WO2014102417A1 - USE OF BICYCLIC DERIVATIVES OF 1-DEOXYGALACTONOJIRIMYCIN IN THE PRODUCTION OF A MEDICAMENT FOR THE TREATMENT OF DISEASES ASSOCIATED WITH MUTANT HUMAN LYSOSOMAL GALACTOSIDASE ß-ENZYMES - Google Patents

USE OF BICYCLIC DERIVATIVES OF 1-DEOXYGALACTONOJIRIMYCIN IN THE PRODUCTION OF A MEDICAMENT FOR THE TREATMENT OF DISEASES ASSOCIATED WITH MUTANT HUMAN LYSOSOMAL GALACTOSIDASE ß-ENZYMES Download PDF

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WO2014102417A1
WO2014102417A1 PCT/ES2013/070883 ES2013070883W WO2014102417A1 WO 2014102417 A1 WO2014102417 A1 WO 2014102417A1 ES 2013070883 W ES2013070883 W ES 2013070883W WO 2014102417 A1 WO2014102417 A1 WO 2014102417A1
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compound
galactosidase
treatment
general formula
enzymes
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PCT/ES2013/070883
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Spanish (es)
French (fr)
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José Manuel GARCÍA FERNÁNDEZ
Carmen Ortiz Mellet
Nanba EIJI
Higaki KATSUMI
Suzuki Yoshiyuki
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Consejo Superior De Investigaciones Científicas (Csic)
Universidad De Sevilla
International University Of Health And Wellfare (Iuhw)
National Universtity Corporation Tottori University
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Publication of WO2014102417A1 publication Critical patent/WO2014102417A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4365—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system having sulfur as a ring hetero atom, e.g. ticlopidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention is directed to the pharmaceutical sector, with applications for the treatment of lysosomal storage diseases related to mutations of lysosomal acid ⁇ -glucosidase.
  • the object of the invention is the use of a compound of formula (I)
  • R represents an aliphatic chain, linear or branched, which is selected from the group consisting of saturated hydrocarbon chain C Ci 6 , and unsaturated hydrocarbon chain C 2 -Ci 6 , for the manufacture of a medicament for the treatment of diseases related to ⁇ - human mutant lysosomal galactosidase enzymes.
  • Lysosomal storage disorders are a group of diseases resulting from the abnormal metabolism of several substrates that do not degrade and accumulate in lysosomes, leading to a series of phenotypes that include megalovisceralia, neurological pathologies, skeletal injuries and premature death. . These diseases are the result of mutations in the genes that encode enzymes involved in the degradation process.
  • symptomatic therapies are available for these patients, differing two therapeutic strategies: substrate reduction therapy, based on the inhibition of substrate production using inhibitors of the enzymes involved in its biosynthesis, and enzyme replacement therapy, based on the exogenous administration of recombinant active enzymes.
  • Gaucher's disease this therapy costs between $ 100,000 and $ 750,000 a year, and is not very effective for cases that show involvement of the central nervous system.
  • ⁇ -galactosidosis hereditary deficiency of lysosomal acid ⁇ -galactosidase causes two clinically distinct diseases in humans, GM1 gangliosidosis and Morquio B disease. Both are the result of mutations. in the GLB1 gene that lead to a wrong protein folding. The mode of inheritance is autosomal recessive.
  • GM1 gangliosidosis is a generalized neurosomatic disease that appears mainly in early childhood, and rarely in childhood or in young adults.
  • Morquio B disease is a rare bone disease without involvement of the central nervous system.
  • glycoconjugates In patients with these clinical phenotypes glycoconjugates accumulate with terminal ⁇ -galactose residues in tissues and urine.
  • the GM1 ganglioside and its Asian derivative GA1 accumulate in the brain in the case of GM1 gangliosidosis.
  • High amounts of oligosaccharides derived from keratane sulfate or glycoproteins in visceral organs and urine are detected in both patients with GM1 gangliosidosis and in patients suffering from Morquio B disease. More than 160 different point mutations have been identified in the gene encoding ⁇ -galactosidase.
  • the mutations lead to significant defects in the folding of the protein during translation in the endoplasmic reticulum, resulting in a reduction in the transport of the enzyme to the lysosome (degradation mediated by cellular quality control machinery).
  • the clinical form of the disease depends on the mutation in the acid ⁇ -galactosidase presented by the patient, and its severity is related to the residual enzymatic activity.
  • Enzyme replacement therapy for cases in which the central nervous system is affected has low or no efficacy, since recombinant enzymes do not cross the blood brain barrier. Thus, there is a large number of patients for whom there is no treatment or its effectiveness is very low. In recent years it has been described that some inhibitors of these glycosidases enzymes are capable of binding to the active site and stabilizing the appropriate folding, being able to act as "pharmacological chaperones" that facilitate the transport of the catalytically active form to lysosomes. Thus, the development of compounds with pharmacological chaperone activity has been postulated as a possible therapeutic strategy for the treatment of lysosomal storage diseases, and of particular interest for those clinical manifestations of the disease that involve the central nervous system.
  • these compounds used at subinhibitory concentrations have been shown to act as mutant ⁇ -galactosidase chaperones responsible for GM1 and Morquio B gangliosidosis disease (US 2006/0100241; W02004 / 037373).
  • these types of compounds generally behave as inhibitors of broad-spectrum glycosidases, simultaneously inhibiting several glycosidases, which represents a serious drawback for clinical applications.
  • valienamine such as N-octyl-4-epi ⁇ -valienamine (NOEV), which does not have a chemical structure of iminoaz ⁇ car, have also been shown to act as pharmacological chaperones for mutant enzymes responsible for these lysosomal storage diseases.
  • NOEV N-octyl-4-epi ⁇ -valienamine
  • a problem with this therapeutic strategy based on the use of pharmacological chaperones for the treatment of Morquio B disease and GM1 Gangliosidosis is that the compounds known to date are effective for reduced number of mutations in the gene encoding ⁇ -galactosidase. Therefore, and given the great variability of these patients, it is necessary to develop compounds that have a broad spectrum of action with respect to these mutations to enable the development of drugs for widespread use.
  • the present invention relates to the use of a compound of general formula (I) or one of its salts
  • R represents an aliphatic chain, linear or branched, which is selected from the group consisting of saturated hydrocarbon chain C Ci 6 , and unsaturated hydrocarbon chain
  • C2-C16 for the preparation of a medicament for the treatment of the deficiency of the active form of the enzyme ⁇ -galactosidase in humans, which is related to diseases such as GM1 gangliosidosis and Morquio B disease.
  • condensed bicyclic compounds (six members / five members) derived from 1-deoxygalactonojirimycin in which the bridgehead nitrogen atom is part of an isothiourea functional group that, in turn, carries a hydrophobic substituent, behave as specific lysosomal ⁇ -galactosidase inhibitors capable of acting as pharmacological chaperones at concentrations lower than those of inhibition, with the significant advantage over other glycomimetics, including other sp 2 iminoaz ⁇ cares, that do not present toxicity and that their chaperone activity is effective for a wide range of different mutations, including G190D, R201C, R201 H, V216A, D332N, Y444C, R457Q, R590H, I51T, R148T, L155R, R208C, D214Y, C230Y, L264S, N266S, W273R, K346N, S434L, G438
  • the chemical structure of the compounds of the invention allows them to cross the blood brain barrier, so their use in the manufacture of drugs for the treatment of lysosomal storage diseases with implications in the central nervous system, such as GM1 gangliosidosis, is especially interest.
  • saturated hydrocarbon chain refers, in the present invention, to aliphatic, linear or branched chains, having 1 to 16 carbon atoms, for example, methyl, ethyl, n-propyl, / ' -propyl, n -butyl, tere-butyl, sec-butyl, n-pentyl, n-hexyl, etc.
