puede llevarse a cabo mediante métodos convencionales conocidos por los expertos en la materia.Also, within the scope of this invention the use of the prodrugs of the compounds are found agonists of the invention. The term "prodrug" such as used here includes any compound derived from a agonist compound of the invention, for example, esters, including carboxylic acid esters, amino acid esters, phosphate esters, sulphonate esters of metal salts, etc., carbamates, amides, etc., which, when administered to an individual is capable of providing, directly or indirectly, said compound agonist of the invention in said individual. Advantageously said derivative is a compound that increases the bioavailability of agonist compound when administered to an individual or that enhances the release of it in a biological compartment. The preparation of said prodrug can be carried out by conventional methods known to experts in the matter.
Tal como se utiliza en la presente invención el término "enfermedades tumorales" se refiere a patologías creadas por el crecimiento de células tumorales humanas o animales, y de forma más concreta nos referimos, a título ilustrativo y sin que limite el alcance de la invención, al melanoma, cáncer de mama, cáncer de pulmón, cáncer de próstata, cáncer de sistema nervioso central y sarcoma. Así, otro objeto particular de la presente invención lo constituye la utilización de un compuesto de la invención en la elaboración de medicamentos o composiciones farmacéuticas para la prevención y/o tratamiento de enfermedades tumorales humanas pertenecientes, a título ilustrativo y sin que limite el alcance de la invención, al siguiente grupo: melanoma, cáncer de mama, cáncer de pulmón, cáncer de próstata, cáncer de sistema nervioso central y sarcoma.As used in the present invention the term "tumor diseases" refers to pathologies created by the growth of human or animal tumor cells, and more specifically we refer, for illustrative purposes and without that limits the scope of the invention, to melanoma, breast cancer, lung cancer, prostate cancer, nervous system cancer Central and sarcoma. Thus, another particular object of the present invention constitutes the use of a compound of the invention in the preparation of medicaments or compositions pharmaceuticals for the prevention and / or treatment of diseases human tumor belonging, by way of illustration and without limit the scope of the invention to the following group: melanoma, breast cancer, lung cancer, prostate cancer, cancer central nervous system and sarcoma.
Otro objeto de la presente invención lo constituye una composición farmacéutica, en adelante composición farmacéutica de la presente invención, que comprende una cantidad terapéuticamente efectiva de un compuesto o agente agonista del receptor opioide delta, junto con, opcionalmente, uno o más adyuvantes y/o vehículos farmacéuticamente aceptables. Dicha composición terapéutica es particularmente útil frente a células tumorales humanas y animales.Another object of the present invention is constitutes a pharmaceutical composition, hereinafter composition pharmaceutical of the present invention, which comprises an amount therapeutically effective of a compound or agonist agent of the Delta opioid receptor, along with, optionally, one or more pharmaceutically acceptable adjuvants and / or vehicles. Bliss therapeutic composition is particularly useful against cells Human and animal tumors.
Otro objeto particular de la presente invención lo constituye la composición farmacéutica de la invención en la que el compuesto agonista pertenece al siguiente grupo, ya sean de estructura peptídica o no: compuestos de diarilmetilpiperazina, TAN-67, derivados tetracíclicos de piridina y pirazina, pirrolooctahidroisoquinolonas, el compuesto BW373U86, el compuesto SNC 80, DPDPE, deltorfina I y II, biphalin, el péptido DADLE y la metadona.Another particular object of the present invention it is constituted by the pharmaceutical composition of the invention in the that the agonist compound belongs to the following group, whether peptide structure or not: diarylmethylpiperazine compounds, TAN-67, tetracyclic derivatives of pyridine and pyrazine, pyrrolooctahydroisoquinolones, compound BW373U86, the compound SNC 80, DPDPE, deltorfina I and II, biphalin, the peptide DADLE and methadone.
Otro objeto particular de la presente invención lo constituye la composición farmacéutica de la invención en la que el compuesto es el agonista D-Pen-D-Pen-encefalina (DPDPE).Another particular object of the present invention it is constituted by the pharmaceutical composition of the invention in the that the compound is the agonist D-Pen-D-Pen-enkephalin (DPDPE).
En el sentido utilizado en esta descripción, la expresión "vehículo farmacéuticamente aceptable" se refiere a aquellas sustancias, o combinación de sustancias, conocidas en el sector farmacéutico, utilizadas en la elaboración de formas farmacéuticas de administración e incluye adyuvantes, sólidos o líquidos, disolventes, tensioactivos, etc.In the sense used in this description, the "pharmaceutically acceptable vehicle" refers to those substances, or combination of substances, known in the pharmaceutical sector, used in the elaboration of forms pharmaceutical administration and includes adjuvants, solids or liquids, solvents, surfactants, etc.
Si se desea, dicha composición farmacéutica puede contener, además, uno o más agentes terapéuticos que, eventualmente, potencien la acción terapéutica de dicho compuesto agonista o bien que incrementen su espectro de acción.If desired, said pharmaceutical composition it may also contain one or more therapeutic agents that, eventually, enhance the therapeutic action of said compound agonist or that increase their spectrum of action.