  • the saturated hydrocarbon chain has between 1 and 8 carbon atoms. More preferably it is n-butyl.
  • the saturated hydrocarbon chain may be optionally substituted by one or more substituents such as halogen, hydroxyl, azide, carboxylic acid or a group substituted or not selected from amino, amide, carboxylic ester, ether, thiol, acylamino or carboxyamide.
  • substituents such as halogen, hydroxyl, azide, carboxylic acid or a group substituted or not selected from amino, amide, carboxylic ester, ether, thiol, acylamino or carboxyamide.
  • unsaturated hydrocarbon chain refers, in the present invention, to unsaturated, linear or branched aliphatic chains, having from 2 to 16 atoms of carbon, and having between 1 or more unsaturations independently selected between double and triple bonds, for example, vinyl, allyl, 2-propynyl, 1,3-pentadiinyl, but-1- in-3-inyl, etc.
  • the unsaturated hydrocarbon chains may be optionally substituted by one or more substituents such as halogen, hydroxyl, azide, carboxylic acid or a substituted or unsubstituted group selected from amino, amide, carboxylic ester, ether, thiol, acylamino or carboxyamide.
  • Compounds derived from the 1-deoxygalactonojirimycin of the invention include six-membered / five-membered condensed bicycles having a bridgehead nitrogen atom that is part of a cyclic isothiourea functionality.
  • a preferred embodiment of the present invention comprises the use of a compound of general formula (I) where R is a saturated hydrocarbon chain.
  • R is a linear chain.
  • R is a hydrocarbon chain of 1 to 8 carbon atoms.
  • R is butyl, and therefore the compound of general formula (I) is 5 / V, 6S - (/ V-butyliminomethylidene) -6-thio-1-deoxygalactonojirimycin (6S-NBI-DGJ).
  • compositions intended for the treatment of diseases related to the malfunction of the lysosomal ⁇ -galactosidase enzyme in a human subject, comprising in its formulation at least one compound as described above, in any of its variants.
  • said composition may comprise another active ingredient and a pharmaceutically acceptable carrier.
  • compositions are the vehicles known to those skilled in the art. Brief description of the figure
  • Figure 1 Data corresponding to 24 mutations, compared with wild ⁇ -galactosidase (WT) and with a reference enzyme (mock).
  • the compounds of the invention can be prepared following the synthetic procedure developed by Garc ⁇ a Fernandez et al. (J. M. Garc ⁇ a Fernández, et al., Chem. Commun., 2012, 48, 6514-6516).
  • Human fibroblasts were grown in Dulbecco-modified Eagle medium supplemented with antibiotics (streptomycin and penicillin) and 10% fetal bovine serum in the absence and in the presence of increasing concentrations of 6S-NBI-DGJ for 96 h. After this time the enzyme activity in lysates was measured as described in the example!
  • COS7 cells were transfected with plasmids containing complementary DNA (cDNA) from wild ⁇ -galactosidase (WT) and from mutants associated with ⁇ -galactosidase deficiency diseases using as Lipofectamine 2000 vector (Higaki K, Linjing L, Bahrudin U, Okuzawa S, Takamura A, Yamamoto K et al. Hum Mutat 32: 843-852, 2011).
  • cDNA complementary DNA
  • WT wild ⁇ -galactosidase
  • mutants associated with ⁇ -galactosidase deficiency diseases using as Lipofectamine 2000 vector (Higaki K, Linjing L, Bahrudin U, Okuzawa S, Takamura A, Yamamoto K et al. Hum Mutat 32: 843-852, 2011).
  • the cells were exposed to fresh culture medium in the absence or in the presence of 6S-NBI-DGJ at concentrations 20 ⁇ and 80 ⁇ , after which the activity of ⁇ -galactosidase in lysates was determined as determined. described in the previous examples. 88 different mutants were tested.
  • 6S-NBI-DGJ The possible toxicity of 6S-NBI-DGJ in human fibroblasts was determined by the lactate dehydrogenase (LHD; Wako) assay in the supernatant of the corresponding cell cultures. No toxic effect was found at concentrations of up to 600 ⁇ of this compound.

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Abstract

The invention relates to a use of a compound of formula (I), or one of the salts thereof, wherein: R represents a linear or branched aliphatic chain selected from the group consisting of a C1-C16 saturated hydrocarbonated chain and a C2-C16 unsaturated hydrocarbonated chain, in the production of a medicament for the treatment of diseases associated with mutant lysosomal ß-galactosidase enzymes in humans.

Description

UTILIZACIÓN DE DERIVADOS BICÍCLICOS DE 1 -DESOXIGALACTONOJIRIMICINA EN LA PREPARACIÓN DE UN MEDICAMENTO PARA EL TRATAMIENTO DE ENFERMEDADES RELACIONADAS CON β- ENZIMAS GALACTOSI DASAS  USE OF BICYCLIC DERIVATIVES OF 1-DESOXIGALACTONOJIRIMICIN IN THE PREPARATION OF A MEDICINAL PRODUCT FOR THE TREATMENT OF ILLNESSES RELATED TO β- GALACTOSI DASAS ENZYMES
LISOSÓMICAS MUTANTES HUMANAS  HUMAN MUTAN LISOSOMICS
DESCRIPCIÓN DESCRIPTION
Sector y objeto de la invención La presente invención va dirigida al sector farmacéutico, con aplicaciones destinadas al tratamiento de enfermedades de almacenamiento lisosomal relacionadas con mutaciones de la β-glucosidasa ácida lisosómica. Sector and object of the invention The present invention is directed to the pharmaceutical sector, with applications for the treatment of lysosomal storage diseases related to mutations of lysosomal acid β-glucosidase.
El objeto de la invención es el uso de un compuesto de fórmula (I)  The object of the invention is the use of a compound of formula (I)
Figure imgf000003_0001
o una de sus sales, donde:
Figure imgf000003_0001
or one of its salts, where:
R representa una cadena alifática, lineal o ramificada, que se selecciona del grupo que consiste en cadena hidrocarbonada saturada C Ci6, y cadena hidrocarbonada insaturada C2-Ci6, para la fabricación de un medicamento destinado al tratamiento de enfermedades relacionadas con β- enzimas galactosidasas lisosómicas mutantes humanas. R represents an aliphatic chain, linear or branched, which is selected from the group consisting of saturated hydrocarbon chain C Ci 6 , and unsaturated hydrocarbon chain C 2 -Ci 6 , for the manufacture of a medicament for the treatment of diseases related to β- human mutant lysosomal galactosidase enzymes.
Estado de la técnica Los trastornos de almacenamiento lisosómico son un grupo de enfermedades resultantes del metabolismo anormal de varios sustratos que no se degradan y se acumulan en los lisosomas, conduciendo a una serie de fenotipos que incluyen megalovisceralia, patologías neurológicas, lesiones esqueléticas y muerte prematura. Estas enfermedades son el resultado de mutaciones en los genes que codifican enzimas implicadas en el proceso de degradación. Actualmente sólo hay disponibles terapias sintomáticas para estos enfermos, diferenciándose dos estrategias terapéuticas: la terapia de reducción de sustrato, basada la inhibición de la producción de sustrato usando inhibidores de las enzimas implicadas en su biosíntesis, y la terapia de reemplazamiento enzimático, basada en la administración exógena de enzimas activas recombinantes. Para el trastorno de almacenamiento lisosómico más predominante, la enfermedad de Gaucher, esta terapia cuesta entre 100.000 y 750.000 dólares al año, y no es muy eficaz para los casos que muestran implicación del sistema nervioso central. State of the art Lysosomal storage disorders are a group of diseases resulting from the abnormal metabolism of several substrates that do not degrade and accumulate in lysosomes, leading to a series of phenotypes that include megalovisceralia, neurological pathologies, skeletal injuries and premature death. . These diseases are the result of mutations in the genes that encode enzymes involved in the degradation process. Currently, only symptomatic therapies are available for these patients, differing two therapeutic strategies: substrate reduction therapy, based on the inhibition of substrate production using inhibitors of the enzymes involved in its biosynthesis, and enzyme replacement therapy, based on the exogenous administration of recombinant active enzymes. For the most predominant lysosomal storage disorder, Gaucher's disease, this therapy costs between $ 100,000 and $ 750,000 a year, and is not very effective for cases that show involvement of the central nervous system.
Dentro de este tipo de patologías de almacenamiento lisosómico, la deficiencia hereditaria de β-galactosidasa ácida lisosómica (β-galactosidosis), causa dos enfermedades clínicamente distintas en seres humanos, la gangliosidosis GM1 y la enfermedad de Morquio B. Ambas son el resultado de mutaciones en el gen GLB1 que conducen a un plegamiento proteico erróneo. El modo de herencia es recesivo autosómico. La gangliosidosis GM1 es una enfermedad neurosomática generalizada que aparece principalmente en la primera infancia, y raramente en la niñez o en adultos jóvenes. La enfermedad de Morquio B es una rara enfermedad ósea sin implicación del sistema nervioso central. En pacientes con estos fenotipos clínicos se acumulan glicoconjugados con restos de β-galactosa terminales en los tejidos y orina. El gangliósido GM1 y su derivado asiálico GA1 se acumulan en el cerebro en el caso de la gangliosidosis GM1. Tanto en pacientes con gangliosidosis GM1 como en pacientes que padecen la enfermedad de Morquio B se detectan altas cantidades de oligosacáridos derivados de sulfato de queratano o glicoproteínas en órganos viscerales y orina. Se han identificado más de 160 mutaciones puntuales diferentes en el gen que codifica la β-galactosidasa. Las mutaciones conducen a defectos significativos en el plegamiento de la proteína durante la traducción en el retículo endoplasmático, dando como resultado una reducción del transporte de la enzima al lisosoma (degradación mediada por la maquinaria celular de control de calidad). La forma clínica de la enfermedad depende de la mutación en la β- galactosidasa ácida que presenta el enfermo, y su gravedad se relaciona con la actividad enzimática residual. Within this type of lysosomal storage pathologies, hereditary deficiency of lysosomal acid β-galactosidase (β-galactosidosis) causes two clinically distinct diseases in humans, GM1 gangliosidosis and Morquio B disease. Both are the result of mutations. in the GLB1 gene that lead to a wrong protein folding. The mode of inheritance is autosomal recessive. GM1 gangliosidosis is a generalized neurosomatic disease that appears mainly in early childhood, and rarely in childhood or in young adults. Morquio B disease is a rare bone disease without involvement of the central nervous system. In patients with these clinical phenotypes glycoconjugates accumulate with terminal β-galactose residues in tissues and urine. The GM1 ganglioside and its Asian derivative GA1 accumulate in the brain in the case of GM1 gangliosidosis. High amounts of oligosaccharides derived from keratane sulfate or glycoproteins in visceral organs and urine are detected in both patients with GM1 gangliosidosis and in patients suffering from Morquio B disease. More than 160 different point mutations have been identified in the gene encoding β-galactosidase. The mutations lead to significant defects in the folding of the protein during translation in the endoplasmic reticulum, resulting in a reduction in the transport of the enzyme to the lysosome (degradation mediated by cellular quality control machinery). The clinical form of the disease depends on the mutation in the acid β-galactosidase presented by the patient, and its severity is related to the residual enzymatic activity.
La terapia de reemplazamiento enzimático para los casos en los que el sistema nervioso central se ve afectado presenta una eficacia baja o nula, ya que las enzimas recombinantes no atraviesan la barrera hematoencefálica. De este modo, existe un gran número de pacientes para los cuales no existe tratamiento o la efectividad del mismo es muy baja. En los últimos años se ha descrito que algunos inhibidores de estas enzimas glicosidasas son capaces de unirse al sitio activo y estabilizar el plegamiento apropiado, pudiendo actuar como "chaperonas farmacológicas" que facilitan el transporte de la forma catalíticamente activa a los lisosomas. De este modo, el desarrollo de compuestos con actividad de chaperonas farmacológica se ha postulado como una posible estrategia terapéutica para el tratamiento de enfermedades de almacenamiento lisosomal, y de particular interés para aquellas manifestaciones clínicas de la enfermedad que involucran al sistema nervioso central. Algunos alcaloides polihidroxilados naturales y sintéticos estructuralmente relacionados con los azúcares (glicomiméticos) que incorporan un nitrógeno endocíclico de tipo amina (hibridación sp3) exhiben una actividad inhibidora significativa frente a glicosidasas. En algunos casos, se ha demostrado que estos compuestos usados a concentraciones subinhibidoras actúan como chaperonas de β-galactosidasa mutantes responsables de la enfermedad de gangliosidosis GM1 y de Morquio B (US 2006/0100241 ; W02004/037373). Sin embargo, estos tipos de compuestos se comportan en general como inhibidores de glicosidasas de amplio espectro, inhibiendo simultáneamente varias glicosidasas, lo que representa un inconveniente serio para aplicaciones clínicas. Esta falta de selectividad de acción frente a glicosidasas ha sido corregida con el desarrollo de compuestos donde el nitrógeno endocíclico de tipo amina se ha transformado en un nitrógeno de tipo pseudoamida (como por ejemplo, un grupo carbamato, tiocarbamato, isourea, isotiourea, urea, tiourea o guanidina), con hibridación sp2 (en adelante, iminoazúcares sp2). De este modo, se han desarrollado iminoazúcares sp2 de notable actividad chaperona y que muestran gran selectividad frente a β-glucocerebrosidasa, α-galactosidasa o β- galactosidasa (WO2010046517A1 ; C. Ortiz Mellet, J. M. García Fernández, Y. Suzuki, et al., Chem. Commun., 2012, 48, 6514-6516) Enzyme replacement therapy for cases in which the central nervous system is affected has low or no efficacy, since recombinant enzymes do not cross the blood brain barrier. Thus, there is a large number of patients for whom there is no treatment or its effectiveness is very low. In recent years it has been described that some inhibitors of these glycosidases enzymes are capable of binding to the active site and stabilizing the appropriate folding, being able to act as "pharmacological chaperones" that facilitate the transport of the catalytically active form to lysosomes. Thus, the development of compounds with pharmacological chaperone activity has been postulated as a possible therapeutic strategy for the treatment of lysosomal storage diseases, and of particular interest for those clinical manifestations of the disease that involve the central nervous system. Some natural and synthetic polyhydroxylated alkaloids structurally related to sugars (glycomimetics) that incorporate an amine-type endocyclic nitrogen (sp 3 hybridization) exhibit significant inhibitory activity against glycosidases. In some cases, these compounds used at subinhibitory concentrations have been shown to act as mutant β-galactosidase chaperones responsible for GM1 and Morquio B gangliosidosis disease (US 2006/0100241; W02004 / 037373). However, these types of compounds generally behave as inhibitors of broad-spectrum glycosidases, simultaneously inhibiting several glycosidases, which represents a serious drawback for clinical applications. This lack of selectivity of action against glycosidases has been corrected with the development of compounds where amine-type endocyclic nitrogen has been transformed into a pseudoamide-type nitrogen (such as a carbamate, thiocarbamate, isourea, isothiourea, urea group, thiourea or guanidine), with sp 2 hybridization (hereinafter, iminoazúcares sp 2 ). Thus, sp 2 iminoazúres of remarkable chaperone activity have been developed and show great selectivity against β-glucocerebrosidase, α-galactosidase or β-galactosidase (WO2010046517A1; C. Ortiz Mellet, JM García Fernández, Y. Suzuki, et al ., Chem. Commun., 2012, 48, 6514-6516)
Compuestos derivados de valienamina como N-octil-4-epi^-valienamina (NOEV), que no presenta estructura química de iminoazúcar, también han demostrado actuar como chaperonas farmacológicas para enzimas mutantes responsables de estas enfermedades de almacenamiento lisosómico. Compounds derived from valienamine such as N-octyl-4-epi ^ -valienamine (NOEV), which does not have a chemical structure of iminoazúcar, have also been shown to act as pharmacological chaperones for mutant enzymes responsible for these lysosomal storage diseases.