Dicha composición farmacéutica puede ser utilizada para prevenir y/o tratar enfermedades tumorales humanas o animales.Said pharmaceutical composition may be used to prevent and / or treat human tumor diseases or animals.
El compuesto agonista estará presente en la composición farmacéutica en una cantidad terapéuticamente eficaz, es decir, en una cantidad apropiada para ejercer su efecto terapéutico. En una realización particular, la composición farmacéutica proporcionada por esta invención, contiene entre 0,01% y 99,99% en peso de un compuesto agonista y sus mezclas, y puede presentarse en cualquier forma farmacéutica de administración apropiada en función de la vía de administración elegida, por ejemplo, oral, parenteral o tópica. Una revisión de las distintas formas farmacéuticas de administración de fármacos y de sus procedimientos de preparación puede encontrarse, por ejemplo, en el Tratado de Farmacia Galénica, C. Faulí i Trillo, 1ª edición, 1993, Luzán 5, S.A. de Ediciones.The agonist compound will be present in the pharmaceutical composition in a therapeutically effective amount, that is, in an appropriate amount to exert its effect therapeutic. In a particular embodiment, the composition Pharmaceutical provided by this invention, contains between 0.01% and 99.99% by weight of an agonist compound and mixtures thereof, and may present in any pharmaceutical form of administration appropriate depending on the route of administration chosen, by example, oral, parenteral or topical. A review of the different pharmaceutical forms of drug administration and their preparation procedures can be found, for example, in the Treaty of Farmacia Galenica, C. Faulí i Trillo, 1st edition, 1993, Luzán 5, S.A. of Editions.
Otro objeto de la presente invención lo constituye el uso de la composición farmacéutica de la invención en el tratamiento de un mamífero, preferentemente un ser humano, afectado por una enfermedad tumoral, en adelante uso de la composición farmacéutica de la presente invención, consistente en la administración de dicha composición terapéutica que reduce la progresión tumoral, por ejemplo, la migración, extravasación y asentamiento de células tumorales en localizaciones distintas de la original (metástasis).Another object of the present invention is constitutes the use of the pharmaceutical composition of the invention in the treatment of a mammal, preferably a human being, affected by a tumor disease, hereinafter use of the pharmaceutical composition of the present invention, consisting of the administration of said therapeutic composition that reduces the tumor progression, for example, migration, extravasation and settlement of tumor cells in locations other than the original (metastasis).
El uso de la composición farmacéutica de la invención puede por tanto ser útil para el tratamiento de pacientes con distintos tipos de cáncer, a título ilustrativo y sin que limite el alcance de la invención, el melanoma, cáncer de mama, cáncer de pulmón, cáncer de próstata, cáncer de sistema nervioso central y sarcoma.The use of the pharmaceutical composition of the invention may therefore be useful for the treatment of patients with different types of cancer, for illustrative purposes and without limit the scope of the invention, melanoma, breast cancer, lung cancer, prostate cancer, nervous system cancer Central and sarcoma.
De esta manera, la invención proporciona un método para prevenir y/o tratar enfermedades tumorales en humanos que comprende la etapa de administrar a un ser humano, con necesidad de tratamiento, una cantidad terapéuticamente eficaz de una composición farmacéutica proporcionada por esta invención.In this way, the invention provides a method to prevent and / or treat tumor diseases in humans which includes the stage of administering to a human being, with need for treatment, a therapeutically effective amount of a pharmaceutical composition provided by this invention.
En resumen, la presente invención considera la utilización de ciertos fármacos opiáceos no sólo como analgésicos sino también como agentes anti-tumorales, debido a la modulación cruzada por los agonistas del receptor opioide delta sobre la cascada de transducción CXCL12-CXCR4 que está implicada en la progresión tumoral y generación de metástasis.In summary, the present invention considers the use of certain opioid drugs not only as analgesics but also as anti-tumor agents, due to cross modulation by delta opioid receptor agonists about the CXCL12-CXCR4 transduction cascade that is involved in tumor progression and generation of metastasis.
Figura 1Figure one
Las células fueron permeabilizadas, fijadas y teñidas como se describe en materiales y métodos con anticuerpos anti-CXCR4 o anti-receptor opiáceo delta. Como control de tinción, las células fueron incubadas con un anticuerpo secundario anti-conejo marcado con FITC o con un anticuerpo secundario anti-ratón marcado con FITC. La expresión de los receptores se puede visualizar en las imágenes B (expresión de CXCR4) y D (expresión del DOR), mientras que A and C representan las células controles.The cells were permeabilized, fixed and stained as described in materials and methods with antibodies anti-CXCR4 or opioid anti-receptor delta. As a staining control, the cells were incubated with a secondary anti-rabbit antibody labeled with FITC or with a secondary anti-mouse labeled antibody with FITC. The expression of the receptors can be visualized in the images B (expression of CXCR4) and D (expression of DOR), while that A and C represent the control cells.