Un problema con el que se encuentra esta estrategia terapeútica basada en el uso de chaperonas farmacológicas para el tratamiento de la enfermedad de Morquio B y de la Gangliosidosis GM1 es que los compuestos conocidos hasta la fecha son efectivos para un número reducido de mutaciones en el gen que codifica la β-galactosidasa. Por tanto, y dada la gran variabilidad de las mismas que presentan los enfermos, es necesario el desarrollo de compuestos que tengan un amplio espectro de acción respecto a estas mutaciones para posibilitar el desarrollo de medicamentos de uso generalizado. A problem with this therapeutic strategy based on the use of pharmacological chaperones for the treatment of Morquio B disease and GM1 Gangliosidosis is that the compounds known to date are effective for reduced number of mutations in the gene encoding β-galactosidase. Therefore, and given the great variability of these patients, it is necessary to develop compounds that have a broad spectrum of action with respect to these mutations to enable the development of drugs for widespread use.
Existe, por la tanto, la necesidad de desarrollar moléculas con una alta especificad de unión a la β-galactosidasa, con una elevada relación de actividad chaperona frente a actividad inhibidora y que sean efectivas para un amplio espectro de enzimas β- galactosidasas mutantes. There is, therefore, the need to develop molecules with a high specificity of binding to β-galactosidase, with a high ratio of chaperone activity to inhibitory activity and that are effective for a broad spectrum of mutant β-galactosidase enzymes.
Si bien se han descrito tanto iminoazúcares como iminoazúcares sp2 con actividad como chaperonas farmacológicas frente a mutantes de la β-galactosidasa asociados a las enfermedades de gangliosidosis GM1 y de Morquio B conteniendo en su estructura un anillo de piperidina polihidroxilado con un patrón de sustitución que corresponde con el de la D-glucosa o la D-galactosa (es decir, son derivados de la nojirimicina o de la galactonojirimicina), no existían datos que permitiesen predecir que derivados bicíclicos de 1-desoxigalactonojirimicina pudieran actuar como chaperonas efectivas de amplio rango de mutantes, incluyendo mutaciones para las que no se conocían compuestos con actividad chaperona. Although both iminoazúcares and iminoazúres sp 2 have been described with activity as pharmacological chaperones against mutants of β-galactosidase associated with GM1 and Morquio B gangliosidosis diseases containing in their structure a polyhydroxylated piperidine ring with a replacement pattern that corresponds to that of D-glucose or D-galactose (that is, they are derivatives of nojirimycin or galactonojirimycin), there were no data that could predict that bicyclic derivatives of 1-deoxygalactonojirimycin could act as effective chaperones of wide range of mutants, including mutations for which compounds with chaperone activity were not known.
Descripción detallada de la invención Detailed description of the invention
La presente invención se refiere al uso de un compuesto de fórmula general (I) o una de sus sales The present invention relates to the use of a compound of general formula (I) or one of its salts
Figure imgf000006_0001
dónde
Figure imgf000006_0001
where
R representa una cadena alifática, lineal o ramificada, que se selecciona del grupo que consiste en cadena hidrocarbonada saturada C Ci6, y cadena hidrocarbonada insaturadaR represents an aliphatic chain, linear or branched, which is selected from the group consisting of saturated hydrocarbon chain C Ci 6 , and unsaturated hydrocarbon chain
C2-C16, para la elaboración de un medicamento destinado al tratamiento de la deficiencia de la forma activa de la enzima β-galactosidasa en el ser humano, que se relaciona con enfermedades tales como la gangliosidosis GM1 y la enfermedad de Morquio B. En la presente invención se ha encontrado que compuestos bicíclicos condensados (seis miembros/cinco miembros) derivados de 1-desoxigalactonojirimicina en los que el átomo de nitrógeno cabeza de puente forma parte de un grupo funcional isotiourea que, a su vez, porta un sustituyente de naturaleza hidrófoba, se comportan como inhibidores específicos de la β-galactosidasa lisosomal capaces de actuar como chaperonas farmacológicas a concentraciones inferiores a las de inhibición, con la ventaja significativa respecto a otros glicomiméticos, incluidos otros iminoazúcares sp2, de que no presentan toxicidad y que su actividad chaperona es efectiva para un rango amplio de mutaciones diferentes, entre las que se incluyen las G190D, R201C, R201 H, V216A, D332N, Y444C, R457Q, R590H, I51T, R148T, L155R, R208C, D214Y, C230Y, L264S, N266S, W273R, K346N, S434L, G438E, R482H, D491Y, E632G y D640E, que son relativamente frecuentes en varios fenotipos clínicos. Para algunas de estas mutaciones (I51T, R148T, L155R, R208C, D214Y, C230Y, L264S, N266S, W273R, K346N, S434L, G438E, R482H, D491Y, E632G y D640E), algunos compuestos bien conocidos como activadores de la β-galactosidasa, tales como NOEV, no mostraron sin embargo ninguna actividad. C2-C16, for the preparation of a medicament for the treatment of the deficiency of the active form of the enzyme β-galactosidase in humans, which is related to diseases such as GM1 gangliosidosis and Morquio B disease. In the present invention it has been found that condensed bicyclic compounds (six members / five members) derived from 1-deoxygalactonojirimycin in which the bridgehead nitrogen atom is part of an isothiourea functional group that, in turn, carries a hydrophobic substituent, behave as specific lysosomal β-galactosidase inhibitors capable of acting as pharmacological chaperones at concentrations lower than those of inhibition, with the significant advantage over other glycomimetics, including other sp 2 iminoazúcares, that do not present toxicity and that their chaperone activity is effective for a wide range of different mutations, including G190D, R201C, R201 H, V216A, D332N, Y444C, R457Q, R590H, I51T, R148T, L155R, R208C, D214Y, C230Y, L264S, N266S, W273R, K346N, S434L, G438E, R482H40, D491 and E6, relatively frequent Several clinical phenotypes. For some of these mutations (I51T, R148T, L155R, R208C, D214Y, C230Y, L264S, N266S, W273R, K346N, S434L, G438E, R482H, D491Y, E632G and D640E), some compounds well known as β-galactose activators , such as NOEV, however, showed no activity.
La estructura química de los compuestos de la invención les permite atravesar la barrera hematoencefálica, por lo que su uso en la fabricación de medicamentos destinados al tratamiento de enfermedades de almacenamiento lisosomal con implicaciones en el sistema nervioso central, como la gangliosidosis GM1 , es de especial interés. The chemical structure of the compounds of the invention allows them to cross the blood brain barrier, so their use in the manufacture of drugs for the treatment of lysosomal storage diseases with implications in the central nervous system, such as GM1 gangliosidosis, is especially interest.
El término "cadena hidrocarbonada saturada" se refiere, en la presente invención, a cadenas alifáticas, lineales o ramificadas, que tienen de 1 a 16 átomos de carbono, por ejemplo, metilo, etilo, n-propilo, /'-propilo, n-butilo, tere-butilo, sec-butilo, n-pentilo, n-hexilo, etc. Preferiblemente la cadena hidrocarbonada saturada tiene entre 1 y 8 átomos de carbono. Más preferiblemente es n-butilo. La cadena hidrocarbonada saturada puede estar opcionalmente sustituida por uno o más sustituyentes tales como halógeno, hidroxilo, azida, ácido carboxílico o un grupo sustituido o no seleccionado de entre amino, amida, éster carboxílico, éter, tiol, acilamino o carboxiamida. The term "saturated hydrocarbon chain" refers, in the present invention, to aliphatic, linear or branched chains, having 1 to 16 carbon atoms, for example, methyl, ethyl, n-propyl, / ' -propyl, n -butyl, tere-butyl, sec-butyl, n-pentyl, n-hexyl, etc. Preferably the saturated hydrocarbon chain has between 1 and 8 carbon atoms. More preferably it is n-butyl. The saturated hydrocarbon chain may be optionally substituted by one or more substituents such as halogen, hydroxyl, azide, carboxylic acid or a group substituted or not selected from amino, amide, carboxylic ester, ether, thiol, acylamino or carboxyamide.
El término "cadena hidrocarbonada insaturada" se refiere, en la presente invención, a cadenas alifáticas insaturadas, lineales o ramificadas, que tienen de 2 a 16 átomos de carbono, y que poseen entre 1 o más insaturaciones elegidas de forma independiente entre dobles y triples enlaces, por ejemplo, vinilo, alilo, 2-propinilo, 1 ,3-pentadiinilo, but-1- en-3-inilo, etc. Las cadenas hidrocarbonadas insaturadas pueden estar opcionalmente sustituidas por uno o más sustituyentes tales como halógeno, hidroxilo, azida, ácido carboxílico o un grupo sustituido o no sustituido seleccionado de entre amino, amida, éster carboxílico, éter, tiol, acilamino o carboxiamida. The term "unsaturated hydrocarbon chain" refers, in the present invention, to unsaturated, linear or branched aliphatic chains, having from 2 to 16 atoms of carbon, and having between 1 or more unsaturations independently selected between double and triple bonds, for example, vinyl, allyl, 2-propynyl, 1,3-pentadiinyl, but-1- in-3-inyl, etc. The unsaturated hydrocarbon chains may be optionally substituted by one or more substituents such as halogen, hydroxyl, azide, carboxylic acid or a substituted or unsubstituted group selected from amino, amide, carboxylic ester, ether, thiol, acylamino or carboxyamide.
Los compuestos derivados de la 1-desoxigalactonojirimicina de la invención, según se representa en la fórmula general (I), incluyen biciclos condensados de seis miembros/cinco miembros que tienen un átomo de nitrógeno cabeza de puente que es parte de una funcionalidad isotiourea cíclica. Por "funcionalidad isotiourea cíclica", se define un grupo de fórmula general N-C(=NR)S, en el que el átomo de nitrógeno que no porta el doble enlace y el átomo de azufre forman parte de un ciclo y en el que el átomo de nitrógeno exocíclico porta como sustituyente una cadena hidrocarbonada tal y como se ha definido en los párrafos anteriores. Compounds derived from the 1-deoxygalactonojirimycin of the invention, as represented in the general formula (I), include six-membered / five-membered condensed bicycles having a bridgehead nitrogen atom that is part of a cyclic isothiourea functionality. By "cyclic isothiourea functionality", a group of general formula NC (= NR) S is defined, in which the nitrogen atom that does not carry the double bond and the sulfur atom are part of a cycle and in which the atom of exocyclic nitrogen carries as a substituent a hydrocarbon chain as defined in the preceding paragraphs.
Una realización preferida de la presente invención, comprende el uso de un compuesto de fórmula general (I) donde R es una cadena hidrocarbonada saturada. En una realización preferida de la invención R es una cadena lineal. A preferred embodiment of the present invention comprises the use of a compound of general formula (I) where R is a saturated hydrocarbon chain. In a preferred embodiment of the invention R is a linear chain.
En una realización preferida de la invención R es una cadena hidrocarbonada de 1 a 8 átomos de carbono. En otra realización más preferida R es butilo, y por tanto el compuesto de fórmula general (I) es 5/V,6S-(/V-butiliminometiliden)-6-tio-1-desoxigalactonojirimicina (6S-NBI-DGJ). In a preferred embodiment of the invention R is a hydrocarbon chain of 1 to 8 carbon atoms. In another more preferred embodiment R is butyl, and therefore the compound of general formula (I) is 5 / V, 6S - (/ V-butyliminomethylidene) -6-thio-1-deoxygalactonojirimycin (6S-NBI-DGJ).
Otro aspecto de la invención se refiere a una composición farmacéutica, destinada al tratamiento de enfermedades relacionadas con el mal funcionamiento de la enzima β- galactosidasa lisosómica en un sujeto humano, que comprende en su formulación al menos un compuesto como el descrito anteriormente, en cualquiera de sus variantes. Opcionalmente dicha composición puede comprender otro principio activo y un vehículo farmacéuticamente aceptable. Another aspect of the invention relates to a pharmaceutical composition, intended for the treatment of diseases related to the malfunction of the lysosomal β-galactosidase enzyme in a human subject, comprising in its formulation at least one compound as described above, in any of its variants. Optionally said composition may comprise another active ingredient and a pharmaceutically acceptable carrier.
Los "vehículos farmacéuticamente aceptables" que pueden ser utilizados en dichas composiciones son los vehículos conocidos por los técnicos en la materia. Breve descripción de la figura The "pharmaceutically acceptable vehicles" that can be used in said compositions are the vehicles known to those skilled in the art. Brief description of the figure
Figura 1 : Datos correspondientes a 24 mutaciones, en comparación con la β-galactosidasa silvestre (WT) y con una enzima de referencia (mock). Figure 1: Data corresponding to 24 mutations, compared with wild β-galactosidase (WT) and with a reference enzyme (mock).
Modo de realización de la invención Embodiment of the invention
Los compuestos de la invención pueden ser preparados siguiendo el procedimiento sintético desarrollado por García Fernandez et al. (J. M. García Fernández, et al., Chem. Commun., 2012, 48, 6514-6516).  The compounds of the invention can be prepared following the synthetic procedure developed by García Fernandez et al. (J. M. García Fernández, et al., Chem. Commun., 2012, 48, 6514-6516).
Ejemplo 1 Example 1
Inhibición selectiva in vitro de 3-galactosidasa lisosomal humana por 5N$S-(N'- butiliminometilideno)-6-tio-1-desoxigalactono¡irimicina (6S-NBI-DGJ).  Selective in vitro inhibition of human lysosomal 3-galactosidase by 5N $ S- (N'-butyliminomethylidene) -6-thio-1-deoxygalactonoimirimicin (6S-NBI-DGJ).
En primer lugar, se determinaron las actividades de varias glicosidasas lisosomales humanas, en concreto de β-glucosidasa (β-glucocerebrosidasa), α-glucosidasa, β- galactosidasa, α-galactosidasa, y hexosaminidasa en lisados celulares usando el correspondiente D-glicopiranósido conjugado con 4-metilumbeliferona como sustrato (A.M. Vaccaro, M. Muschilli, M. Tatti, R. Salvioli, E. Gallozzi, K. Suzuki. Clin. Biochem. 20: 429- 43, 1987). Brevemente, se incubaron 4 μΙ_ de lisados celulares a 37 °C con 8 μΙ_ de disolución sustrato en tampón citrato 0, 1 M, pH 5,2, suplementada con taurocolato de sodio (0,8% p/v). Se terminó la reacción añadiendo 1 ,0 ml_ de tampon de glicina-hidróxido de sodio 0,2 M (pH 10,7). Se definió una unidad de actividad enzimática como nmoles de 4- metilumbeliferona liberados por hora. First, the activities of several human lysosomal glycosidases were determined, in particular of β-glucosidase (β-glucocerebrosidase), α-glucosidase, β-galactosidase, α-galactosidase, and hexosaminidase in cell lysates using the corresponding conjugated D-glycopyranoside with 4-methylumbelliferone as a substrate (AM Vaccaro, M. Muschilli, M. Tatti, R. Salvioli, E. Gallozzi, K. Suzuki. Clin. Biochem. 20: 429-43, 1987). Briefly, 4 μΙ_ of cell lysates were incubated at 37 ° C with 8 μΙ_ of substrate solution in 0.1 M citrate buffer, pH 5.2, supplemented with sodium taurocholate (0.8% w / v). The reaction was terminated by adding 1.0 ml_ of 0.2 M sodium glycine-hydroxide buffer (pH 10.7). One unit of enzymatic activity was defined as nmoles of 4- methylumbeliferone released per hour.