Figura 2Figure 2
Las células fueron activadas con forscolina y los ligandos como se describe en materiales y métodos. Los niveles de AMPc fueron determinados a continuación. Las barras representan el porcentaje de inhibición del AMPc vs control (células pretratadas con forscolina y no expuestas a ligandos). Como se muestran en el gráfico, dosis de DPDPE del orden de 10^{-7} M reducen significativamente los niveles de AMPc. Los datos fueron recogidos a partir de cuatro experimentos independientes. Los datos se expresan como valores medios más desviación estándar. Se consideraron diferencias significativas para valores de P menores que 0.05.Cells were activated with forscolin and ligands as described in materials and methods. CAMP levels were determined below. The bars represent the percentage of cAMP vs. control inhibition (cells pretreated with forscolin and not exposed to ligands). As shown in the graph, doses of DPDPE of the order of 10-7 M significantly reduce cAMP levels. Data were collected from four independent experiments. Data are expressed as mean values plus standard deviation. Significant differences were considered for P values less than 0.05.
Figura 3Figure 3
A) Perfil del ciclo celular de la línea B16 tratada con DPDPE durante 24, 48 y 72 horas. B) Las células crecieron bajo las condiciones descritas en ausencia y en presencia de DPDPE (10^{-5} M) de 24 a 72 horas. Las barras representan el número medio células por placa. Los datos son representativos de 4 experimentos independientes.A) B16 line cell cycle profile treated with DPDPE for 24, 48 and 72 hours. B) The cells grew under the conditions described in absence and in presence DPDPE (10-5 M) from 24 to 72 hours. The bars represent the Average number of cells per plate. The data are representative of 4 independent experiments
Figura 4Figure 4
Las células B16 fueron incubadas con CXCL12 marcado. Después de los tiempos indicados se cuantificó el I^{125}-CXCL12 unido a la superficie. Los datos son expresados como cuentas por minuto (cpm), procedentes de tres ensayos independientes. No se observa ninguna diferencia estadísticamente significativa entre los diferentes grupos.B16 cells were incubated with CXCL12 marked. After the indicated times, the I125-CXCL12 attached to the surface. The data are expressed as counts per minute (cpm), coming from three independent trials. No difference is observed Statistically significant between the different groups.
Figura 5Figure 5
A) Células B16 marcadas con BCECF fueron sembradas en placas cubiertas tratadas con fibronectina. A una concentración de DPDPE 10^{-3} M se observa un efecto similar al que se observa con una concentración de AMD3100 10^{-6} M. Los datos se expresan como luminiscencia a partir de al menos tres experimentos por grupo. B) Migración polarizada a través de matrigel de células B16 hacia CXCL12 en ausencia o en presencia de las sustancias indicadas. De nuevo, se puede observar una reducción de la movilidad celular, en este caso en la migración, después del tratamiento de las células B16 de melanoma con DPDPE. Los datos proceden de 3 experimentos, cada uno de ellos realizado por triplicado.A) B16 cells marked with BCECF were seeded in covered plates treated with fibronectin. To one DPDPE concentration 10-3 M shows an effect similar to which is observed with a concentration of AMD3100 10-6 M. Los data are expressed as luminescence from at least three Experiments by group. B) Polarized migration through Matrix of B16 cells to CXCL12 in the absence or presence of The indicated substances. Again, a reduction can be observed of cell mobility, in this case in migration, after B16 melanoma cell treatment with DPDPE. The data They come from 3 experiments, each of them performed by triplicate.
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Figura 6Figure 6
Células B16 marcadas con CFDA fueron inyectadas en ratones C57B1/6. Las interacciones de rodamiento de las células con la microvasculatura de la piel fueron observadas después de inyección subcutánea de CXCL12. Se estudiaron las interacciones de rodamiento de células B16 de melanoma pretratadas con DPDPE en el mismo suelo vascular. Cada par de datos conectados (líneas punteadas) representan el rodamiento de células no tratadas con DPDPE (izquierda) y células tratadas (derecha). La línea llena y los puntos de datos corresponden a la media de rodamiento en nueve vasos de cuatro ratones (P = 0.027, prueba t-student pareada).B16 cells marked with CFDA were injected in C57B1 / 6 mice. The bearing interactions of the cells with the microvasculature of the skin were observed after subcutaneous injection of CXCL12. The interactions of B16 melanoma cell bearing pretreated with DPDPE in the same vascular floor. Each pair of connected data (lines dotted) represent the bearing of untreated cells with DPDPE (left) and treated cells (right). The full line and the data points correspond to the average of bearing in nine vessels of four mice (P = 0.027, test paired t-student).