Seguidamente, para explorar el efecto del compuesto 6S-NBI-DGJ sobre las diferentes glicosidasas lisosomales, se cultivaron células de controles sanos durante 4 días en ausencia o presencia de concentraciones crecientes del compuesto. Después de la exposición, se rascaron en H20 enfriada con hielo (106/ml_) y se lisaron mediante sonicación. Se retiraron los materiales insolubles mediante centrifugación a 12.000 g durante 10 min a 4 °C y se midieron las actividades de las enzimas en lisados celulares. Los resultados indicaron que el compuesto 6S-NBI-DGJ inhibe selectivamente y de manera competitiva la β-galactosidasa, con un valor de concentración necesaria para alcanzar un 50% de inhibición (IC50) de 32 μΜ. A esta concentración, la actividad del resto de enzimas lisosomales humanas determinadas se afectó en menos de un 2%. Next, to explore the effect of compound 6S-NBI-DGJ on the different lysosomal glycosidases, healthy control cells were cultured for 4 days in the absence or presence of increasing concentrations of the compound. After exposure, were scraped in H 2 0 cooled with ice (10 6 / ml_) and lysed by sonication. Insoluble materials were removed by centrifugation at 12,000 g for 10 min at 4 ° C and enzyme activities in cell lysates were measured. The results indicated that the 6S-NBI-DGJ compound selectively and competitively inhibits β-galactosidase, with a concentration value necessary to achieve a 50% inhibition (IC 50 ) of 32 μΜ. At this concentration, the activity of the rest of determined human lysosomal enzymes was affected in less than 2%.
Ejemplo 2 Example 2
Estabilización in vitro de 3-galactosidasa lisosomal humana frente a la desnaturalización inducida por calentamiento en presencia de 6S-NBI-DGJ. In vitro stabilization of human lysosomal 3-galactosidase against heating-induced denaturation in the presence of 6S-NBI-DGJ.
Para evaluar el potencial del compuesto 6S-NBI-DGJ como chaperona farmacológica se determinó su capacidad para proteger la β-galactosidasa lisosomal humana frente a la degradación inducida por calor a pH neutro. Para ello, los lisados celulares se incubaron en ausencia y en presencia de concentraciones crecientes de 6S-NBI-DGJ en tampón citrato 0.1 M (pH 7) a 48 °C. La incubación se terminó mediante adición de dos volúmenes de tampón citrato 0.1 M (pH 4.5) y se midió entonces la actividad de la β-galactosidasa como en el ejemplo anterior. En ausencia de 6S-NBI-DGJ, la actividad de la enzima cayó a menos del 20% del valor inicial tras 20 minutos de incubación. En presencia de 6S-NBI- DGJ se observó la supresión de la degradación de la enzima de manera dependiente de la dosis, con valores residuales de actividad de la enzima tras 20 minutos de incubación que permanecieron entre el 80% y el 100% para concentraciones de 6S-NBI-DG entre 20 μΜ y 160 μΜ. To assess the potential of compound 6S-NBI-DGJ as a pharmacological chaperone, its ability to protect human lysosomal β-galactosidase was determined against heat-induced degradation at neutral pH. For this, cell lysates were incubated in the absence and in the presence of increasing concentrations of 6S-NBI-DGJ in 0.1 M citrate buffer (pH 7) at 48 ° C. Incubation was terminated by the addition of two volumes of 0.1 M citrate buffer (pH 4.5) and the activity of β-galactosidase was then measured as in the previous example. In the absence of 6S-NBI-DGJ, the enzyme activity dropped to less than 20% of the initial value after 20 minutes of incubation. In the presence of 6S-NBI-DGJ the suppression of the degradation of the enzyme was observed in a dose-dependent manner, with residual values of enzyme activity after 20 minutes of incubation that remained between 80% and 100% for concentrations 6S-NBI-DG between 20 μΜ and 160 μΜ.
Ejemplo 3 Example 3
Potenciación in vitro de la actividad de mutantes de la 3-galactosidasa asociados a enfermedades de deficiencia de esta enzima por 6S-NBI-DGJ en fibroblastos humanos. Se cultivaron fibroblastos cutáneos derivados de personas sanas y de pacientes con deficiencia de la β-galactosidasa presentando las mutaciones I51T/I51T, I51TVY316C, I51T/R457Q, G190D/G190D, R201C/R201C, G438E/G438E, R457Q/R457Q y R59H/R59H, siguiendo el procedimiento descrito (Iwasaki H, Watanabe H, lida M, Ogawa S, Tabe M, Higaki K et al. Brain Dev 28:482-486, 2006). Los fibroblastos humanos de cultivaron en medio de Eagle modificado por Dulbecco suplementado con antibióticos (estreptomicina y penicilina) y suero bovino fetal al 10% en ausencia y en presencia de concentraciones crecientes de 6S-NBI-DGJ durante 96 h. Tras este tiempo se midió la actividad de la enzima en lisados como se describe en el ejemplo! Los resultados indicaron que 6S-NBI-DGJ incrementó de manera estadísticamente significativa la actividad de la enzima para las mutaciones I51T/I51T, I51T7Y316C, I51T/R457Q, G190D/G190D, R201 C/R201C, G438E/G438E y R457Q/R457Q a concentraciones 20 μΜ (incremento entre 1.3 y 3 veces) y 80 μΜ (incremento entre 2 y 5 veces). La única excepción en esta serie fue la enzima con la mutación R59H/R59H. Ejemplo 4 In vitro potentiation of the activity of 3-galactosidase mutants associated with diseases of deficiency of this enzyme by 6S-NBI-DGJ in human fibroblasts. Dermal fibroblasts derived from healthy people and patients with β-galactosidase deficiency were cultured presenting the mutations I51T / I51T, I51TVY316C, I51T / R457Q, G190D / G190D, R201C / R201C, G438E / G438E, R457Q / R457H / R457Q / R457Q / R457Q / R457H / R457Q , following the procedure described (Iwasaki H, Watanabe H, lida M, Ogawa S, Tabe M, Higaki K et al. Brain Dev 28: 482-486, 2006). Human fibroblasts were grown in Dulbecco-modified Eagle medium supplemented with antibiotics (streptomycin and penicillin) and 10% fetal bovine serum in the absence and in the presence of increasing concentrations of 6S-NBI-DGJ for 96 h. After this time the enzyme activity in lysates was measured as described in the example! The results indicated that 6S-NBI-DGJ statistically significantly increased enzyme activity for mutations I51T / I51T, I51T7Y316C, I51T / R457Q, G190D / G190D, R201 C / R201C, G438E / G438E and R457Q / R457Q at concentrations 20 μΜ (increase between 1.3 and 3 times) and 80 μΜ (increase between 2 and 5 times). The only exception in this series was the enzyme with the R59H / R59H mutation. Example 4
Potenciación in vitro de la actividad de mutantes de la 3-galactosidasa recombinante humana asociados a enfermedades de deficiencia de esta enzima por 6S-NBI-DGJ en células COS7. Células COS7 se transfectaron con plásmidos conteniendo ADN complementario (cADN) de la β-galactosidasa silvestre (WT) y de mutantes asociados a enfermedades de deficiencia de la β-galactosidasa usando como vector Lipofectamine 2000 (Higaki K, Linjing L, Bahrudin U, Okuzawa S, Takamura A, Yamamoto K et al. Hum Mutat 32:843-852, 2011). Tras 5 horas de incubación, las células se expusieron a medio de cultivo fresco en ausencia o en presencia de 6S-NBI-DGJ a concentraciones 20 μΜ y 80 μΜ, tras lo cual se determinó la actividad de la β-galactosidasa en lisados como se describe en los ejemplos anteriores. Se ensayaron 88 mutantes diferentes. Los resultados indicaron un incremento significativo en 24 de estos mutantes: G190D, R201C, R201 H, V216A, D332N, Y444C, R457Q, R590H, I51T, R148T, L155R, R208C, D214Y, C230Y, L264S, N266S, W273R, K346N, S434L, G438E, R482H, D491Y, E632G, D640E. Los datos correspondientes a estas 24 mutaciones, en comparación con la b-galactosidasa silvestre (WT) y con una enzima de referencia irrelevante (mock) se recogen en la Figura 1.  In vitro potentiation of the activity of mutants of human recombinant 3-galactosidase associated with diseases of deficiency of this enzyme by 6S-NBI-DGJ in COS7 cells. COS7 cells were transfected with plasmids containing complementary DNA (cDNA) from wild β-galactosidase (WT) and from mutants associated with β-galactosidase deficiency diseases using as Lipofectamine 2000 vector (Higaki K, Linjing L, Bahrudin U, Okuzawa S, Takamura A, Yamamoto K et al. Hum Mutat 32: 843-852, 2011). After 5 hours of incubation, the cells were exposed to fresh culture medium in the absence or in the presence of 6S-NBI-DGJ at concentrations 20 μΜ and 80 μΜ, after which the activity of β-galactosidase in lysates was determined as determined. described in the previous examples. 88 different mutants were tested. The results indicated a significant increase in 24 of these mutants: G190D, R201C, R201 H, V216A, D332N, Y444C, R457Q, R590H, I51T, R148T, L155R, R208C, D214Y, C230Y, L264S, N266S, W273R34, W273R344 , G438E, R482H, D491Y, E632G, D640E. The data corresponding to these 24 mutations, compared with wild b-galactosidase (WT) and with an irrelevant reference enzyme (mock) are collected in Figure 1.
En las siguientes mutaciones no se observó incremento de actividad significativo a las concentraciones estudiadas: In the following mutations no significant increase in activity was observed at the concentrations studied:
S54N, Y83C, Y83H, E131 K, L173P, Y199C, R201Y, Q255H, N318H, Y324C, D332E,N484K,G494S, R590C, R49C, S54I, R59C, R59H, R68W, T82M, F107L, R121S, G123R, G134V, P136S, R148C, R148S,D151V,W161 G, L162S, G178R, 1181 K, V240M, R263S, Y270D, G272D, W273L, H281Y,Y316C,T329A, Y333H, Y347C, R351X, Q408P, T420K, T420P, L422R, V439G, D441 N, R442Q, D448V, R457X, M480V, R482C, D491 N, G494C, T500A, W509C, P549L, G554E, K578R, G579D, Y591 N, Y591 C. S54N, Y83C, Y83H, E131 K, L173P, Y199C, R201Y, Q255H, N318H, Y324C, D332E, N484K, G494S, R590C, R49C, S54I, R59C, R59H, R68W, T82M, F10134, R123, R133 , R148C, R148S, D151V, W161 G, L162S, G178R, 1181 K, V240M, R263S, Y270D, G272D, W273L, H281Y, Y316C, T329A, Y333H, Y347C, R351X, Q408P, T420, T420, T420, T420, T4204 N, R442Q, D448V, R457X, M480V, R482C, D491 N, G494C, T500A, W509C, P549L, G554E, K578R, G579D, Y591 N, Y591 C.
Es importante destacar que no existe hasta el momento ningún otro compuesto que haya demostrado comportarse como activador de β-galactosidasa muíante en un rango tan amplio de mutaciones asociadas a enfermedades de deficiencia de esta enzima. Por ejemplo, en 16 de las 24 mutaciones para las que el compuesto incrementa significativamente la actividad (I51T, R148T, L155R, R208C, D214Y, C230Y, L264S, N266S, W273R, K346N, S434L, G438E, R482H, D491Y, E632G, D640E), el NOEV, un compuesto ampliamente estudiado como activador de β-galactosidasa muíante, no mostró ninguna activad en un ensayo realizado en paralelo. It is important to note that there is no other compound that has proven to behave as a activator of mutant β-galactosidase in such a range so far. wide mutations associated with deficiency diseases of this enzyme. For example, in 16 of the 24 mutations for which the compound significantly increases activity (I51T, R148T, L155R, R208C, D214Y, C230Y, L264S, N266S, W273R, K346N, S434L, G438E, R482H, D491Y, E632G, D640G, D640G ), NOEV, a compound widely studied as a mutant β-galactosidase activator, showed no activeness in a parallel trial.
Ejemplo 5 Example 5
Ausencia de toxicidad del compuesto 6S-NBI-DGJ  Absence of toxicity of compound 6S-NBI-DGJ
La posible toxicidad de 6S-NBI-DGJ en fibroblastos humanos se determinó mediante el ensayo de la lactato deshidrogenasa (LHD; Wako) en el sobrenadante de los correspondientes cultivos celulares. No se encontró efecto tóxico a concentraciones de hasta 600 μΜ de este compuesto. The possible toxicity of 6S-NBI-DGJ in human fibroblasts was determined by the lactate dehydrogenase (LHD; Wako) assay in the supernatant of the corresponding cell cultures. No toxic effect was found at concentrations of up to 600 μΜ of this compound.
Ejemplo 6 Example 6
Supresión de la acumulación de gangliósido GM1 en fibroblastos de pacientes de enfermedades asociadas a la deficiencia de la 3-galactosidasa por el compuesto 6S-NBI- DGJ  Suppression of the accumulation of ganglioside GM1 in fibroblasts of patients with diseases associated with 3-galactosidase deficiency due to compound 6S-NBI-DGJ
La deficiencia de la β-galactosidasa lisosomal humana conduce a la acumulación del gangliósido GM1 en las células del paciente. Esta es, de hecho, la causa primera de la patogénesis en la gangliosidosis GM1. Cuando se cultivaron fibroblastos de enfermos homozigóticos para las mutaciones 151 T y R201 C en medio suplementado con gangliósido GM1 , se observó su acumulación de manera importante. El tratamiento con 6S-NBI-DGJ (80 mM) suprimió esta acumulación, reduciendo sus niveles a menos del 50% del valor inicial en ausencia del compuesto. En un ensayo comparativo, el compuesto de referencia NOEV presentó una eficacia similar en fibroblastos con la mutación R201C, pero fue ineficaz en el caso de la mutación 151 T. Deficiency of human lysosomal β-galactosidase leads to the accumulation of the GM1 ganglioside in the patient's cells. This is, in fact, the leading cause of pathogenesis in GM1 gangliosidosis. When fibroblasts from homozygous patients were cultured for mutations 151 T and R201 C in medium supplemented with GM1 ganglioside, their accumulation was observed significantly. Treatment with 6S-NBI-DGJ (80 mM) suppressed this accumulation, reducing its levels to less than 50% of the initial value in the absence of the compound. In a comparative trial, the NOEV reference compound showed similar efficacy in fibroblasts with the R201C mutation, but was ineffective in the case of the 151 T mutation.