Figura 7Figure 7
A) Células B16 fueron inyectadas por vía intravenosa en ratones singénicos C57/BL6. El gráfico muestra el número de puntos de metástasis presentes en la superficie de los pulmones en ratones C57/BL6 tratados o no con DPDPE 7 días después de la inoculación de células tumorales. En comparación con los controles, se observa una reducción de más del 50% en los animales tratados con DPDPE. El experimento fue repetido 3 veces con de 7 a 10 ratones por grupo. B) Efecto del DPDPE en ensayos de metástasis experimental de melanoma sobre pulmones con ratones SCID. Como el número de focos metastásicos no pudo ser contado en los ratones control, se muestra una estimación de la superficie afectada por la metástasis. De forma similar a los resultados obtenidos usando ratones C57/BL6, hay una gran reducción en la formación de metástasis debido al tratamiento con DPDPE. C) Imágenes representativas de pulmones en ratones C57BL/6 tratados o no tratados con DPDPE, una semana después de la inyección de células de melanoma B16 F10.A) B16 cells were injected via intravenous in syngeneic mice C57 / BL6. The graph shows the number of metastatic points present on the surface of the lungs in C57 / BL6 mice treated or not with DPDPE 7 days later of inoculation of tumor cells. In comparison with the controls, a reduction of more than 50% is observed in animals treated with DPDPE. The experiment was repeated 3 times with 7 to 10 mice per group. B) Effect of DPDPE in metastasis trials Experimental melanoma on lungs with SCID mice. As the number of metastatic foci could not be counted in mice control, an estimate of the area affected by the metastasis. Similar to the results obtained using C57 / BL6 mice, there is a large reduction in the formation of metastasis due to treatment with DPDPE. C) Images representative of lungs in C57BL / 6 mice treated or not treated with DPDPE, one week after cell injection of melanoma B16 F10.
Propósitos adicionales, ventajas y características de la invención podrán desarrollarse en el estado de la técnica o ser aprendidos por la práctica del invento. A través de los métodos descritos y demandas no se intenta excluir otros rasgos técnicos, componentes, aditivos o etapas del proceso que surjan como resultado del desarrollo de la técnica. Los siguientes ejemplos e ilustraciones no intentan limitar la presente invención.Additional purposes, advantages and characteristics of the invention may be developed in the state of the technique or be learned by the practice of the invention. TO through the described methods and demands no attempt is made to exclude other technical features, components, additives or process steps that arise as a result of the development of the technique. The following examples and illustrations do not attempt to limit this invention.
Se realizaron ensayos con melanoma de ratón, tanto in vivo como in vitro. En primer lugar, se buscó si las células B16 de melanoma de ratón expresaban los receptores del agonista DPDPE y CXCR4 (marcaje inumnoquímico). Células de melanoma ratón B16 (colección americana de cultivos tipo ATTC N° CRL-6475; Manassas, VA) fueron crecidas en medio Dulbecco-Eagle modificado (Sigma, St. Louis, MO), suplementado con un 10% de suero bovino fetal inactivado por calor (FBS), penicilina y estreptomicina. Los anticuerpos monoclonales (mAb) anti-CXCR4 habían sido obtenidos con anterioridad por los mismos inventores (Vila-Coro, A.J. et al. 1999); el anticuerpo policlonal de conejo anti-receptor opioide delta se obtuvo de Oncogene Research (San Diego, CA); el anticuerpo marcado con isotiocianato de fluoresceína (FITC) y el anticuerpo marcado anti-conejo (FITC) fueron suministrados por BD Biosciences Pharmingen (San Diego, CA). El BCECF "Cell Tracker" de tinción procedía de Molecular Probes (Eugene, OR).Trials with mouse melanoma were performed, both in vivo and in vitro . First, it was sought whether mouse melanoma B16 cells expressed the DPDPE and CXCR4 agonist receptors (immunochemical labeling). Mouse B16 melanoma cells (American collection of ATTC type cultures No. CRL-6475; Manassas, VA) were grown in modified Dulbecco-Eagle medium (Sigma, St. Louis, MO), supplemented with 10% inactivated fetal bovine serum by heat (FBS), penicillin and streptomycin. The anti-CXCR4 monoclonal antibodies (mAb) had been previously obtained by the same inventors (Vila-Coro, AJ et al . 1999); Rabbit polyclonal anti-opioid receptor delta antibody was obtained from Oncogene Research (San Diego, CA); fluorescein isothiocyanate labeled antibody (FITC) and anti-rabbit labeled antibody (FITC) were supplied by BD Biosciences Pharmingen (San Diego, CA). The BCECF "Cell Tracker" staining came from Molecular Probes (Eugene, OR).
Las células B16 fueron sembradas en pocillos con fibronectina adherida, fijadas con paraformaldehido al 4% (15 minutos a temperatura ambiente), permeabilizadas con 0.01% Triton X100 (5 minutos), lavadas tres veces con PBS-0.01% Tween, y bloqueadas con PBS-BSA (1 hora a temperatura ambiente). Entonces, fueron incubadas con anticuerpo anti-CXCR4 (1:50) o con anticuerpo anti-receptor opioide delta (1:100), junto con el segundo anticuerpo FITC-anti-ratón (1:200) o con FITC-anti-conejo (1:100), respectivamente. Las células fueron lavadas y la expresión del receptor fue evaluada mediante microscopía confocal de fluorescencia. Los resultados obtenidos confirmaron, efectivamente, que ambos receptores se expresan en las células B16 de melanoma (Figura 1).B16 cells were seeded in wells with adhered fibronectin, fixed with 4% paraformaldehyde (15 minutes at room temperature), permeabilized with 0.01% Triton X100 (5 minutes), washed three times with PBS-0.01% Tween, and blocked with PBS-BSA (1 hour at room temperature). Then, they were incubated with antibody anti-CXCR4 (1:50) or with antibody delta opioid anti-receptor (1: 100), along with the second FITC-anti-mouse antibody (1: 200) or with FITC-anti-rabbit (1: 100), respectively. The cells were washed and the expression of the recipient was evaluated by confocal microscopy of fluorescence. The results obtained effectively confirmed that both receptors are expressed in melanoma B16 cells (Figure 1).
A continuación, se estudió si el agonista DPDPE podría desensibilizar el receptor CXCR4 en células B16 de melanoma (modulación cruzada) mediante ensayos de AMPc. Para ello, las células B16 fueron incubadas con forscolina 10 mM (3 minutos a 37°C) para incrementar la producción de AMPc; posteriormente fueron estimuladas con los agonistas CXCL12 (50 nM), DPDPE (10^{-5} M), o con ambos (10 minutos a 37°C). Los niveles de AMPc fueron determinados con el paquete comercial AMPc Direct Immunoassay Kit (Calbiochem, Darmstadt, Germany). En presencia de DPDPE los niveles de AMPc fueron similares a los que existían en células sin estimular, de lo que se concluye que el DPDPE desensibiliza CXCR4 (Figura 2).Next, it was studied whether the DPDPE agonist could desensitize the CXCR4 receptor in B16 melanoma cells (cross modulation) by cAMP assays. To do this, the B16 cells were incubated with 10 mM forscolin (3 minutes at 37 ° C) to increase cAMP production; subsequently they were stimulated with the CXCL12 agonists (50 nM), DPDPE (10-5 M), or with both (10 minutes at 37 ° C). CAMP levels were determined with the AMPc Direct Immunoassay Kit commercial package (Calbiochem, Darmstadt, Germany). In the presence of DPDPE levels cAMP were similar to those that existed in cells without stimulate, from which it is concluded that DPDPE desensitizes CXCR4 (Figure 2).
Seguidamente, se valoró la posible citotoxicidad del DPDPE sobre las células (Figura 3), pero no se observó ningún efecto citotóxico. Para ello, se usó un paquete comercial para determinar la proliferación celular (Beckman Coulter, Hialeah, FL) y analizar los posibles efectos de este agonista opiáceo en el ciclo celular de las células B16. Las células fueron plantadas en una placa de 6 pocillos, incubadas durante entre 24 y 72 horas en medio DMEM completo, solas o con DPDPE a concentraciones de hasta 10^{-5} M. Posteriormente, se recogieron las células, se trataron de acuerdo al protocolo proporcionado, y el ciclo celular se analizó mediante citometría de flujo.Next, the possible cytotoxicity was assessed of DPDPE on the cells (Figure 3), but no cytotoxic effect For this, a commercial package was used to determine cell proliferation (Beckman Coulter, Hialeah, FL) and analyze the possible effects of this opioid agonist on the B16 cell cell cycle. The cells were planted in a 6-well plate, incubated for 24 to 72 hours in complete DMEM medium, alone or with DPDPE at concentrations up to 10-5 M. Subsequently, the cells were collected, treated according to the protocol provided, and the cell cycle is analyzed by flow cytometry.
Seguidamente, para determinar si el efecto observado se debía a la internalización de CXCR4, se llevó a cabo un ensayo de unión a la superficie en células B16, y más concretamente sobre células intactas para marcar sólo los receptores presentes en la superficie celular. Así, las células (500.000 células/pocillo) fueron sembradas en una placa de 24 pocillos y expuestas a DPDPE (10 -5 M) durante 15 a 30 minutos a 37°C. Para el resto del experimento, la temperatura se mantuvo a 4°C. Las células fueron entonces tratadas con ^{125}I CXCL12 0.15 nM (2000 Ci/mmol; Amersham Pharmacia) durante 2 horas a 4°C con una agitación suave. La unión no específica se evaluó mediante la incubación de las células conjuntamente con CXCL12 no marcado (500 nM) en el medio de unión. Las células fueron entonces lavadas 3 veces cuidadosamente con PBS y recogidas en tubos. La cantidad de ^{125}I CXCL12 se cuantificó para cada una de las condiciones con un contador de centelleo \gamma. Como se muestra en la Figura 4, las células pretratadas con DPDPE no presentaron diferencias significativas en las cantidades de ^{125}I CXCL12 unido en la superficie celular, lo que indica que la alteración de la respuesta de CXCR4 por DPDPE no se debe a la internalización del receptor.Next, to determine if the effect observed due to the internalization of CXCR4, was carried out a surface binding assay in B16 cells, and more specifically on intact cells to mark only the receptors present on the cell surface. So the cells (500,000 cells / well) were seeded on a plate of 24 wells and exposed to DPDPE (10-5 M) for 15 to 30 minutes at 37 ° C For the rest of the experiment, the temperature was maintained at 4 ° C The cells were then treated with125 I CXCL12 0.15 nM (2000 Ci / mmol; Amersham Pharmacia) for 2 hours at 4 ° C with a gentle agitation Non-specific binding was assessed by incubation of the cells together with unlabeled CXCL12 (500 nM) in the joining means. The cells were then washed 3 Sometimes carefully with PBS and collected in tubes. The amount of 125 I CXCL12 was quantified for each of the conditions with a scintillation counter γ. As shown in Figure 4, cells pretreated with DPDPE did not show differences significant in the amounts of125I CXCL12 bound in the cell surface, indicating that the alteration of the response of CXCR4 by DPDPE is not due to internalization of the receiver.