Ejemplo 7 Example 7
Efecto del compuesto 6S-NBI-DGJ en el tejido cerebral y en otros órganos de ratones que expresan la mutación R201 C en la 3-galactosidasa Para obtener una prueba de concepto de la capacidad del compuesto 6S-NBI-DGJ para atravesar la membrana hematoencefálica, así como para activar la β-galactosidasa muíante en diferentes órganos, se aplicaron dosis del mismo (1 mM, 2 mM, 5 mM y 10 mM en agua) a ratones que expresan la β-galactosidasa humana con la mutación R201 C por vía oral durante una semana. Cuando se usaron concentraciones de 1 mM y 2 mM, se observó un aumento de más de dos veces de la actividad de la enzima en lisados del corazón, riñon y pulmones, y de 1 ,2 veces en lisados del cerebro. A concentraciones de 5 mM y 10 mM la actividad en lisados del cortex cerebral y del tallo cerebral aumentó en más de cuatro veces. Además, se observó una reducción muy notable de la acumulación de gangliósido GM1 en los lisosomas. Los datos demuestran de manera inequívoca que el compuesto 6S-NBI-DGJ atraviesa la barrera hematoencefálica, aumenta la actividad de la β-galactosidasa muíante y produce una mejora significativa en la paíología cerebral asociada a la deficiencia de la β-galacíosidasa. Effect of compound 6S-NBI-DGJ on brain tissue and other organs of mice expressing the R201 C mutation in 3-galactosidase To obtain a proof of concept of the ability of the 6S-NBI-DGJ compound to cross the blood brain membrane, as well as to activate the mutant β-galactosidase in different organs, doses thereof (1 mM, 2 mM, 5 mM and 10 mM in water) to mice expressing human β-galactosidase with the R201 C mutation orally for one week. When concentrations of 1 mM and 2 mM were used, an increase of more than twice the activity of the enzyme was observed in lysates of the heart, kidney and lungs, and 1, 2 times in lysates of the brain. At concentrations of 5 mM and 10 mM the activity in lysates of the cerebral cortex and brain stem increased by more than four times. In addition, a very notable reduction in the accumulation of GM1 ganglioside in lysosomes was observed. The data unequivocally demonstrates that the compound 6S-NBI-DGJ crosses the blood-brain barrier, increases the activity of the mutant β-galactosidase and produces a significant improvement in cerebral pathology associated with the β-galaciosidase deficiency.

Claims

REIVINDICACIONES
1.- Uso de un compuesto de fórmula (I) 1.- Use of a compound of formula (I)
Figure imgf000014_0001
o una de sus sales, donde:
Figure imgf000014_0001
or one of its salts, where:
R representa una cadena alifática, lineal o ramificada, que se selecciona del grupo que consiste en cadena hidrocarbonada saturada C Ci6, y cadena hidrocarbonada insaturada en la preparación de un medicamento para el tratamiento de enfermedades relacionadas con enzimas β-galactosidasas lisosómicas mutantes en humanos. R represents an aliphatic chain, linear or branched, which is selected from the group consisting of saturated hydrocarbon chain C Ci 6 , and unsaturated hydrocarbon chain in the preparation of a medicament for the treatment of diseases related to mutant lysosomal β-galactosidase enzymes in humans .
2. - Uso de un compuesto se fórmula general (I) según la reivindicación 1 , caracterizado porque R es una cadena hidrocarbonada saturada C Ci6. 2. - Use of a compound with general formula (I) according to claim 1, characterized in that R is a saturated C Ci 6 hydrocarbon chain.
3. - Uso de un compuesto de fórmula general (I) según una cualquiera de las reivindicaciones anteriores, caracterizado porque R es una cadena hidrocarbonada lineal. 3. - Use of a compound of general formula (I) according to any one of the preceding claims, characterized in that R is a linear hydrocarbon chain.
4.- Uso de un compuesto de según la reivindicación 3, caracterizado porque dicho compuesto es 5/V,6S-(/V-butiliminometiliden)-6-tio-1-desoxigalactonojirimicina. 4.- Use of a compound according to claim 3, characterized in that said compound is 5/V,6S-(/V-butyliminomethylidene)-6-thio-1-deoxygalactonojirimycin.
5. - Uso de un compuesto de fórmula general (I) según una cualquiera de las reivindicaciones 1 a 4 en la preparación de un medicamento para el tratamiento de la enfermedad Morquio B en humanos. 5. - Use of a compound of general formula (I) according to any one of claims 1 to 4 in the preparation of a medication for the treatment of Morquio B disease in humans.
6. - Uso de un compuesto de fórmula general (I) según una cualquiera de las reivindicaciones 1 a 4, en la preparación de un medicamento para el tratamiento de la enfermedad Gangliosidosis GM 1 en humanos. 6. - Use of a compound of general formula (I) according to any one of claims 1 to 4, in the preparation of a medication for the treatment of the GM 1 Gangliosidosis disease in humans.
7. - Uso de un compuesto de fórmula general (I) según una cualquiera de las reivindicaciones 1 a 6, en la preparación de un medicamento para el tratamiento de enfermedades relacionadas con enzimas β-galactosidasas lisosómicas mutantes humanas seleccionadas entre algunos de los siguientes mutantes: G190D, R201C, R201 H, V216A, D332N, Y444C, R457Q, R590H, I51T, R148T, L155R, R208C, D214Y, C230Y, L264S, N266S, W273R, K346N, S434L, G438E, R482H, D491Y, E632G y D640E. 7. - Use of a compound of general formula (I) according to any one of claims 1 to 6, in the preparation of a medication for the treatment of diseases related to human mutant lysosomal β-galactosidase enzymes selected from some of the following mutants: G190D, R201C, R201 H, V216A, D332N, Y444C, R457Q, R590H, I51T, R148T, L155R, R208C, D214Y, C230Y, L264S, N266S , W273R, K346N, S434L, G438E, R482H, D491Y, E632G and D640E.
8. - Uso de un compuesto de fórmula general (I) según la reivindicación 7, caracterizado porque las enzimas β-galactosidasas lisosómicas mutantes humanas se seleccionan entre algunos de los siguientes mutantes: I51T, R148T, L155R, R208C, D214Y, C230Y, L264S, N266S, W273R, K346N, S434L, G438E, R482H, D491Y, E632G y D640E. 8. - Use of a compound of general formula (I) according to claim 7, characterized in that the human mutant lysosomal β-galactosidase enzymes are selected from some of the following mutants: I51T, R148T, L155R, R208C, D214Y, C230Y, L264S , N266S, W273R, K346N, S434L, G438E, R482H, D491Y, E632G and D640E.
9. - Composición farmacéutica para el tratamiento de enfermedades relacionadas con enzimas β-galactosidasas lisosómicas mutantes en humanos que comprende un compuesto de fórmula general (I)9. - Pharmaceutical composition for the treatment of diseases related to mutant lysosomal β-galactosidase enzymes in humans comprising a compound of general formula (I)
10.- Composición farmacéutica según la reivindicación 9, caracterizada porque que adicionalmente comprende otro principio activo. 10.- Pharmaceutical composition according to claim 9, characterized in that it additionally comprises another active ingredient.
11.- Composición farmacéutica según las reivindicaciones 9 y 10 caracterizada porque comprende un vehículo farmacéuticamente aceptable. 11.- Pharmaceutical composition according to claims 9 and 10 characterized in that it comprises a pharmaceutically acceptable vehicle.
PCT/ES2013/070883 2012-12-26 2013-12-17 USE OF BICYCLIC DERIVATIVES OF 1-DEOXYGALACTONOJIRIMYCIN IN THE PRODUCTION OF A MEDICAMENT FOR THE TREATMENT OF DISEASES ASSOCIATED WITH MUTANT HUMAN LYSOSOMAL GALACTOSIDASE ß-ENZYMES WO2014102417A1 (en)

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Citations (2)

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WO2004037373A2 (en) * 2002-10-21 2004-05-06 The Scripps Research Institute CHEMICAL CHAPERONES AND THEIR EFFECT UPON THE CELLULAR ACTIVITY OF β-GLUCOSIDASE
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