Se realizaron ensayos in vitro para determinar el efecto del DPDPE tras la desensibilización de CXCR4 en la adhesión, invasión y migración de las células tumorales. Para el ensayo de adhesión celular se prepararon placas de cultivo celular de 96 pocillos de BD BioScience durante la noche a 4°C con 20 \mug/ml de fibronectina y los ligandos. Las placas fueron lavadas con PBS y bloqueadas con 0.5% BSA durante 1 hora a 37°C. Las células B16, previamente marcadas con BCECF, se sembraron (2 x 10^{5}/pocillo) en 200 \mul de PBS para su adhesión (10 minutos, 37°C). Las células no adheridas se eliminaron mediante un lavado suave con PBS; la adhesión celular se determinó con un luminómetro tal como se describió por otros autores (Chen, C. et al. 2004). La luminiscencia es directamente proporcional al número de células por pocillo. Para el ensayo concreto de la invención las células fueron sembradas en placas recubiertas con CXCL12/fibronectina en presencia o en ausencia de DPDPE, observándose que el nivel de adhesión celular se redujo en presencia de DPDPE y en un nivel dependiente de la dosis (Figura 5a). In vitro assays were performed to determine the effect of DPDPE after desensitization of CXCR4 on the adhesion, invasion and migration of tumor cells. For cell adhesion assay, 96-well cell culture plates of BD BioScience were prepared overnight at 4 ° C with 20 µg / ml fibronectin and ligands. The plates were washed with PBS and blocked with 0.5% BSA for 1 hour at 37 ° C. B16 cells, previously labeled with BCECF, were seeded (2 x 10 5 / well) in 200 µl of PBS for adhesion (10 minutes, 37 ° C). Non-adhered cells were removed by gentle washing with PBS; Cell adhesion was determined with a luminometer as described by other authors (Chen, C. et al . 2004). The luminescence is directly proportional to the number of cells per well. For the specific assay of the invention the cells were seeded on plates coated with CXCL12 / fibronectin in the presence or absence of DPDPE, it being observed that the level of cell adhesion was reduced in the presence of DPDPE and at a dose dependent level (Figure 5a ).
Los ensayos de migración se realizaron en cámaras de invasión de matrigel (BD, Biocat, San José, CA) por triplicado. Las células B16 (10^{5} células/100 \mul) se añadieron al compartimiento superior de la cámara y los ligandos fueron añadidos a la cámara inferior para promover la migración celular a través del matrigel. Las placas se incubaron a 37°C durante 22 horas. Las células que no migraron se eliminaron de la parte superior del matrigel con un bastoncillo de algodón. Después del ensayo de migración, el matrigel fue digerido con dispasa 15 minutos a 37°C y las células emigrantes fueron contabilizadas después de la completa digestión del matrigel por citometría de flujo (Bartolome, R.A. et al, 2004). Como se esperaba, el CXCL12 indujo la migración de las células B16. Sin embargo, en presencia de concentraciones de 10^{-5} a 10^{-3} de DPDPE la migración inducida por CXCL12 fue inhibida de una manera dependiente de dosis por DPDPE (Figura 5b).Migration tests were performed in matrigel invasion chambers (BD, Biocat, San José, CA) in triplicate. B16 cells (10 5 cells / 100 µl) were added to the upper chamber compartment and ligands were added to the lower chamber to promote cell migration through the matrigel. The plates were incubated at 37 ° C for 22 hours. Cells that did not migrate were removed from the top of the matrigel with a cotton swab. After the migration test, the matrigel was digested 15 minutes at 37 ° C and the emigrating cells were counted after the complete digestion of the matrigel by flow cytometry (Bartolome, RA et al , 2004). As expected, CXCL12 induced the migration of B16 cells. However, in the presence of concentrations of 10-5 to 10-3 DPDPE, migration induced by CXCL12 was inhibited in a dose-dependent manner by DPDPE (Figure 5b).
Los inventores también analizaron el efecto del DPDPE en el rodamiento in vivo de células de melanoma B16, primer paso crítico de la adhesión de las células sanguíneas a las paredes del endotelio vascular. La alta expresión de CXCL12 en órganos como hígado y pulmón podría explicar la alta frecuencia de metástasis en esos órganos (hipótesis semilla-suelo). Mediante microscopía intravital se estudió la interacción de células B16 de melanoma con la microvasculatura de la oreja en presencia o en ausencia del agonista DPDPE (Ludwig, R.J. 2004). El ratón fue anestesiado mediante inyección intraperitoneal de ketamina (Schwabe-Curamed, Karlsruhe, Germany) y xilacina (Rompun, Bayer, Leverkusen, Germany), y colocado en una manta homotérmica. Se preparó para microcirugía la arteria carótida común derecha; se insertó un catéter para la inyección retrograda de las células. La oreja izquierda del ratón, que recibió 50 \mul de 1.25 \muM de CXCL12, fue colocada suavemente entre un microscopio y un soporte. La arquitectura vascular y las células marcadas con fluorescencia (10 \muM CFDA-SE, Molecular Probes, Eugene, OR) se visualizaron durante su paso por los vasos bajo una epi-iluminación fluorescente usando un sistema de filtro multibanda (XF 53, Omega Optical, Brattleboro, VT). Se obtuvieron imágenes continuas de la microcirculación con una cámara 1/3'' DSP 3-CCD (DXC-390, Sony, Cologne, Germany), montada en un microscopio 32 Zeiss modificado (Axioteck Vario 100 HD, Zeiss) que estaba equipado con un objetivo de inmersión saltwater 10x (Nikon, Düsseldorf, Germany). Las imágenes fueron almacenadas digitalmente usando MediaStudio Pro7 (Ulead, Kaarst, Germany). Las velocidades de las células en segmentos de vasos individuales fueron determinadas mediante un análisis off-line. Las células fueron consideradas no interactuantes si se movían a la velocidad media del flujo sanguíneo, o rodantes si se movían a velocidades menores.The inventors also analyzed the effect of DPDPE on the in vivo bearing of B16 melanoma cells, the first critical step in the adhesion of blood cells to the walls of the vascular endothelium. The high expression of CXCL12 in organs such as liver and lung could explain the high frequency of metastases in those organs (seed-soil hypothesis). The interaction of melanoma B16 cells with the microvasculature of the ear in the presence or absence of the DPDPE agonist was studied by intravital microscopy (Ludwig, RJ 2004). The mouse was anesthetized by intraperitoneal injection of ketamine (Schwabe-Curamed, Karlsruhe, Germany) and xylazine (Rompun, Bayer, Leverkusen, Germany), and placed in a homothermal blanket. The right common carotid artery was prepared for microsurgery; a catheter was inserted for retrograde injection of the cells. The left ear of the mouse, which received 50 µl of 1.25 µM of CXCL12, was gently placed between a microscope and a support. Vascular architecture and fluorescently labeled cells (10 µM CFDA-SE, Molecular Probes, Eugene, OR) were visualized during their passage through the vessels under fluorescent epi-illumination using a multiband filter system (XF 53, Omega Optical , Brattleboro, VT). Continuous images of the microcirculation were obtained with a 1/3 '' DSP 3-CCD camera (DXC-390, Sony, Cologne, Germany), mounted on a modified Zeiss 32 microscope (Axioteck Vario 100 HD, Zeiss) that was equipped with a 10x saltwater immersion lens (Nikon, Düsseldorf, Germany). The images were stored digitally using MediaStudio Pro7 (Ulead, Kaarst, Germany). Cell speeds in individual vessel segments were determined by an off-line analysis. Cells were considered non-interacting if they moved at average blood flow velocity, or rolling if they moved at lower speeds.
Después de la inyección subcutánea de CXCL12 se observó un incremento del rodamiento respecto al observado espontáneamente. No obstante, después del tratamiento con DPDPE el rodamiendo se redujo de una manera significativa (3.9+/-5.1%) (Figura 6). Por tanto, el tratamiento con el agonista DPDPE también afecta las interacciones celulares dependientes de CXCL12 in vivo, impidiendo que las células cancerosas interactúen con las paredes vasculares.After subcutaneous injection of CXCL12 an increase in bearing was observed compared to that observed spontaneously. However, after treatment with DPDPE, rolling was reduced significantly (3.9 +/- 5.1%) (Figure 6). Therefore, treatment with the DPDPE agonist also affects CXCL12-dependent cell interactions in vivo , preventing cancer cells from interacting with vascular walls.
Como última etapa, se procedió al estudio del efecto del agonista DPDPE in vivo, observando la aparición de metástasis de pulmón en hembras de ratón inyectadas con células de melanoma B16 con y sin DPDPE. Así, se construyó un modelo experimental, inyectando células B16 de melanoma singénicas en ratones C57BL/6. Se utilizaron hembras de ratón (6 a 8 semanas de edad) C57BL/6 (Charles River); y hembras de ratón con inmunodeficiencias severas combinadas y deficientes en NK más adelante (CB17 SCID beige de Harlan, Chicago, IL). Los animales fueron mantenidos a temperatura constante (23°C) y humedad (50%-60%) con acceso libre a dieta estándar y agua. Todas estas experiencias con animales se realizaron observando la legislación española.As a last stage, the effect of the DPDPE agonist was studied in vivo , observing the appearance of lung metastases in mouse females injected with B16 melanoma cells with and without DPDPE. Thus, an experimental model was constructed, injecting syngeneic B16 melanoma cells into C57BL / 6 mice. Mouse females (6 to 8 weeks old) C57BL / 6 (Charles River) were used; and mouse females with severe immunodeficiencies combined and deficient in NK later (CB17 SCID beige from Harlan, Chicago, IL). The animals were kept at constant temperature (23 ° C) and humidity (50% -60%) with free access to standard diet and water. All these experiences with animals were carried out observing the Spanish legislation.
En el primer día, células B16 de melanoma fueron inyectadas en la vena de la cola del ratón (10^{5} células/ratón C57BL/6 y 10^{4} células/ratón CB17 SCID en un volumen de 250 \mul). Desde el día 1 al día 3, los ratones recibieron una inyección intraperitoneal diaria de DPDPE (10^{-4} M; 500 \mul); los animales control fueron tratados con PBS. La primera inyección de DPDPE fue aplicada justo antes de la inyección de las células. Las dosis de DPDPE utilizadas fueron las establecidas para promover la analgesia en el ratón (Heyman, J.S et al, 1987).On the first day, B16 melanoma cells were injected into the mouse tail vein (10 5 cells / mouse C57BL / 6 and 10 4 cells / mouse CB17 SCID in a volume of 250 µl) . From day 1 to day 3, the mice received a daily intraperitoneal injection of DPDPE (10-4M; 500 µl); Control animals were treated with PBS. The first DPDPE injection was applied just before the injection of the cells. The doses of DPDPE used were those established to promote mouse analgesia (Heyman, JS et al , 1987).
Los animales fueron sacrificados 7 días después de la inoculación de células B16 para analizar la propagación de los tumores. Los pulmones fueron lavados con PBS y fijados con una solución de Bouin durante 24 h. A continuación, se contaron los nódulos metastásicos en C57BL/6 con microscopía. Los experimentos fueron realizados por triplicado, con 10 animales en cada grupo.The animals were sacrificed 7 days later of inoculation of B16 cells to analyze the spread of tumors The lungs were washed with PBS and fixed with a Bouin solution for 24 h. Next, the metastatic nodules in C57BL / 6 with microscopy. The experiments They were performed in triplicate, with 10 animals in each group.
Como resultado, en el día 7 después de la inoculación de células tumorales in vivo, se hicieron visibles focos metastásicos en la superficie de los pulmones de todos los ratones. Sin embargo, en el ratón tratado con DPDPE el número de nódulos era significativamente más bajo que en el grupo control (40 \pm 27 nódulos/pulmón vs 150 \pm 40 nódulos/pulmón) (Figura 7).As a result, on day 7 after inoculation of tumor cells in vivo , metastatic foci became visible on the surface of the lungs of all mice. However, in the mouse treated with DPDPE the number of nodules was significantly lower than in the control group (40 ± 27 nodules / lung vs 150 ± 40 nodules / lung) (Figure 7).
Como se ha visto que los opiáceos modifican la respuesta inmune y podrían estar implicados en el fenómeno observado, para excluir la participación del sistema inmune, se realizaron experiencias en la cepa inmunocomprometida SCID. En el ratón SCID, debido a la gran masa tumoral, la superficie afectada por la metástasis fue estimada independientemente por dos investigadores. Los experimentos fueron realizados por triplicado, con 10 animales en cada grupo. En los ratones de esta cepa, como cabría esperar, la formación y la propagación de metástasis se desarrolló más rápidamente que en ratones C57BL/6. Los resultados muestran que en un ratón sin tratar el 50\pm18% del pulmón resultó afectado por metástasis. En ratones tratados con DPDPE, sin embargo, aunque los pulmones aparecían también con metástasis, se estimó que sólo el 7\pm4% del pulmón estaba afectado. Estos datos muestran que el DPDPE reduce la progresión de las metástasis sin a intervención de la respuesta inmune.As it has been seen that opiates modify the immune response and could be involved in the phenomenon observed, to exclude the involvement of the immune system, it performed experiences in the immunocompromised strain SCID. At SCID mouse, due to the large tumor mass, the affected surface by metastasis it was estimated independently by two researchers. The experiments were performed in triplicate, with 10 animals in each group. In mice of this strain, as would be expected, the formation and spread of metastases were developed faster than in C57BL / 6 mice. The results show that in an untreated mouse 50 ± 18% of the lung It was affected by metastasis. In mice treated with DPDPE, without However, although the lungs also appeared with metastasis, they estimated that only 7 ± 4% of the lung was affected. These dates show that DPDPE reduces the progression of metastases without immune response intervention.
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HATZOGLOU, A. et al.: "The antiproliferative effect of opioid receptor agonists on the T47D human breast cancer cell line, is partiallly mediated through opioid receptors". European Journal of Pharmacology, 1996, vol. 296, páginas 199-207, todo el documento. * |
KNAPP, R.J. et al.: "Properties of TAN-67, a nonpeptidic d-opioid receptor agonist, at cloned human d- and u-opioid receptors". European Journal of Pharmacology, 1995, vol. 291, páginas 129-134, todo el documento. * |
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